Veröffentlichungen

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  Poerschke, S., Oeljeklaus, S., Cruz-Zaragoza, L. D., Schenzielorz, A., Dahal, D., Hillen, H. S., Das, H., Kremer, L. S., Valpadashi, A., Breuer, M., Sattmann, J., Richter-Dennerlein, R., Warscheid, B., Dennerlein, S., & Rehling, P. (2024). Identification of TMEM126A as OXA1L-interacting Protein Reveals Cotranslational Quality Control in Mitochondria. Molecular Cell, 84(2), 345-358.e5. https://doi.org/10.1016/j.molcel.2023.12.013
  • [ Abstract ]
  • [ DOI ]
  Cellular proteostasis requires transport of polypeptides across membranes. Although defective transport processes trigger cytosolic rescue and quality control mechanisms that clear translocases and membranes from unproductive cargo, proteins that are synthesized within mitochondria are not accessible to these mechanisms. Mitochondrial-encoded proteins are inserted cotranslationally into the inner membrane by the conserved insertase OXA1L. Here, we identify TMEM126A as a OXA1L-interacting protein. TMEM126A associates with mitochondrial ribosomes and translation products. Loss of TMEM126A leads to the destabilization of mitochondrial translation products, triggering an inner membrane quality control process, in which newly synthesized proteins are degraded by the mitochondrial iAAA protease. Our data reveal that TMEM126A cooperates with OXA1L in protein insertion into the membrane. Upon loss of TMEM126A, the cargo-blocked OXA1L insertase complexes undergo proteolytic clearance by the iAAA protease machinery together with its cargo.
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  Chen, W. W., Rodrigues, T. A., Wendscheck, D., Pedrosa, A. G., Yang, C., Francisco, T., Möcklinghoff, T., Zografakis, A., Nunes-Silva, B., Avraham, R. E., Silva, A. R., Ferreira, M. J., Das, H., Bender, J., Oeljeklaus, S., Sondhi, V., Schuldiner, M., Zalckvar, E., Hofmann, K., … Warscheid, B. (2024). PEX39 facilitates the peroxisomal import of PTS2 proteins. https://doi.org/10.1101/2024.04.30.591961
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  von Känel, C., Oeljeklaus, S., Wenger, C., Stettler, P., Harsman, A., Warscheid, B., & Schneider, A. (2024). Intermembrane Space-Localized TbTim15 Is an Essential Subunit of the Single Mitochondrial Inner Membrane Protein Translocase of Trypanosomes. Molecular Microbiology. https://doi.org/10.1111/mmi.15262
  • [ Abstract ]
  • [ DOI ]
  All mitochondria import {\($>$\)95% of their proteins from the cytosol. This process is mediated by protein translocases in the mitochondrial membranes, whose subunits are generally highly conserved. Most eukaryotes have two inner membrane protein translocases (TIMs) that are specialized to import either presequence-containing or mitochondrial carrier proteins. In contrast, the parasitic protozoan Trypanosoma brucei has a single TIM complex consisting of one conserved and five unique subunits. Here, we identify candidates for new subunits of the TIM or the presequence translocase-associated motor (PAM) using a protein-protein interaction network of previously characterized TIM and PAM subunits. This analysis reveals that the trypanosomal TIM complex contains an additional trypanosomatid-specific subunit, designated TbTim15. TbTim15 is associated with the TIM complex, lacks transmembrane domains, and localizes to the intermembrane space. TbTim15 is essential for procyclic and bloodstream forms of trypanosomes. It contains two twin CX(9)C motifs and mediates import of both presequence-containing and mitochondrial carrier proteins. While the precise function of TbTim15 in mitochondrial protein import is unknown, our results are consistent with the notion that it may function as an import receptor for the non-canonical trypanosomal TIM complex.
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  Steinmetz, T. D., Thomas, J., Reimann, L., Himmelreich, A.-K., Schulz, S. R., Golombek, F., Castiglione, K., Jäck, H.-M., Brodesser, S., Warscheid, B., & Mielenz, D. (2024). Identification of TFG- and Autophagy-Regulated Proteins and Glycerophospholipids in B Cells. Journal of Proteome Research, 23(5), 1615-1633. https://doi.org/10.1021/acs.jproteome.3c00713
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  Autophagy supervises the proteostasis and survival of B lymphocytic cells. Trk-fused gene (TFG) promotes autophagosome-lysosome flux in murine CH12 B cells, as well as their survival. Hence, quantitative proteomics of CH12tfgKO and WT B cells in combination with lysosomal inhibition should identify proteins that are prone to lysosomal degradation and contribute to autophagy and B cell survival. Lysosome inhibition via NH(4)Cl unexpectedly reduced a number of proteins but increased a large cluster of translational, ribosomal, and mitochondrial proteins, independent of TFG. Hence, we propose a role for lysosomes in ribophagy in B cells. TFG-regulated proteins include CD74, BCL10, or the immunoglobulin JCHAIN. Gene ontology (GO) analysis reveals that proteins regulated by TFG alone, or in concert with lysosomes, localize to mitochondria and membrane-bound organelles. Likewise, TFG regulates the abundance of metabolic enzymes, such as ALDOC and the fatty acid-activating enzyme ACOT9. To test consequently for a function of TFG in lipid metabolism, we performed shotgun lipidomics of glycerophospholipids. Total phosphatidylglycerol is more abundant in CH12tfgKO B cells. Several glycerophospholipid species with similar acyl side chains, such as 36:2 phosphatidylethanolamine and 36:2 phosphatidylinositol, show a dysequilibrium. We suggest a role for TFG in lipid homeostasis, mitochondrial functions, translation, and metabolism in B cells.
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  Jonak, K., Suppanz, I., Bender, J., Chacinska, A., Warscheid, B., & Topf, U. (2024). Ageing-Dependent Thiol Oxidation Reveals Early Oxidation of Proteins with Core Proteostasis Functions. Life Science Alliance, 7(5), Article 5. https://doi.org/10.26508/lsa.202302300
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  Oxidative post-translational modifications of protein thiols are well recognized as a readily occurring alteration of proteins, which can modify their function and thus control cellular processes. The development of techniques enabling the site-specific assessment of protein thiol oxidation on a proteome-wide scale significantly expanded the number of known oxidation-sensitive protein thiols. However, lacking behind are large-scale data on the redox state of proteins during ageing, a physiological process accompanied by increased levels of endogenous oxidants. Here, we present the landscape of protein thiol oxidation in chronologically aged wild-type Saccharomyces cerevisiae in a time-dependent manner. Our data determine early-oxidation targets in key biological processes governing the de novo production of proteins, protein folding, and degradation, and indicate a hierarchy of cellular responses affected by a reversible redox modification. Comparison with existing datasets in yeast, nematode, fruit fly, and mouse reveals the evolutionary conservation of these oxidation targets. To facilitate accessibility, we integrated the cross-species comparison into the newly developed OxiAge Database.

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  Guhathakurta, S., Erdogdu, N. U., Hoffmann, J. J., Grzadzielewska, I., Schendzielorz, A., Seyfferth, J., Maa}rtensson, C. U., Corrado, M., Karoutas, A., Warscheid, B., Pfanner, N., Becker, T., & Akhtar, A. (2023). COX17 Acetylation via MOF-KANSL Complex Promotes Mitochondrial Integrity and Function. Nature Metabolism, 5(11), 1931-1952. https://doi.org/10.1038/s42255-023-00904-w
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  Reversible acetylation of mitochondrial proteins is a regulatory mechanism central to adaptive metabolic responses. Yet, how such functionally relevant protein acetylation is achieved remains unexplored. Here we reveal an unprecedented role of the MYST family lysine acetyltransferase MOF in energy metabolism via mitochondrial protein acetylation. Loss of MOF-KANSL complex members leads to mitochondrial defects including fragmentation, reduced cristae density and impaired mitochondrial electron transport chain complex IV integrity in primary mouse embryonic fibroblasts. We demonstrate COX17, a complex IV assembly factor, as a bona fide acetylation target of MOF. Loss of COX17 or expression of its non-acetylatable mutant phenocopies the mitochondrial defects observed upon MOF depletion. The acetylation-mimetic COX17 rescues these defects and maintains complex IV activity even in the absence of MOF, suggesting an activatory role of mitochondrial electron transport chain protein acetylation. Fibroblasts from patients with MOF syndrome who have intellectual disability also revealed respiratory defects that could be restored by alternative oxidase, acetylation-mimetic COX17 or mitochondrially targeted MOF. Overall, our findings highlight the critical role of MOF-KANSL complex in mitochondrial physiology and provide new insights into MOF syndrome.
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  Fischer, S., Bürgi, J., Gabay-Maskit, S., Maier, R., Mastalski, T., Yifrach, E., Obarska-Kosinska, A., Rudowitz, M., Erdmann, R., Platta, H. W., Wilmanns, M., Schuldiner, M., Zalckvar, E., Oeljeklaus, S., Drepper, F., & Warscheid, B. (2023). Phosphorylation of the Receptor Protein Pex5p Modulates Import of Proteins into Peroxisomes. Biological Chemistry, 404(2-3), 135-155. https://doi.org/10.1515/hsz-2022-0168
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  Peroxisomes are organelles with vital functions in metabolism and their dysfunction is associated with human diseases. To fulfill their multiple roles, peroxisomes import nuclear-encoded matrix proteins, most carrying a peroxisomal targeting signal (PTS) 1. The receptor Pex5p recruits PTS1-proteins for import into peroxisomes; whether and how this process is posttranslationally regulated is unknown. Here, we identify 22 phosphorylation sites of Pex5p. Yeast cells expressing phospho-mimicking Pex5p-S507/523D (Pex5p(2D)) show decreased import of GFP with a PTS1. We show that the binding affinity between a PTS1-protein and Pex5p(2D) is reduced. An in vivo analysis of the effect of the phospho-mimicking mutant on PTS1-proteins revealed that import of most, but not all, cargos is affected. The physiological effect of the phosphomimetic mutations correlates with the binding affinity of the corresponding extended PTS1-sequences. Thus, we report a novel Pex5p phosphorylation-dependent mechanism for regulating PTS1-protein import into peroxisomes. In a broader view, this suggests that posttranslational modifications can function in fine-tuning the peroxisomal protein composition and, thus, cellular metabolism.
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  Fricke, A. L., Mühlhäuser, W. W. D., Reimann, L., Zimmermann, J. P., Reichenbach, C., Knapp, B., Peikert, C. D., Heberle, A. M., Faessler, E., Schäuble, S., Hahn, U., Thedieck, K., Radziwill, G., & Warscheid, B. (2023). Phosphoproteomics Profiling Defines a Target Landscape of the Basophilic Protein Kinases AKT, S6K, and RSK in Skeletal Myotubes. Journal of Proteome Research, 22(3), 768-789. https://doi.org/10.1021/acs.jproteome.2c00505
  • [ Abstract ]
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  Phosphorylation-dependent signal transduction plays an important role in regulating the functions and fate of skeletal muscle cells. Central players in the phospho-signaling network are the protein kinases AKT, S6K, and RSK as part of the PI3K-AKT-mTOR-S6K and RAF-MEK-ERK-RSK pathways. However, despite their functional importance, knowledge about their specific targets is incomplete because these kinases share the same basophilic substrate motif RxRxxp[ST]. To address this, we performed a multifaceted quantitative phosphoproteomics study of skeletal myotubes following kinase inhibition. Our data corroborate a cross talk between AKT and RAF, a negative feedback loop of RSK on ERK, and a putative connection between RSK and PI3K signaling. Altogether, we report a kinase target landscape containing 49 so far unknown target sites. AKT, S6K, and RSK phosphorylate numerous proteins involved in muscle development, integrity, and functions, and signaling converges on factors that are central for the skeletal muscle cytoskeleton. Whereas AKT controls insulin signaling and impinges on GTPase signaling, nuclear signaling is characteristic for RSK. Our data further support a role of RSK in glucose metabolism. Shared targets have functions in RNA maturation, stability, and translation, which suggests that these basophilic kinases establish an intricate signaling network to orchestrate and regulate processes involved in translation.
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  Wenger, C., Harsman, A., Niemann, M., Oeljeklaus, S., von Känel, C., Calderaro, S., Warscheid, B., & Schneider, A. (2023). The Mba1 Homologue of Trypanosoma Brucei Is Involved in the Biogenesis of Oxidative Phosphorylation Complexes. Molecular Microbiology, 119(5), 537-550. https://doi.org/10.1111/mmi.15048
  • [ Abstract ]
  • [ DOI ]
  Consistent with other eukaryotes, the Trypanosoma brucei mitochondrial genome encodes mainly hydrophobic core subunits of the oxidative phosphorylation system. These proteins must be co-translationally inserted into the inner mitochondrial membrane and are synthesized by the highly unique trypanosomal mitoribosomes, which have a much higher protein to RNA ratio than any other ribosome. Here, we show that the trypanosomal orthologue of the mitoribosome receptor Mba1 (TbMba1) is essential for normal growth of procyclic trypanosomes but redundant in the bloodstream form, which lacks an oxidative phosphorylation system. Proteomic analyses of TbMba1-depleted mitochondria from procyclic cells revealed reduced levels of many components of the oxidative phosphorylation system, most of which belong to the cytochrome c oxidase (Cox) complex, three subunits of which are mitochondrially encoded. However, the integrity of the mitoribosome and its interaction with the inner membrane were not affected. Pull-down experiments showed that TbMba1 forms a dynamic interaction network that includes the trypanosomal Mdm38/Letm1 orthologue and a trypanosome-specific factor that stabilizes the CoxI and CoxII mRNAs. In summary, our study suggests that the function of Mba1 in the biogenesis of membrane subunits of OXPHOS complexes is conserved among yeast, mammals and trypanosomes, which belong to two eukaryotic supergroups.
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  Gerber, M., Suppanz, I., Oeljeklaus, S., Niemann, M., Käser, S., Warscheid, B., Schneider, A., & Dewar, C. E. (2023). A Msp1-containing Complex Removes Orphaned Proteins in the Mitochondrial Outer Membrane of T. Brucei. Life Science Alliance, 6(11), Article 11. https://doi.org/10.26508/lsa.202302004
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  • [ DOI ]
  The AAA-ATPase Msp1 extracts mislocalised outer membrane proteins and thus contributes to mitochondrial proteostasis. Using pulldown experiments, we show that trypanosomal Msp1 localises to both glycosomes and the mitochondrial outer membrane, where it forms a complex with four outer membrane proteins. The trypanosome-specific pATOM36 mediates complex assembly of {\($\alpha$\)}-helically anchored mitochondrial outer membrane proteins such as protein translocase subunits. Inhibition of their assembly triggers a pathway that results in the proteasomal digestion of unassembled substrates. Using inducible single, double, and triple RNAi cell lines combined with proteomic analyses, we demonstrate that not only Msp1 but also the trypanosomal homolog of the AAA-ATPase VCP are implicated in this quality control pathway. Moreover, in the absence of VCP three out of the four Msp1-interacting mitochondrial proteins are required for efficient proteasomal digestion of pATOM36 substrates, suggesting they act in concert with Msp1. pATOM36 is a functional analog of the yeast mitochondrial import complex complex and possibly of human mitochondrial animal-specific carrier homolog 2, suggesting that similar mitochondrial quality control pathways linked to Msp1 might also exist in yeast and humans.
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  Xu, F., Li, R., von Gromoff, E. D., Drepper, F., Knapp, B., Warscheid, B., Baumeister, R., & Qi, W. (2023). Reprogramming of the Transcriptome after Heat Stress Mediates Heat Hormesis in Caenorhabditis Elegans. Nature Communications, 14(1), 4176. https://doi.org/10.1038/s41467-023-39882-8
  • [ Abstract ]
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  Transient stress experiences not only trigger acute stress responses, but can also have long-lasting effects on cellular functions. In Caenorhabditis elegans, a brief exposure to heat shock during early adulthood extends lifespan and improves stress resistance, a phenomenon known as heat hormesis. Here, we investigated the prolonged effect of hormetic heat stress on the transcriptome of worms and found that the canonical heat shock response is followed by a profound transcriptional reprogramming in the post-stress period. This reprogramming relies on the endoribonuclease ENDU-2 but not the heat shock factor 1. ENDU-2 co-localizes with chromatin and interacts with RNA polymerase II, enabling specific regulation of transcription after the stress period. Failure to activate the post-stress response does not affect the resistance of animals to heat shock but eliminates the beneficial effects of hormetic heat stress. In summary, our work discovers that the RNA-binding protein ENDU-2 mediates the long-term impacts of transient heat stress via reprogramming transcriptome after stress exposure.
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  Becker, F., Fuchs, S., Refisch, L., Drepper, F., Bildl, W., Schulte, U., Liang, S., Heinicke, J. I., Hansen, S. C., Kreutz, C., Warscheid, B., Fakler, B., Mymrikov, E. V., & Hunte, C. (2023). Conformational Regulation and Target-Myristoyl Switch of Calcineurin B Homologous Protein 3. ELife, 12. https://doi.org/10.7554/eLife.83868
  • [ Abstract ]
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  Calcineurin B homologous protein 3 (CHP3) is an EF-hand Ca(2+)-binding protein involved in regulation of cancerogenesis, cardiac hypertrophy, and neuronal development through interactions with sodium/proton exchangers (NHEs) and signalling proteins. While the importance of Ca(2+) binding and myristoylation for CHP3 function has been recognized, the underlying molecular mechanism remained elusive. In this study, we demonstrate that Ca(2+) binding and myristoylation independently affect the conformation and functions of human CHP3. Ca(2+) binding increased local flexibility and hydrophobicity of CHP3 indicative of an open conformation. The Ca(2+)-bound CHP3 exhibited a higher affinity for NHE1 and associated stronger with lipid membranes compared to the Mg(2+)-bound CHP3, which adopted a closed conformation. Myristoylation enhanced the local flexibility of CHP3 and decreased its affinity to NHE1 independently of the bound ion, but did not affect its binding to lipid membranes. The data exclude the proposed Ca(2+)-myristoyl switch for CHP3. Instead, a Ca(2+)-independent exposure of the myristoyl moiety is induced by binding of the target peptide to CHP3 enhancing its association to lipid membranes. We name this novel regulatory mechanism 'target-myristoyl switch'. Collectively, the interplay of Ca(2+) binding, myristoylation, and target binding allows for a context-specific regulation of CHP3 functions.
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  Kim, M., Serwa, R. A., Samluk, L., Suppanz, I., Kodroń, A., Stk epkowski, T. M., Elancheliyan, P., Tsegaye, B., Oeljeklaus, S., Wasilewski, M., Warscheid, B., & Chacinska, A. (2023). Immunoproteasome-Specific Subunit PSMB9 Induction Is Required to Regulate Cellular Proteostasis upon Mitochondrial Dysfunction. Nature Communications, 14(1), 4092. https://doi.org/10.1038/s41467-023-39642-8
  • [ Abstract ]
  • [ DOI ]
  Perturbed cellular protein homeostasis (proteostasis) and mitochondrial dysfunction play an important role in neurodegenerative diseases, however, the interplay between these two phenomena remains unclear. Mitochondrial dysfunction leads to a delay in mitochondrial protein import, causing accumulation of non-imported mitochondrial proteins in the cytosol and challenging proteostasis. Cells respond by increasing proteasome activity and molecular chaperones in yeast and C. elegans. Here, we demonstrate that in human cells mitochondrial dysfunction leads to the upregulation of a chaperone HSPB1 and, interestingly, an immunoproteasome-specific subunit PSMB9. Moreover, PSMB9 expression is dependent on the translation elongation factor EEF1A2. These mechanisms constitute a defense response to preserve cellular proteostasis under mitochondrial stress. Our findings define a mode of proteasomal activation through the change in proteasome composition driven by EEF1A2 and its spatial regulation, and are useful to formulate therapies to prevent neurodegenerative diseases.
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  Das, H., Zografakis, A., Oeljeklaus, S., & Warscheid, B. (2023). Analysis of Yeast Peroxisomes via Spatial Proteomics. Methods in Molecular Biology (Clifton, N.J.), 2643, 13-31. https://doi.org/10.1007/978-1-0716-3048-8_2
  • [ Abstract ]
  • [ DOI ]
  Peroxisomes are ubiquitous organelles with essential functions in numerous cellular processes such as lipid metabolism, detoxification of reactive oxygen species, and signaling. Knowledge of the peroxisomal proteome including multi-localized proteins and, most importantly, changes of its composition induced by altering cellular conditions or impaired peroxisome biogenesis and function is of paramount importance for a holistic view on peroxisomes and their diverse functions in a cellular context. In this chapter, we provide a spatial proteomics protocol specifically tailored to the analysis of the peroxisomal proteome of baker's yeast that enables the definition of the peroxisomal proteome under distinct conditions and to monitor dynamic changes of the proteome including the relocation of individual proteins to a different cellular compartment. The protocol comprises subcellular fractionation by differential centrifugation followed by Nycodenz density gradient centrifugation of a crude peroxisomal fraction, quantitative mass spectrometric measurements of subcellular and density gradient fractions, and advanced computational data analysis, resulting in the establishment of organellar maps on a global scale.

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  Iyer, A., Niemann, M., Serricchio, M., Dewar, C. E., Oeljeklaus, S., Farine, L., Warscheid, B., Schneider, A., & Bütikofer, P. (2022). The Endoplasmic Reticulum Membrane Protein Complex Localizes to the Mitochondrial - Endoplasmic Reticulum Interface and Its Subunits Modulate Phospholipid Biosynthesis in Trypanosoma Brucei. PLoS Pathogens, 18(5), e1009717. https://doi.org/10.1371/journal.ppat.1009717
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  • [ DOI ]
  The endoplasmic reticulum membrane complex (EMC) is a versatile complex that plays a key role in membrane protein biogenesis in the ER. Deletion of the complex has wide-ranging consequences including ER stress, disturbance in lipid transport and organelle tethering, among others. Here we report the function and organization of the evolutionarily conserved EMC (TbEMC) in the highly diverged eukaryote, Trypanosoma brucei. Using (co-) immunoprecipitation experiments in combination with mass spectrometry and whole cell proteomic analyses of parasites after depletion of select TbEMC subunits, we demonstrate that the TbEMC is composed of 9 subunits that are present in a high molecular mass complex localizing to the mitochondrial-endoplasmic reticulum interface. Knocking out or knocking down of single TbEMC subunits led to growth defects of T. brucei procyclic forms in culture. Interestingly, we found that depletion of individual TbEMC subunits lead to disruption of de novo synthesis of phosphatidylcholine (PC) or phosphatidylethanolamine (PE), the two most abundant phospholipid classes in T. brucei. Downregulation of TbEMC1 or TbEMC3 inhibited formation of PC while depletion of TbEMC8 inhibited PE synthesis, pointing to a role of the TbEMC in phospholipid synthesis. In addition, we found that in TbEMC7 knock-out parasites, TbEMC3 is released from the complex, implying that TbEMC7 is essential for the formation or the maintenance of the TbEMC.
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  Schimanski, B., Aeschlimann, S., Stettler, P., Käser, S., Gomez-Fabra Gala, M., Bender, J., Warscheid, B., Vögtle, F.-N., & Schneider, A. (2022). P166 Links Membrane and Intramitochondrial Modules of the Trypanosomal Tripartite Attachment Complex. PLoS Pathogens, 18(6), e1010207. https://doi.org/10.1371/journal.ppat.1010207
  • [ Abstract ]
  • [ DOI ]
  The protist parasite Trypanosoma brucei has a single mitochondrion with a single unit genome termed kinetoplast DNA (kDNA). Faithfull segregation of replicated kDNA is ensured by a complicated structure termed tripartite attachment complex (TAC). The TAC physically links the basal body of the flagellum with the kDNA spanning the two mitochondrial membranes. Here, we characterized p166 as the only known TAC subunit that is anchored in the inner membrane. Its C-terminal transmembrane domain separates the protein into a large N-terminal region that interacts with the kDNA-localized TAC102 and a 34 aa C-tail that binds to the intermembrane space-exposed loop of the integral outer membrane protein TAC60. Whereas the outer membrane region requires four essential subunits for proper TAC function, the inner membrane integral p166, via its interaction with TAC60 and TAC102, would theoretically suffice to bridge the distance between the OM and the kDNA. Surprisingly, non-functional p166 lacking the C-terminal 34 aa still localizes to the TAC region. This suggests the existence of additional TAC-associated proteins which loosely bind to non-functional p166 lacking the C-terminal 34 aa and keep it at the TAC. However, binding of full length p166 to these TAC-associated proteins alone would not be sufficient to withstand the mechanical load imposed by the segregating basal bodies.
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  Dewar, C. E., Oeljeklaus, S., Wenger, C., Warscheid, B., & Schneider, A. (2022). Characterization of a Highly Diverged Mitochondrial ATP Synthase F(o) Subunit in Trypanosoma Brucei. The Journal of Biological Chemistry, 298(4), 101829. https://doi.org/10.1016/j.jbc.2022.101829
  • [ Abstract ]
  • [ DOI ]
  The mitochondrial F(1)F(o) ATP synthase of the parasite Trypanosoma brucei has been previously studied in detail. This unusual enzyme switches direction in functionality during the life cycle of the parasite, acting as an ATP synthase in the insect stages, and as an ATPase to generate mitochondrial membrane potential in the mammalian bloodstream stages. Whereas the trypanosome F(1) moiety is relatively highly conserved in structure and composition, the F(o) subcomplex and the peripheral stalk have been shown to be more variable. Interestingly, a core subunit of the latter, the normally conserved subunit b, has been resistant to identification by sequence alignment or biochemical methods. Here, we identified a 17~kDa mitochondrial protein of the inner membrane, Tb927.8.3070, that is essential for normal growth, efficient oxidative phosphorylation, and membrane potential maintenance. Pull-down experiments and native PAGE analysis indicated that the protein is both associated with the F(1)F(o) ATP synthase and integral to its assembly. In addition, its knockdown reduced the levels of F(o) subunits, but not those of F(1), and disturbed the cell cycle. Finally, analysis of structural homology using the HHpred algorithm showed that this protein has structural similarities to F(o) subunit b of other species, indicating that this subunit may be a highly diverged form of the elusive subunit b.
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  Dewar, C. E., Oeljeklaus, S., Mani, J., Mühlhäuser, W. W. D., von Känel, C., Zimmermann, J., Ochsenreiter, T., Warscheid, B., & Schneider, A. (2022). Mistargeting of Aggregation Prone Mitochondrial Proteins Activates a Nucleus-Mediated Posttranscriptional Quality Control Pathway in Trypanosomes. Nature Communications, 13(1), 3084. https://doi.org/10.1038/s41467-022-30748-z
  • [ Abstract ]
  • [ DOI ]
  Mitochondrial protein import in the parasitic protozoan Trypanosoma brucei is mediated by the atypical outer membrane translocase, ATOM. It consists of seven subunits including ATOM69, the import receptor for hydrophobic proteins. Ablation of ATOM69, but not of any other subunit, triggers a unique quality control pathway resulting in the proteasomal degradation of non-imported mitochondrial proteins. The process requires a protein of unknown function, an E3 ubiquitin ligase and the ubiquitin-like protein (TbUbL1), which all are recruited to the mitochondrion upon ATOM69 depletion. TbUbL1 is a nuclear protein, a fraction of which is released to the cytosol upon triggering of the pathway. Nuclear release is essential as cytosolic TbUbL1 can bind mislocalised mitochondrial proteins and likely transfers them to the proteasome. Mitochondrial quality control has previously been studied in yeast and metazoans. Finding such a pathway in the highly diverged trypanosomes suggests such pathways are an obligate feature of all eukaryotes.
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  Pinter, N., Glätzer, D., Fahrner, M., Fröhlich, K., Johnson, J., Grüning, B. A., Warscheid, B., Drepper, F., Schilling, O., & Föll, M. C. (2022). MaxQuant and MSstats in Galaxy Enable Reproducible Cloud-Based Analysis of Quantitative Proteomics Experiments for Everyone. Journal of Proteome Research, 21(6), 1558-1565. https://doi.org/10.1021/acs.jproteome.2c00051
  • [ Abstract ]
  • [ DOI ]
  Quantitative mass spectrometry-based proteomics has become a high-throughput technology for the identification and quantification of thousands of proteins in complex biological samples. Two frequently used tools, MaxQuant and MSstats, allow for the analysis of raw data and finding proteins with differential abundance between conditions of interest. To enable accessible and reproducible quantitative proteomics analyses in a cloud environment, we have integrated MaxQuant (including TMTpro 16/18plex), Proteomics Quality Control (PTXQC), MSstats, and MSstatsTMT into the open-source Galaxy framework. This enables the web-based analysis of label-free and isobaric labeling proteomics experiments via Galaxy's graphical user interface on public clouds. MaxQuant and MSstats in Galaxy can be applied in conjunction with thousands of existing Galaxy tools and integrated into standardized, sharable workflows. Galaxy tracks all metadata and intermediate results in analysis histories, which can be shared privately for collaborations or publicly, allowing full reproducibility and transparency of published analysis. To further increase accessibility, we provide detailed hands-on training materials. The integration of MaxQuant and MSstats into the Galaxy framework enables their usage in a reproducible way on accessible large computational infrastructures, hence realizing the foundation for high-throughput proteomics data science for everyone.
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  Kors, S., Hacker, C., Bolton, C., Maier, R., Reimann, L., Kitchener, E. J. A., Warscheid, B., Costello, J. L., & Schrader, M. (2022). Regulating Peroxisome-ER Contacts via the ACBD5-VAPB Tether by FFAT Motif Phosphorylation and {{GSK3\($\beta$\)}}. The Journal of Cell Biology, 221(3), Article 3. https://doi.org/10.1083/jcb.202003143
  • [ Abstract ]
  • [ DOI ]
  Peroxisomes and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism. They form membrane contacts through interaction of the peroxisomal membrane protein ACBD5 (acyl-coenzyme A-binding domain protein 5) and the ER-resident protein VAPB (vesicle-associated membrane protein-associated protein B). ACBD5 binds to the major sperm protein domain of VAPB via its FFAT-like (two phenylalanines [FF] in an acidic tract) motif. However, molecular mechanisms, which regulate formation of these membrane contact sites, are unknown. Here, we reveal that peroxisome-ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. We show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT-like motif, which alter interaction with VAPB-and thus peroxisome-ER contact sites-differently. Moreover, we demonstrate that GSK3{\($\beta$\)} (glycogen synthase kinase-3 {\($\beta$\)}) regulates this interaction. Our findings reveal for the first time a molecular mechanism for the regulation of peroxisome-ER contacts in mammalian cells and expand the current model of FFAT motifs and VAP interaction.
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  Thuma, N., Döhler, D., Mielenz, D., Sticht, H., Radtke, D., Reimann, L., Warscheid, B., & Voehringer, D. (2022). A Newly Identified Secreted Larval Antigen Elicits Basophil-Dependent Protective Immunity against N. Brasiliensis Infection. Frontiers in Immunology, 13, 979491. https://doi.org/10.3389/fimmu.2022.979491
  • [ Abstract ]
  • [ DOI ]
  Hookworms infect more that 400 million people and cause significant socio-economic burden on endemic countries. The lack of efficient vaccines and the emergence of anthelminthic drug resistance are of major concern. Free-living hookworm larvae infect their hosts via the skin and live as adult worms in the small intestine where they feed on host tissue and blood. Excretory/secretory (E/S) products, released by helminths as they migrate through their host, are thought to play a key role in facilitating infection and successful establishment of parasitism. However, E/S products can also elicit protective immune responses that might be harnessed for vaccine development. By performing Western blots with serum of Nippostrongylus brasiliensis (Nb) infected mice as a model for human hookworm infection, we identified a largely overlapping set of IgG1- and IgE-reactive antigens in E/S from infective L3 stage larvae. Mass spectrometry analysis led to the identification of a new protein family with 6 paralogues in the Nb genome which we termed Nb-LSA1 for "Nippostrongylus brasiliensis larval secreted protein 1". The recombinantly expressed 17 kDa family member Nb-LSA1a was recognized by antibodies in the serum of Nb immune mice. Immunization of mice with Nb-LSA1a in alum elicited a strong IgG1 response but no detectable antigen-specific IgE. Most importantly, immunized mice were largely protected against a challenge Nb infection. This effect was dependent on the presence of basophils and occurred before the parasites reached the intestine. Therefore, basophils appear to play a critical role for rapid control of infection with L3 stage larvae in mice immunized with a single secreted larval protein. A better understanding of basophil-mediated protective immunity and identification of potent larval antigens of human hookworms could help to develop promising vaccination strategies.

• 
  Schwarz, J. J., Grundmann, L., Kokot, T., Kläsener, K., Fotteler, S., Medgyesi, D., Köhn, M., Reth, M., & Warscheid, B. (2021). Quantitative Proteomics Identifies PTP1B as Modulator of B Cell Antigen Receptor Signaling. Life Science Alliance, 4(11), Article 11. https://doi.org/10.26508/lsa.202101084
  • [ Abstract ]
  • [ DOI ]
  B cell antigen receptor (BCR) signaling is initiated by protein kinases and limited by counteracting phosphatases that currently are less well studied in their regulation of BCR signaling. Here, we used the B cell line Ramos to identify and quantify human B cell signaling components. Specifically, a protein tyrosine phosphatase profiling revealed a high expression of the protein tyrosine phosphatase 1B (PTP1B) in Ramos and human naïve B cells. The loss of PTP1B leads to increased B cell activation. Through substrate trapping in combination with quantitative mass spectrometry, we identified 22 putative substrates or interactors of PTP1B. We validated Ig{\($\alpha$\)}, CD22, PLC{\($\gamma$\)}1/2, CBL, BCAP, and APLP2 as specific substrates of PTP1B in Ramos B cells. The tyrosine kinase BTK and the two adaptor proteins GRB2 and VAV1 were identified as direct binding partners and potential substrates of PTP1B. We showed that PTP1B dephosphorylates the inhibitory receptor protein CD22 at phosphotyrosine 807. We conclude that PTP1B negatively modulates BCR signaling by dephosphorylating distinct phosphotyrosines in B cell-specific receptor proteins and various downstream signaling components.
• 
  Höhfeld, J., Benzing, T., Bloch, W., Fürst, D. O., Gehlert, S., Hesse, M., Hoffmann, B., Hoppe, T., Huesgen, P. F., Köhn, M., Kolanus, W., Merkel, R., Niessen, C. M., Pokrzywa, W., Rinschen, M. M., Wachten, D., & Warscheid, B. (2021). Maintaining Proteostasis under Mechanical Stress. EMBO Reports, 22(8), e52507. https://doi.org/10.15252/embr.202152507
  • [ Abstract ]
  • [ DOI ]
  Cell survival, tissue integrity and organismal health depend on the ability to maintain functional protein networks even under conditions that threaten protein integrity. Protection against such stress conditions involves the adaptation of folding and degradation machineries, which help to preserve the protein network by facilitating the refolding or disposal of damaged proteins. In multicellular organisms, cells are permanently exposed to stress resulting from mechanical forces. Yet, for long time mechanical stress was not recognized as a primary stressor that perturbs protein structure and threatens proteome integrity. The identification and characterization of protein folding and degradation systems, which handle force-unfolded proteins, marks a turning point in this regard. It has become apparent that mechanical stress protection operates during cell differentiation, adhesion and migration and is essential for maintaining tissues such as skeletal muscle, heart and kidney as well as the immune system. Here, we provide an overview of recent advances in our understanding of mechanical stress protection.
• 
  Kostan, J., Pavv siv c, M., Puv z, V., Schwarz, T. C., Drepper, F., Molt, S., Graewert, M. A., Schreiner, C., Sajko, S., van der Ven, P. F. M., Onipe, A., Svergun, D. I., Warscheid, B., Konrat, R., Fürst, D. O., Lenarv civ c, B., & Djinovi’c}}-Carugo, K. (2021). Molecular Basis of F-actin Regulation and Sarcomere Assembly via Myotilin. PLoS Biology, 19(4), e3001148. https://doi.org/10.1371/journal.pbio.3001148
  • [ Abstract ]
  • [ DOI ]
  Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs-the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and {\($\alpha$\)}-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin's structural role in Z-discs.
• 
  Dennerlein, S., Poerschke, S., Oeljeklaus, S., Wang, C., Richter-Dennerlein, R., Sattmann, J., Bauermeister, D., Hanitsch, E., Stoldt, S., Langer, T., Jakobs, S., Warscheid, B., & Rehling, P. (2021). Defining the Interactome of the Human Mitochondrial Ribosome Identifies SMIM4 and TMEM223 as Respiratory Chain Assembly Factors. ELife, 10. https://doi.org/10.7554/eLife.68213
  • [ Abstract ]
  • [ DOI ]
  Human mitochondria express a genome that encodes thirteen core subunits of the oxidative phosphorylation system (OXPHOS). These proteins insert into the inner membrane co-translationally. Therefore, mitochondrial ribosomes engage with the OXA1L-insertase and membrane-associated proteins, which support membrane insertion of translation products and early assembly steps into OXPHOS complexes. To identify ribosome-associated biogenesis factors for the OXPHOS system, we purified ribosomes and associated proteins from mitochondria. We identified TMEM223 as a ribosome-associated protein involved in complex IV biogenesis. TMEM223 stimulates the translation of COX1 mRNA and is a constituent of early COX1 assembly intermediates. Moreover, we show that SMIM4 together with C12ORF73 interacts with newly synthesized cytochrome b to support initial steps of complex III biogenesis in complex with UQCC1 and UQCC2. Our analyses define the interactome of the human mitochondrial ribosome and reveal novel assembly factors for complex III and IV biogenesis that link early assembly stages to the translation machinery.
• 
  Barchiesi, A., Bazzani, V., Jabczynska, A., Borowski, L. S., Oeljeklaus, S., Warscheid, B., Chacinska, A., Szczesny, R. J., & Vascotto, C. (2021). DNA Repair Protein APE1 Degrades Dysfunctional Abasic mRNA in Mitochondria Affecting Oxidative Phosphorylation. Journal of Molecular Biology, 433(18), 167125. https://doi.org/10.1016/j.jmb.2021.167125
  • [ Abstract ]
  • [ DOI ]
  APE1 is a multifunctional protein which plays a central role in the maintenance of nuclear and mitochondrial genomes repairing DNA lesions caused by oxidative and alkylating agents. In addition, it works as a redox signaling protein regulating gene expression by interacting with many transcriptional factors. Apart from these canonical activities, recent studies have shown that APE1 is also enzymatically active on RNA molecules. The present study unveils for the first time a new role of the mitochondrial form of APE1 protein in the metabolism of RNA in mitochondria. Our data demonstrate that APE1 is associated with mitochondrial messenger RNA and exerts endoribonuclease activity on abasic sites. Loss of APE1 results in the accumulation of damaged mitochondrial mRNA species, determining impairment in protein translation and reduced expression of mitochondrial-encoded proteins, finally leading to less efficient mitochondrial respiration. Altogether, our data demonstrate that APE1 plays an active role in the degradation of the mitochondrial mRNA and has a profound impact on mitochondrial well-being.
• 
  Dannenmaier, S., Desroches Altamirano, C., Schüler, L., Zhang, Y., Hummel, J., Milanov, M., Oeljeklaus, S., Koch, H.-G., Rospert, S., Alberti, S., & Warscheid, B. (2021). Quantitative Proteomics Identifies the Universally Conserved ATPase Ola1p as a Positive Regulator of Heat Shock Response in Saccharomyces Cerevisiae. The Journal of Biological Chemistry, 297(5), 101050. https://doi.org/10.1016/j.jbc.2021.101050
  • [ Abstract ]
  • [ DOI ]
  The universally conserved P-loop ATPase Ola1 is implicated in various cellular stress response pathways, as well as in cancer and tumor progression. However, Ola1p functions are divergent between species, and the involved mechanisms are only poorly understood. Here, we studied the role of Ola1p in the heat shock response of the yeast Saccharomyces cerevisiae using a combination of quantitative and pulse labeling-based proteomics approaches, in~vitro studies, and cell-based assays. Our data show that when heat stress is applied to cells lacking Ola1p, the expression of stress-protective proteins is enhanced. During heat stress Ola1p associates with detergent-resistant protein aggregates and rapidly forms assemblies that localize to stress granules. The assembly of Ola1p was also observed in~vitro using purified protein and conditions, which resembled those in living cells. We show that loss of Ola1p results in increased protein ubiquitination of detergent-insoluble aggregates recovered from heat-shocked cells. When cells lacking Ola1p were subsequently relieved from heat stress, reinitiation of translation was delayed, whereas, at the same time, de novo synthesis of central factors required for protein refolding and the clearance of aggregates was enhanced when compared with wild-type cells. The combined data suggest that upon acute heat stress, Ola1p is involved in the stabilization of misfolded proteins, which become sequestered in cytoplasmic stress granules. This function of Ola1p enables cells to resume translation in a timely manner as soon as heat stress is relieved.
• 
  Landwehr, V., Milanov, M., Angebauer, L., Hong, J., Jüngert, G., Hiersemenzel, A., Siebler, A., Schmit, F., Öztürk, Y., Dannenmaier, S., Drepper, F., Warscheid, B., & Koch, H.-G. (2021). The Universally Conserved ATPase YchF Regulates Translation of Leaderless mRNA in Response to Stress Conditions. Frontiers in Molecular Biosciences, 8, 643696. https://doi.org/10.3389/fmolb.2021.643696
  • [ Abstract ]
  • [ DOI ]
  The universally conserved P-loop GTPases control diverse cellular processes, like signal transduction, ribosome assembly, cell motility, and intracellular transport and translation. YchF belongs to the Obg-family of P-loop GTPases and is one of the least characterized member of this family. It is unique because it preferentially hydrolyses ATP rather than GTP, but its physiological role is largely unknown. Studies in different organisms including humans suggest a possible role of YchF in regulating the cellular adaptation to stress conditions. In the current study, we explored the role of YchF in the model organism Escherichia coli. By western blot and promoter fusion experiments, we demonstrate that YchF levels decrease during stress conditions or when cells enter stationary phase. The decline in YchF levels trigger increased stress resistance and cells lacking YchF are resistant to multiple stress conditions, like oxidative stress, replication stress, or translational stress. By in vivo site directed cross-linking we demonstrate that YchF interacts with the translation initiation factor 3 (IF3) and with multiple ribosomal proteins at the surface of the small ribosomal subunit. The absence of YchF enhances the anti-association activity of IF3, stimulates the translation of leaderless mRNAs, and increases the resistance against the endoribonuclease MazF, which generates leaderless mRNAs during stress conditions. In summary, our data identify YchF as a stress-responsive regulator of leaderless mRNA translation.
• 
  Sponga, A., Arolas, J. L., Schwarz, T. C., Jeffries, C. M., Rodriguez Chamorro, A., Kostan, J., Ghisleni, A., Drepper, F., Polyansky, A., De Almeida Ribeiro, E., Pedron, M., Zawadzka-Kazimierczuk, A., Mlynek, G., Peterbauer, T., Doto, P., Schreiner, C., Hollerl, E., Mateos, B., Geist, L., … Djinovi’c}}-Carugo, K. (2021). Order from Disorder in the Sarcomere: FATZ Forms a Fuzzy but Tight Complex and Phase-Separated Condensates with {\($\alpha$\)}-Actinin. Science Advances, 7(22), Article 22. https://doi.org/10.1126/sciadv.abg7653
  • [ Abstract ]
  • [ DOI ]
  In sarcomeres, {\($\alpha$\)}-actinin cross-links actin filaments and anchors them to the Z-disk. FATZ (filamin-, {\($\alpha$\)}-actinin-, and telethonin-binding protein of the Z-disk) proteins interact with {\($\alpha$\)}-actinin and other core Z-disk proteins, contributing to myofibril assembly and maintenance. Here, we report the first structure and its cellular validation of {\($\alpha$\)}-actinin-2 in complex with a Z-disk partner, FATZ-1, which is best described as a conformational ensemble. We show that FATZ-1 forms a tight fuzzy complex with {\($\alpha$\)}-actinin-2 and propose an interaction mechanism via main molecular recognition elements and secondary binding sites. The obtained integrative model reveals a polar architecture of the complex which, in combination with FATZ-1 multivalent scaffold function, might organize interaction partners and stabilize {\($\alpha$\)}-actinin-2 preferential orientation in Z-disk. Last, we uncover FATZ-1 ability to phase-separate and form biomolecular condensates with {\($\alpha$\)}-actinin-2, raising the question whether FATZ proteins can create an interaction hub for Z-disk proteins through membraneless compartmentalization during myofibrillogenesis.
• 
  Pfanner, N., Warscheid, B., & Wiedemann, N. (2021). Author Correction: Mitochondrial Proteins: From Biogenesis to Functional Networks. Nature Reviews. Molecular Cell Biology, 22(5), 367. https://doi.org/10.1038/s41580-021-00361-x
  • [ DOI ]
• 
  Dannenmaier, S., Oeljeklaus, S., & Warscheid, B. (2021). 2nSILAC for Quantitative Proteomics of Prototrophic Baker’s Yeast. In Methods in molecular biology (Clifton, N.J.) (Vols. 2228, pp. 253-270). https://doi.org/10.1007/978-1-0716-1024-4_18
  • [ Abstract ]
  • [ DOI ]
  Stable isotope labeling by amino acids in cell culture (SILAC) combined with high-resolution mass spectrometry is a quantitative strategy for the comparative analysis of (sub)proteomes. It is based on the metabolic incorporation of stable isotope-coded amino acids during growth of cells or organisms. Here, complete labeling of proteins with the amino acid(s) selected for incorporation needs to be guaranteed to enable accurate quantification on a proteomic scale. Wild-type strains of baker's yeast (Saccharomyces cerevisiae ), which is a widely accepted and well-studied eukaryotic model organism, are generally able to synthesize all amino acids on their own (i.e., prototrophic). To render them amenable to SILAC, auxotrophies are introduced by genetic manipulations. We addressed this limitation by developing a generic strategy for complete "native" labeling of prototrophic S. cerevisiae with isotope-coded arginine and lysine, referred to as "2nSILAC". It allows for directly using and screening several genome-wide yeast mutant collections that are easily accessible to the scientific community for functional proteomic studies but are based on prototrophic variants of S. cerevisiae.
• 
  Egert, J., Brombacher, E., Warscheid, B., & Kreutz, C. (2021). DIMA: Data-Driven Selection of an Imputation Algorithm. Journal of Proteome Research, 20(7), 3489-3496. https://doi.org/10.1021/acs.jproteome.1c00119
  • [ Abstract ]
  • [ DOI ]
  Imputation is a prominent strategy when dealing with missing values (MVs) in proteomics data analysis pipelines. However, it is difficult to assess the performance of different imputation methods and varies strongly depending on data characteristics. To overcome this issue, we present the concept of a data-driven selection of an imputation algorithm (DIMA). The performance and broad applicability of DIMA are demonstrated on 142 quantitative proteomics data sets from the PRoteomics IDEntifications (PRIDE) database and on simulated data consisting of 5-50% MVs with different proportions of missing not at random and missing completely at random values. DIMA reliably suggests a high-performing imputation algorithm, which is always among the three best algorithms and results in a root mean square error difference ({\($\Delta$\)}RMSE) {\($\leq$\)} 10% in 80% of the cases. DIMA implementation is available in MATLAB at github.com/kreutz-lab/OmicsData and in R at github.com/kreutz-lab/DIMAR.
• 
  Morgenstern, M., Peikert, C. D., Lübbert, P., Suppanz, I., Klemm, C., Alka, O., Steiert, C., Naumenko, N., Schendzielorz, A., Melchionda, L., Mühlhäuser, W. W. D., Knapp, B., Busch, J. D., Stiller, S. B., Dannenmaier, S., Lindau, C., Licheva, M., Eickhorst, C., Galbusera, R., … Warscheid, B. (2021). Quantitative High-Confidence Human Mitochondrial Proteome and Its Dynamics in Cellular Context. Cell Metabolism, 33(12), 2464-2483.e18. https://doi.org/10.1016/j.cmet.2021.11.001
  • [ Abstract ]
  • [ DOI ]
  Mitochondria are key organelles for cellular energetics, metabolism, signaling, and quality control and have been linked to various diseases. Different views exist on the composition of the human mitochondrial proteome. We classified {\($>$\)8,000 proteins in mitochondrial preparations of human cells and defined a mitochondrial high-confidence proteome of {\($>$\)1,100 proteins (MitoCoP). We identified interactors of translocases, respiratory chain, and ATP synthase assembly factors. The abundance of MitoCoP proteins covers six orders of magnitude and amounts to 7% of the cellular proteome with the chaperones HSP60-HSP10 being the most abundant mitochondrial proteins. MitoCoP dynamics spans three orders of magnitudes, with half-lives from hours to months, and suggests a rapid regulation of biosynthesis and assembly processes. 460 MitoCoP genes are linked to human diseases with a strong prevalence for the central nervous system and metabolism. MitoCoP will provide a high-confidence resource for placing dynamics, functions, and dysfunctions of mitochondria into the cellular context.
• 
  Christopher, J. A., Stadler, C., Martin, C. E., Morgenstern, M., Pan, Y., Betsinger, C. N., Rattray, D. G., Mahdessian, D., Gingras, A.-C., Warscheid, B., Lehtiö, J., Cristea, I. M., Foster, L. J., Emili, A., & Lilley, K. S. (2021). Subcellular Proteomics. Nature Reviews. Methods Primers, 1, 32. https://doi.org/10.1038/s43586-021-00029-y
  • [ Abstract ]
  • [ DOI ]
  The eukaryotic cell is compartmentalized into subcellular niches, including membrane-bound and membrane-less organelles. Proteins localize to these niches to fulfil their function, enabling discreet biological processes to occur in synchrony. Dynamic movement of proteins between niches is essential for cellular processes such as signalling, growth, proliferation, motility and programmed cell death, and mutations causing aberrant protein localization are associated with a wide range of diseases. Determining the location of proteins in different cell states and cell types and how proteins relocalize following perturbation is important for understanding their functions, related cellular processes and pathologies associated with their mislocalization. In this Primer, we cover the major spatial proteomics methods for determining the location, distribution and abundance of proteins within subcellular structures. These technologies include fluorescent imaging, protein proximity labelling, organelle purification and cell-wide biochemical fractionation. We describe their workflows, data outputs and applications in exploring different cell biological scenarios, and discuss their main limitations. Finally, we describe emerging technologies and identify areas that require technological innovation to allow better characterization of the spatial proteome.
• 
  Rout, S., Oeljeklaus, S., Makki, A., Tachezy, J., Warscheid, B., & Schneider, A. (2021). Determinism and Contingencies Shaped the Evolution of Mitochondrial Protein Import. Proceedings of the National Academy of Sciences of the United States of America, 118(6), Article 6. https://doi.org/10.1073/pnas.2017774118
  • [ Abstract ]
  • [ DOI ]
  Mitochondrial protein import requires outer membrane receptors that evolved independently in different lineages. Here we used quantitative proteomics and in vitro binding assays to investigate the substrate preferences of ATOM46 and ATOM69, the two mitochondrial import receptors of Trypanosoma brucei The results show that ATOM46 prefers presequence-containing, hydrophilic proteins that lack transmembrane domains (TMDs), whereas ATOM69 prefers presequence-lacking, hydrophobic substrates that have TMDs. Thus, the ATOM46/yeast Tom20 and the ATOM69/yeast Tom70 pairs have similar substrate preferences. However, ATOM46 mainly uses electrostatic, and Tom20 hydrophobic, interactions for substrate binding. In vivo replacement of T. brucei ATOM46 by yeast Tom20 did not restore import. However, replacement of ATOM69 by the recently discovered Tom36 receptor of Trichomonas hydrogenosomes, while not allowing for growth, restored import of a large subset of trypanosomal proteins that lack TMDs. Thus, even though ATOM69 and Tom36 share the same domain structure and topology, they have different substrate preferences. The study establishes complementation experiments, combined with quantitative proteomics, as a highly versatile and sensitive method to compare in vivo preferences of protein import receptors. Moreover, it illustrates the role determinism and contingencies played in the evolution of mitochondrial protein import receptors.

• 
  Gomkale, R., Cruz-Zaragoza, L. D., Suppanz, I., Guiard, B., Montoya, J., Callegari, S., Pacheu-Grau, D., Warscheid, B., & Rehling, P. (2020). Defining the Substrate Spectrum of the TIM22 Complex Identifies Pyruvate Carrier Subunits as Unconventional Cargos. Current Biology : CB, 30(6), 1119-1127.e5. https://doi.org/10.1016/j.cub.2020.01.024
  • [ Abstract ]
  • [ DOI ]
  In mitochondria, the carrier translocase (TIM22 complex) facilitates membrane insertion of multi-spanning proteins with internal targeting signals into the inner membrane [1-3]. Tom70, a subunit of TOM complex, represents the major receptor for these precursors [2, 4-6]. After transport across the outer membrane, the hydrophobic carriers engage with the small TIM protein complex composed of Tim9 and Tim10 for transport across the intermembrane space (IMS) toward the TIM22 complex [7-12]. Tim22 represents the pore-forming core unit of the complex [13, 14]. Only a small subset of TIM22 cargo molecules, containing four or six transmembrane spans, have been experimentally defined. Here, we used a tim22 temperature-conditional mutant to define the TIM22 substrate spectrum. Along with carrier-like cargo proteins, we identified subunits of the mitochondrial pyruvate carrier (MPC) as unconventional TIM22 cargos. MPC proteins represent substrates with atypical topology for this transport pathway. In agreement with this, a patient affected in TIM22 function displays reduced MPC levels. Our findings broaden the repertoire of carrier pathway substrates and challenge current concepts of TIM22-mediated transport processes.
• 
  Reimann, L., Schwäble, A. N., Fricke, A. L., Mühlhäuser, W. W. D., Leber, Y., Lohanadan, K., Puchinger, M. G., Schäuble, S., Faessler, E., Wiese, H., Reichenbach, C., Knapp, B., Peikert, C. D., Drepper, F., Hahn, U., Kreutz, C., van der Ven, P. F. M., Radziwill, G., Djinovi’c}}-Carugo, K., … Warscheid, B. (2020). Phosphoproteomics Identifies Dual-Site Phosphorylation in an Extended Basophilic Motif Regulating FILIP1-mediated Degradation of Filamin-C. Communications Biology, 3(1), 253. https://doi.org/10.1038/s42003-020-0982-5
  • [ Abstract ]
  • [ DOI ]
  The PI3K/Akt pathway promotes skeletal muscle growth and myogenic differentiation. Although its importance in skeletal muscle biology is well documented, many of its substrates remain to be identified. We here studied PI3K/Akt signaling in contracting skeletal muscle cells by quantitative phosphoproteomics. We identified the extended basophilic phosphosite motif RxRxxp[S/T]xxp[S/T] in various proteins including filamin-C (FLNc). Importantly, this extended motif, located in a unique insert in Ig-like domain 20 of FLNc, is doubly phosphorylated. The protein kinases responsible for this dual-site phosphorylation are Akt and PKC{\($\alpha$\)}. Proximity proteomics and interaction analysis identified filamin A-interacting protein 1 (FILIP1) as direct FLNc binding partner. FILIP1 binding induces filamin degradation, thereby negatively regulating its function. Here, dual-site phosphorylation of FLNc not only reduces FILIP1 binding, providing a mechanism to shield FLNc from FILIP1-mediated degradation, but also enables fast dynamics of FLNc necessary for its function as signaling adaptor in cross-striated muscle cells.
• 
  Meyer, S. J., Böser, A., Korn, M. A., Koller, C., Bertocci, B., Reimann, L., Warscheid, B., & Nitschke, L. (2020). Cullin 3 Is Crucial for Pro-B Cell Proliferation, Interacts with CD22, and Controls CD22 Internalization on B Cells. Journal of Immunology (Baltimore, Md. : 1950), 204(12), 3360-3374. https://doi.org/10.4049/jimmunol.1900925
  • [ Abstract ]
  • [ DOI ]
  B lymphocytes are important players of the adaptive immune system. However, not just activation of B cells but also regulation of B cell signaling is important to prevent hyperactivity and dysregulation of the immune response. Different mechanisms and proteins contribute to this balance. One of these is CD22, a member of the Siglec family. It is an inhibitory coreceptor of the BCR and inhibits B cell activation. Upon BCR stimulation, CD22-dependent inhibition of BCR signaling results in a decreased calcium mobilization. Although some CD22 binding partners have already been identified, the knowledge about the CD22 interactome is still incomplete. In this study, quantitative affinity purification-mass spectrometry enabled the delineation of the CD22 interactome in the B cell line DT40. These data will clarify molecular mechanisms and CD22 signaling events after BCR activation and revealed several new CD22-associated proteins. One new identified interaction partner is the E3 ubiquitin ligase cullin 3, which was revealed to regulate CD22 surface expression and clathrin-dependent CD22 internalization after BCR stimulation. Furthermore cullin 3 was identified to be important for B lymphocytes in general. B cell-specific cullin 3-deficient mice show reduced developing B cells in the bone marrow and a severe pro-B cell proliferation defect. Mature B cells in the periphery are also reduced and characterized by increased CD22 expression and additionally by preactivated and apoptotic phenotypes. The findings reveal novel functions of cullin 3 in B lymphocytes, namely regulating CD22 surface expression and internalization after B cell activation, as well as promoting proliferation of pro-B cells.
• 
  Lill, P., Hansen, T., Wendscheck, D., Klink, B. U., Jeziorek, T., Vismpas, D., Miehling, J., Bender, J., Schummer, A., Drepper, F., Girzalsky, W., Warscheid, B., Erdmann, R., & Gatsogiannis, C. (2020). Towards the Molecular Architecture of the Peroxisomal Receptor Docking Complex. Proceedings of the National Academy of Sciences of the United States of America, 117(52), 33216-33224. https://doi.org/10.1073/pnas.2009502117
  • [ Abstract ]
  • [ DOI ]
  Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the docking protein Pex14p that is tightly associated with Pex17p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo. The mechanistic principles, however, are unclear, mainly because structures of higher-order assemblies are still lacking. Here, using an integrative approach, we provide the structural characterization of the major components of the peroxisomal docking complex Pex14p/Pex17p, in a native bilayer environment, and reveal its subunit organization. Our data show that three copies of Pex14p and a single copy of Pex17p assemble to form a 20-nm rod-like particle. The different subunits are arranged in a parallel manner, showing interactions along their complete sequences and providing receptor binding sites on both membrane sides. The long rod facing the cytosol is mainly formed by the predicted coiled-coil domains of Pex14p and Pex17p, possibly providing the necessary structural support for the formation of the import pore. Further implications of Pex14p/Pex17p for formation of the peroxisomal translocon are discussed.
• 
  Pacheu-Grau, D., Wasilewski, M., Oeljeklaus, S., Gibhardt, C. S., Aich, A., Chudenkova, M., Dennerlein, S., Deckers, M., Bogeski, I., Warscheid, B., Chacinska, A., & Rehling, P. (2020). COA6 Facilitates Cytochrome c Oxidase Biogenesis as Thiol-reductase for Copper Metallochaperones in Mitochondria. Journal of Molecular Biology, 432(7), 2067-2079. https://doi.org/10.1016/j.jmb.2020.01.036
  • [ Abstract ]
  • [ DOI ]
  The mitochondrial cytochrome c oxidase, the terminal enzyme of the respiratory chain, contains heme and copper centers for electron transfer. The conserved COX2 subunit contains the Cu(A) site, a binuclear copper center. The copper chaperones SCO1, SCO2, and COA6, are required for Cu(A) center formation. Loss of function of these chaperones and the concomitant cytochrome c oxidase deficiency cause severe human disorders. Here we analyzed the molecular function of COA6 and the consequences of COA6 deficiency for mitochondria. Our analyses show that loss of COA6 causes combined complex I and complex IV deficiency and impacts membrane potential-driven protein transport across the inner membrane. We demonstrate that COA6 acts as a thiol-reductase to reduce disulfide bridges of critical cysteine residues in SCO1 and SCO2. Cysteines within the CX(3)CX(N)H domain of SCO2 mediate its interaction with COA6 but are dispensable for SCO2-SCO1 interaction. Our analyses define COA6 as thiol-reductase, which is essential for Cu(A) biogenesis.
• 
  Schummer, A., Maier, R., Gabay-Maskit, S., Hansen, T., Mühlhäuser, W. W. D., Suppanz, I., Fadel, A., Schuldiner, M., Girzalsky, W., Oeljeklaus, S., Zalckvar, E., Erdmann, R., & Warscheid, B. (2020). Pex14p Phosphorylation Modulates Import of Citrate Synthase 2 Into Peroxisomes in Saccharomyces Cerevisiae. Frontiers in Cell and Developmental Biology, 8, 549451. https://doi.org/10.3389/fcell.2020.549451
  • [ Abstract ]
  • [ DOI ]
  The peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores. The central role of Pex14p in peroxisomal matrix protein import processes renders it an obvious target for regulatory mechanisms such as protein phosphorylation. To explore this possibility, we examined the state of Pex14p phosphorylation in Saccharomyces cerevisiae. Phos-tag-SDS-PAGE of Pex14p affinity-purified from solubilized membranes revealed Pex14p as multi-phosphorylated protein. Using mass spectrometry, we identified 16 phosphorylation sites, with phosphorylation hot spots located in the N- and C-terminal regions of Pex14p. Analysis of phosphomimicking and non-phosphorylatable variants of Pex14p revealed a decreased import of GFP carrying a peroxisomal targeting signal type 1, indicating a functional relevance of Pex14p phosphorylation in peroxisomal matrix protein import. We show that this effect can be ascribed to the phosphomimicking mutation at serine 266 of Pex14p (Pex14p-S266D). We further screened the subcellular distribution of 23 native GFP-tagged peroxisomal matrix proteins by high-content fluorescence microscopy. Only Cit2p, the peroxisomal isoform of citrate synthase, was affected in the Pex14p-S266D mutant, showing increased cytosolic localization. Cit2p is part of the glyoxylate cycle, which is required for the production of essential carbohydrates when yeast is grown on non-fermentable carbon sources. Pex14p-S266 phosphosite mutants showed reversed growth phenotypes in oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Overexpression of Cit2p rescued the growth phenotype of yeast cells expressing Pex14p-S266D in oleic acid. Our data indicate that phosphorylation of Pex14p at S266 provides a mechanism for controlling the peroxisomal import of Cit2p, which helps S. cerevisiae cells to adjust their carbohydrate metabolism according to the nutritional conditions.
• 
  von Känel, C., Muñoz-Gómez, S. A., Oeljeklaus, S., Wenger, C., Warscheid, B., Wideman, J. G., Harsman, A., & Schneider, A. (2020). Homologue Replacement in the Import Motor of the Mitochondrial Inner Membrane of Trypanosomes. ELife, 9. https://doi.org/10.7554/eLife.52560
  • [ Abstract ]
  • [ DOI ]
  Many mitochondrial proteins contain N-terminal presequences that direct them to the organelle. The main driving force for their translocation across the inner membrane is provided by the presequence translocase-associated motor (PAM) which contains the J-protein Pam18. Here, we show that in the PAM of Trypanosoma brucei the function of Pam18 has been replaced by the non-orthologous euglenozoan-specific J-protein TbPam27. TbPam27 is specifically required for the import of mitochondrial presequence-containing but not for carrier proteins. Similar to yeast Pam18, TbPam27 requires an intact J-domain to function. Surprisingly, T. brucei still contains a bona fide Pam18 orthologue that, while essential for normal growth, is not involved in protein import. Thus, during evolution of kinetoplastids, Pam18 has been replaced by TbPam27. We propose that this replacement is linked to the transition from two ancestral and functionally distinct TIM complexes, found in most eukaryotes, to the single bifunctional TIM complex present in trypanosomes.
• 
  Drepper, F., Biernat, J., Kaniyappan, S., Meyer, H. E., Mandelkow, E. M., Warscheid, B., & Mandelkow, E. (2020). A Combinatorial Native MS and LC-MS/MS Approach Reveals High Intrinsic Phosphorylation of Human Tau but Minimal Levels of Other Key Modifications. The Journal of Biological Chemistry, 295(52), 18213-18225. https://doi.org/10.1074/jbc.RA120.015882
  • [ Abstract ]
  • [ DOI ]
  Abnormal changes of neuronal Tau protein, such as phosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer's disease. Abnormal phosphorylation is thought to precede aggregation and therefore to promote aggregation, but the nature and extent of phosphorylation remain ill-defined. Tau contains {\($\sim$\)}85 potential phosphorylation sites, which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, methodological limitations (e.g. in MS of phosphopeptides, or antibodies against phosphoepitopes) led to conflicting results regarding the extent of Tau phosphorylation in cells. Here we present results from a new approach based on native MS of intact Tau expressed in eukaryotic cells (Sf9). The extent of phosphorylation is heterogeneous, up to {\($\sim$\)}20 phosphates per molecule distributed over 51 sites. The medium phosphorylated fraction P(m) showed overall occupancies of {\($\sim$\)}8 P(i) (\textpm} 5) with a bell-shaped distribution; the highly phosphorylated fraction P(h) had 14 P(i) (\textpm} 6). The distribution of sites was highly asymmetric (with 71\% of all P-sites in the C-terminal half of Tau). All sites were on Ser or Thr residues, but none were on Tyr. Other known posttranslational modifications were near or below our detection limit (e.g. acetylation, ubiquitination). These findings suggest that normal cellular Tau shows a remarkably high extent of phosphorylation, whereas other modifications are nearly absent. This implies that abnormal phosphorylations at certain sites may not affect the extent of phosphorylation significantly and do not represent hyperphosphorylation. By implication, the pathological aggregation of Tau is not likely a consequence of high phosphorylation.

• 
  Maa}rtensson, C. U., Priesnitz, C., Song, J., Ellenrieder, L., Doan, K. N., Boos, F., Floerchinger, A., Zufall, N., Oeljeklaus, S., Warscheid, B., & Becker, T. (2019). Mitochondrial Protein Translocation-Associated Degradation. Nature, 569(7758), 679-683. https://doi.org/10.1038/s41586-019-1227-y
  • [ Abstract ]
  • [ DOI ]
  Mitochondrial biogenesis and functions depend on the import of precursor proteins via the 'translocase of the outer membrane' (TOM complex). Defects in protein import lead to an accumulation of mitochondrial precursor proteins that induces a range of cellular stress responses. However, constitutive quality-control mechanisms that clear trapped precursor proteins from the TOM channel under non-stress conditions have remained unknown. Here we report that in Saccharomyces cerevisiae Ubx2, which functions in endoplasmic reticulum-associated degradation, is crucial for this quality-control process. A pool of Ubx2 binds to the TOM complex to recruit the AAA ATPase Cdc48 for removal of arrested precursor proteins from the TOM channel. This mitochondrial protein translocation-associated degradation (mitoTAD) pathway continuously monitors the TOM complex under non-stress conditions to prevent clogging of the TOM channel with precursor proteins. The mitoTAD pathway ensures that mitochondria maintain their full protein-import capacity, and protects cells against proteotoxic stress induced by impaired transport of proteins into mitochondria.
• 
  Pfanner, N., Warscheid, B., & Wiedemann, N. (2019). Mitochondrial Proteins: From Biogenesis to Functional Networks. Nature Reviews. Molecular Cell Biology, 20(5), 267-284. https://doi.org/10.1038/s41580-018-0092-0
  • [ Abstract ]
  • [ DOI ]
  Mitochondria are essential for the viability of eukaryotic cells as they perform crucial functions in bioenergetics, metabolism and signalling and have been associated with numerous diseases. Recent functional and proteomic studies have revealed the remarkable complexity of mitochondrial protein organization. Protein machineries with diverse functions such as protein translocation, respiration, metabolite transport, protein quality control and the control of membrane architecture interact with each other in dynamic networks. In this Review, we discuss the emerging role of the mitochondrial protein import machinery as a key organizer of these mitochondrial protein networks. The preprotein translocases that reside on the mitochondrial membranes not only function during organelle biogenesis to deliver newly synthesized proteins to their final mitochondrial destination but also cooperate with numerous other mitochondrial protein complexes that perform a wide range of functions. Moreover, these protein networks form membrane contact sites, for example, with the endoplasmic reticulum, that are key for integration of mitochondria with cellular function, and defects in protein import can lead to diseases.
• 
  Jauss, B., Petriman, N.-A., Drepper, F., Franz, L., Sachelaru, I., Welte, T., Steinberg, R., Warscheid, B., & Koch, H.-G. (2019). Noncompetitive Binding of PpiD and YidC to the SecYEG Translocon Expands the Global View on the SecYEG Interactome in Escherichia Coli. The Journal of Biological Chemistry, 294(50), 19167-19183. https://doi.org/10.1074/jbc.RA119.010686
  • [ Abstract ]
  • [ DOI ]
  The SecYEG translocon constitutes the major protein transport channel in bacteria and transfers an enormous variety of different secretory and inner-membrane proteins. The minimal core of the SecYEG translocon consists of three inner-membrane proteins, SecY, SecE, and SecG, which, together with appropriate targeting factors, are sufficient for protein transport in vitro However, in vivo the SecYEG translocon has been shown to associate with multiple partner proteins, likely allowing the SecYEG translocon to process its diverse substrates. To obtain a global view on SecYEG plasticity in Escherichia coli, here we performed a quantitative interaction proteomic analysis, which identified several known SecYEG-interacting proteins, verified the interaction of SecYEG with quality-control proteins, and revealed several previously unknown putative SecYEG-interacting proteins. Surprisingly, we found that the chaperone complex PpiD/YfgM is the most prominent interaction partner of SecYEG. Detailed analyses of the PpiD-SecY interaction by site-directed cross-linking revealed that PpiD and the established SecY partner protein YidC use almost completely-overlapping binding sites on SecY. Both PpiD and YidC contacted the lateral gate, the plug domain, and the periplasmic cavity of SecY. However, quantitative MS and cross-linking analyses revealed that despite having almost identical binding sites, their binding to SecY is noncompetitive. This observation suggests that the SecYEG translocon forms different substrate-independent subassemblies in which SecYEG either associates with YidC or with the PpiD/YfgM complex. In summary, the results of this study indicate that the PpiD/YfgM chaperone complex is a primary interaction partner of the SecYEG translocon.
• 
  Hörner, M., Eble, J., Yousefi, O. S., Schwarz, J., Warscheid, B., Weber, W., & Schamel, W. W. A. (2019). Light-Controlled Affinity Purification of Protein Complexes Exemplified by the Resting ZAP70 Interactome. Frontiers in Immunology, 10, 226. https://doi.org/10.3389/fimmu.2019.00226
  • [ Abstract ]
  • [ DOI ]
  Multiprotein complexes control the behavior of cells, such as of lymphocytes of the immune system. Methods to affinity purify protein complexes and to determine their interactome by mass spectrometry are thus widely used. One drawback of these methods is the presence of false positives. In fact, the elution of the protein of interest (POI) is achieved by changing the biochemical properties of the buffer, so that unspecifically bound proteins (the false positives) may also elute. Here, we developed an optogenetics-derived and light-controlled affinity purification method based on the light-regulated reversible protein interaction between phytochrome B (PhyB) and its phytochrome interacting factor 6 (PIF6). We engineered a truncated variant of PIF6 comprising only 22 amino acids that can be genetically fused to the POI as an affinity tag. Thereby the POI can be purified with PhyB-functionalized resin material using 660 nm light for binding and washing, and 740 nm light for elution. Far-red light-induced elution is effective but very mild as the same buffer is used for the wash and elution. As proof-of-concept, we expressed PIF-tagged variants of the tyrosine kinase ZAP70 in ZAP70-deficient Jurkat T cells, purified ZAP70 and associating proteins using our light-controlled system, and identified the interaction partners by quantitative mass spectrometry. Using unstimulated T cells, we were able to detect the known interaction partners, and could filter out all other proteins.
• 
  Kalel, V. C., Li, M., Gaussmann, S., Delhommel, F., Schäfer, A.-B., Tippler, B., Jung, M., Maier, R., Oeljeklaus, S., Schliebs, W., Warscheid, B., Sattler, M., & Erdmann, R. (2019). Evolutionary Divergent PEX3 Is Essential for Glycosome Biogenesis and Survival of Trypanosomatid Parasites. Biochimica Et Biophysica Acta. Molecular Cell Research, 1866(12), 118520. https://doi.org/10.1016/j.bbamcr.2019.07.015
  • [ Abstract ]
  • [ DOI ]
  Trypanosomatid parasites cause devastating African sleeping sickness, Chagas disease, and Leishmaniasis that affect about 18 million people worldwide. Recently, we showed that the biogenesis of glycosomes could be the "Achilles' heel" of trypanosomatids suitable for the development of new therapies against trypanosomiases. This was shown for inhibitors of the import machinery of matrix proteins, while the distinct machinery for the topogenesis of glycosomal membrane proteins evaded investigation due to the lack of a druggable interface. Here we report on the identification of the highly divergent trypanosomal PEX3, a central component of the transport machinery of peroxisomal membrane proteins and the master regulator of peroxisome biogenesis. The trypanosomatid PEX3 shows very low degree of conservation and its identification was made possible by a combinatory approach identifying of PEX19-interacting proteins and secondary structure homology screening. The trypanosomal PEX3 localizes to glycosomes and directly interacts with the membrane protein import receptor PEX19. RNAi-studies revealed that the PEX3 is essential and that its depletion results in mislocalization of glycosomal proteins to the cytosol and a severe growth defect. Comparison of the parasites and human PEX3-PEX19 interface disclosed differences that might be accessible for drug development. The absolute requirement for biogenesis of glycosomes and its structural distinction from its human counterpart make PEX3 a prime drug target for the development of novel therapies against trypanosomiases. The identification paves the way for future drug development targeting PEX3, and for the analysis of additional partners involved in this crucial step of glycosome biogenesis.
• 
  El Magraoui, F., Brinkmeier, R., Mastalski, T., Hupperich, A., Strehl, C., Schwerter, D., Girzalsky, W., Meyer, H. E., Warscheid, B., Erdmann, R., & Platta, H. W. (2019). The Deubiquitination of the PTS1-import Receptor Pex5p Is Required for Peroxisomal Matrix Protein Import. Biochimica Et Biophysica Acta. Molecular Cell Research, 1866(2), 199-213. https://doi.org/10.1016/j.bbamcr.2018.11.002
  • [ Abstract ]
  • [ DOI ]
  Peroxisomal biogenesis depends on the correct import of matrix proteins into the lumen of the organelle. Most peroxisomal matrix proteins harbor the peroxisomal targeting-type 1 (PTS1), which is recognized by the soluble PTS1-receptor Pex5p in the cytosol. Pex5p ferries the PTS1-proteins to the peroxisomal membrane and releases them into the lumen. Finally, the PTS1-receptor is monoubiquitinated on the conserved cysteine 6 in Saccharomyces cerevisiae. The monoubiquitinated Pex5p is recognized by the peroxisomal export machinery and is retrotranslocated into the cytosol for further rounds of protein import. However, the functional relevance of deubiquitination has not yet been addressed. In this study, we have analyzed a Pex5p-truncation lacking Cys6 [({\($\Delta$\)}6)Pex5p], a construct with a ubiquitin-moiety genetically fused to the truncation [Ub-({\($\Delta$\)}6)Pex5p], as well as a construct with a reduced susceptibility to deubiquitination [Ub(G75/76A)-({\($\Delta$\)}6)Pex5p]. While the ({\($\Delta$\)}6)Pex5p-truncation is not functional, the Ub-({\($\Delta$\)}6)Pex5p chimeric protein can facilitate matrix protein import. In contrast, the Ub(G75/76A)-({\($\Delta$\)}6)Pex5p chimera exhibits a complete PTS1-import defect. The data show for the first time that not only ubiquitination but also deubiquitination rates are tightly regulated and that efficient deubiquitination of Pex5p is essential for peroxisomal biogenesis.
• 
  Fröbel, J., Blümmel, A.-S., Drepper, F., Warscheid, B., & Müller, M. (2019). Surface-Exposed Domains of TatB Involved in the Structural and Functional Assembly of the Tat Translocase in Escherichia Coli. The Journal of Biological Chemistry, 294(38), 13902-13914. https://doi.org/10.1074/jbc.RA119.009298
  • [ Abstract ]
  • [ DOI ]
  Twin-arginine-dependent translocases transport folded proteins across bacterial, archaeal, and chloroplast membranes. Upon substrate binding, they assemble from hexahelical TatC and single-spanning TatA and TatB membrane proteins. Although structural and functional details of individual Tat subunits have been reported previously, the sequence and dynamics of Tat translocase assembly remain to be determined. Employing the zero-space cross-linker N,N'-dicyclohexylcarbodiimide (DCCD) in combination with LC-MS/MS, we identified as yet unknown intra- and intermolecular contact sites of TatB and TatC. In addition to their established intramembrane binding sites, both proteins were thus found to contact each other through the soluble N terminus of TatC and the interhelical linker region around the conserved glutamyl residue Glu(49) of TatB from Escherichia coli Functional analyses suggested that by interacting with the TatC N terminus, TatB improves the formation of a proficient substrate recognition site of TatC. The Glu(49) region of TatB was found also to contact distinct downstream sites of a neighboring TatB molecule and to thereby mediate oligomerization of TatB within the TatBC receptor complex. Finally, we show that global DCCD-mediated cross-linking of TatB and TatC in membrane vesicles or, alternatively, creating covalently linked TatC oligomers prevents TatA from occupying a position close to the TatBC-bound substrate. Collectively, our results are consistent with a circular arrangement of the TatB and TatC units within the TatBC receptor complex and with TatA entering the interior TatBC-binding cavity through lateral gates between TatBC protomers.
• 
  Eichenberger, C., Oeljeklaus, S., Bruggisser, J., Mani, J., Haenni, B., Kaurov, I., Niemann, M., Zuber, B., Lukev s, J., Hashimi, H., Warscheid, B., Schimanski, B., & Schneider, A. (2019). The Highly Diverged Trypanosomal MICOS Complex Is Organized in a Nonessential Integral Membrane and an Essential Peripheral Module. Molecular Microbiology, 112(6), 1731-1743. https://doi.org/10.1111/mmi.14389
  • [ Abstract ]
  • [ DOI ]
  The mitochondrial contact site and cristae organization system (MICOS) mediates the formation of cristae, invaginations in the mitochondrial inner membrane. The highly diverged MICOS complex of the parasitic protist Trypanosoma brucei consists of nine subunits. Except for two Mic10-like and a Mic60-like protein, all subunits are specific for kinetoplastids. Here, we determined on a proteome-wide scale how ablation of individual MICOS subunits affects the levels of the other subunits. The results reveal co-regulation of TbMic10-1, TbMic10-2, TbMic16 and TbMic60, suggesting that these nonessential, integral inner membrane proteins form an interdependent network. Moreover, the ablation of TbMic34 and TbMic32 reveals another network consisting of the essential, intermembrane space-localized TbMic20, TbMic32, TbMic34 and TbMic40, all of which are peripherally associated with the inner membrane. The downregulation of TbMic20, TbMic32 and TbMic34 also interferes with mitochondrial protein import and reduces the size of the TbMic10-containing complexes. Thus, the diverged MICOS of trypanosomes contains two subcomplexes: a nonessential membrane-integrated one, organized around the conserved Mic10 and Mic60, that mediates cristae formation, and an essential membrane-peripheral one consisting of four kinetoplastid-specific subunits, that is required for import of intermembrane space proteins.

• 
  Lorenzi, I., Oeljeklaus, S., Aich, A., Ronsör, C., Callegari, S., Dudek, J., Warscheid, B., Dennerlein, S., & Rehling, P. (2018). The Mitochondrial TMEM177 Associates with COX20 during COX2 Biogenesis. Biochimica Et Biophysica Acta. Molecular Cell Research, 1865(2), 323-333. https://doi.org/10.1016/j.bbamcr.2017.11.010
  • [ Abstract ]
  • [ DOI ]
  The three mitochondrial-encoded proteins, COX1, COX2, and COX3, form the core of the cytochrome c oxidase. Upon synthesis, COX2 engages with COX20 in the inner mitochondrial membrane, a scaffold protein that recruits metallochaperones for copper delivery to the Cu(A)-Site of COX2. Here we identified the human protein, TMEM177 as a constituent of the COX20 interaction network. Loss or increase in the amount of TMEM177 affects COX20 abundance leading to reduced or increased COX20 levels respectively. TMEM177 associates with newly synthesized COX2 and SCO2 in a COX20-dependent manner. Our data shows that by unbalancing the amount of TMEM177, newly synthesized COX2 accumulates in a COX20-associated state. We conclude that TMEM177 promotes assembly of COX2 at the level of Cu(A)-site formation.
• 
  González Montoro, A., Auffarth, K., Hönscher, C., Bohnert, M., Becker, T., Warscheid, B., Reggiori, F., van der Laan, M., Fröhlich, F., & Ungermann, C. (2018). Vps39 Interacts with Tom40 to Establish One of Two Functionally Distinct Vacuole-Mitochondria Contact Sites. Developmental Cell, 45(5), 621-636.e7. https://doi.org/10.1016/j.devcel.2018.05.011
  • [ Abstract ]
  • [ DOI ]
  The extensive subcellular network of membrane contact sites plays central roles in organelle biogenesis and communication, yet the precise contributions of the involved machineries remain largely enigmatic. The yeast vacuole forms a membrane contact site with mitochondria, called vacuolar and mitochondrial patch (vCLAMP). Formation of vCLAMPs involves the vacuolar Rab GTPase Ypt7 and the Ypt7-interacting Vps39 subunit of the HOPS tethering complex. Here, we uncover the general preprotein translocase of the outer membrane (TOM) subunit Tom40 as the direct binding partner of Vps39 on mitochondria. We identify Vps39 mutants defective in TOM binding, but functional for HOPS. Cells that cannot form vCLAMPs show reduced growth under stress conditions and impaired survival upon starvation. Unexpectedly, our mutant analysis revealed the existence of two functionally independent vacuole-mitochondria MCSs: one formed by the Ypt7-Vps39-Tom40 tether and a second one by Vps13-Mcp1, which is redundant with ER-mitochondrial contacts formed by ERMES.
• 
  Dannenmaier, S., Stiller, S. B., Morgenstern, M., Lübbert, P., Oeljeklaus, S., Wiedemann, N., & Warscheid, B. (2018). Complete Native Stable Isotope Labeling by Amino Acids of Saccharomyces Cerevisiae for Global Proteomic Analysis. Analytical Chemistry, 90(17), 10501-10509. https://doi.org/10.1021/acs.analchem.8b02557
  • [ Abstract ]
  • [ DOI ]
  Knowledge about the functions of individual proteins on a system-wide level is crucial to fully understand molecular mechanisms underlying cellular processes. A considerable part of the proteome across all organisms is still poorly characterized. Mass spectrometry is an efficient technology for the global study of proteins. One of the most prominent methods for accurate proteome-wide comparative quantification is stable isotope labeling by amino acids in cell culture (SILAC). However, application of SILAC to prototrophic organisms such as Saccharomyces cerevisiae, also known as baker's yeast, is compromised since they are able to synthesize all amino acids on their own. Here, we describe an advanced strategy, termed 2nSILAC, that allows for in vivo labeling of prototrophic baker's yeast using heavy arginine and lysine under fermentable and respiratory growth conditions, making it a suitable tool for the global study of protein functions. This generic 2nSILAC strategy allows for directly using and systematically screening yeast mutant strain collections available to the scientific community. We exemplarily demonstrate its high potential by analyzing the effects of mitochondrial gene deletions in mitochondrial fractions using quantitative mass spectrometry revealing the role of Coi1 for the assembly of cytochrome c oxidase (respiratory chain complex IV).
• 
  Opaliński, L., Song, J., Priesnitz, C., Wenz, L.-S., Oeljeklaus, S., Warscheid, B., Pfanner, N., & Becker, T. (2018). Recruitment of Cytosolic J-Proteins by TOM Receptors Promotes Mitochondrial Protein Biogenesis. Cell Reports, 25(8), 2036-2043.e5. https://doi.org/10.1016/j.celrep.2018.10.083
  • [ Abstract ]
  • [ DOI ]
  Mitochondria possess elaborate machineries for the import of proteins from the cytosol. Cytosolic factors like Hsp70 chaperones and their co-chaperones, the J-proteins, guide proteins to the mitochondrial surface. The translocase of the mitochondrial outer membrane (TOM) forms the entry gate for preproteins. How the proteins are delivered to mitochondrial preprotein receptors is poorly understood. We identify the cytosolic J-protein Xdj1 as a specific interaction partner of the central receptor Tom22. Tom22 recruits Xdj1 to the mitochondrial surface to promote import of preproteins and assembly of the TOM complex. Additionally, we find that the receptor Tom70 binds a different cytosolic J-protein, Djp1. Our findings suggest that cytosolic J-proteins target distinct TOM receptors and promote the biogenesis of mitochondrial proteins.
• 
  Petriman, N.-A., Jauß, B., Hufnagel, A., Franz, L., Sachelaru, I., Drepper, F., Warscheid, B., & Koch, H.-G. (2018). The Interaction Network of the YidC Insertase with the SecYEG Translocon, SRP and the SRP Receptor FtsY. Scientific Reports, 8(1), 578. https://doi.org/10.1038/s41598-017-19019-w
  • [ Abstract ]
  • [ DOI ]
  YidC/Oxa1/Alb3 are essential proteins that operate independently or cooperatively with the Sec machinery during membrane protein insertion in bacteria, archaea and eukaryotic organelles. Although the interaction between the bacterial SecYEG translocon and YidC has been observed in multiple studies, it is still unknown which domains of YidC are in contact with the SecYEG translocon. By in vivo and in vitro site-directed and para-formaldehyde cross-linking we identified the auxiliary transmembrane domain 1 of E. coli YidC as a major contact site for SecY and SecG. Additional SecY contacts were observed for the tightly packed globular domain and the C1 loop of YidC, which reveals that the hydrophilic cavity of YidC faces the lateral gate of SecY. Surprisingly, YidC-SecYEG contacts were only observed when YidC and SecYEG were present at about stoichiometric concentrations, suggesting that the YidC-SecYEG contact in vivo is either very transient or only observed for a very small SecYEG sub-population. This is different for the YidC-SRP and YidC-FtsY interaction, which involves the C1 loop of YidC and is efficiently observed even at sub-stoichiometric concentrations of SRP/FtsY. In summary, our data provide a first detailed view on how YidC interacts with the SecYEG translocon and the SRP-targeting machinery.
• 
  Topf, U., Suppanz, I., Samluk, L., Wrobel, L., Böser, A., Sakowska, P., Knapp, B., Pietrzyk, M. K., Chacinska, A., & Warscheid, B. (2018). Quantitative Proteomics Identifies Redox Switches for Global Translation Modulation by Mitochondrially Produced Reactive Oxygen Species. Nature Communications, 9(1), 324. https://doi.org/10.1038/s41467-017-02694-8
  • [ Abstract ]
  • [ DOI ]
  The generation of reactive oxygen species (ROS) is inevitably linked to life. However, the precise role of ROS in signalling and specific targets is largely unknown. We perform a global proteomic analysis to delineate the yeast redoxome to a depth of more than 4,300 unique cysteine residues in over 2,200 proteins. Mapping of redox-active thiols in proteins exposed to exogenous or endogenous mitochondria-derived oxidative stress reveals ROS-sensitive sites in several components of the translation apparatus. Mitochondria are the major source of cellular ROS. We demonstrate that increased levels of intracellular ROS caused by dysfunctional mitochondria serve as a signal to attenuate global protein synthesis. Hence, we propose a universal mechanism that controls protein synthesis by inducing reversible changes in the translation machinery upon modulating the redox status of proteins involved in translation. This crosstalk between mitochondria and protein synthesis may have an important contribution to pathologies caused by dysfunctional mitochondria.
• 
  Kaurov, I., Vancová, M., Schimanski, B., Cadena, L. R., Heller, J. rí, Bíl’y}}, T. s, Potv ev sil, D., Eichenberger, C., Bruce, H., Oeljeklaus, S., Warscheid, B., Zdráhal, Z. ek, Schneider, A., Lukev s, J., & Hashimi, H. (2018). The Diverged Trypanosome MICOS Complex as a Hub for Mitochondrial Cristae Shaping and Protein Import. Current Biology : CB, 28(21), 3393-3407.e5. https://doi.org/10.1016/j.cub.2018.09.008
  • [ Abstract ]
  • [ DOI ]
  The mitochondrial contact site and cristae organization system (MICOS) is a multiprotein complex responsible for cristae formation. Even though cristae are found in all mitochondria capable of oxidative phosphorylation, only Mic10 and Mic60 appear to be conserved throughout eukaryotes. The remaining 4 or 5 known MICOS subunits are specific to the supergroup Opisthokonta, which includes yeast and mammals that are the only organisms in which this complex has been analyzed experimentally. We have isolated the MICOS from Trypanosoma brucei, a member of the supergroup Excavata that is profoundly diverged from opisthokonts. We show that it is required for the maintenance of the unique discoidal cristae that typify excavates, such as euglenids and kinetoplastids, the latter of which include trypanosomes. The trypanosome MICOS consists of 9 subunits, most of which are essential for normal growth. Unlike in opisthokonts, it contains two distinct Mic10 orthologs and an unconventional putative Mic60 that lacks a mitofilin domain. Interestingly, one of the essential trypanosomatid-specific MICOS subunits called TbMic20 is a thioredoxin-like protein that appears to be involved in import of intermembrane space proteins, including respiratory chain complex assembly factors. This result points to trypanosome MICOS coordinating cristae shaping and population of its membrane with proteins involved in respiration, the latter via the catalytic activity of TbMic20. Thus, trypanosome MICOS allows us to define which of its features are conserved in all eukaryotes and decipher those that represent lineage-specific adaptations.
• 
  Yifrach, E., Fischer, S., Oeljeklaus, S., Schuldiner, M., Zalckvar, E., & Warscheid, B. (2018). Defining the Mammalian Peroxisomal Proteome. Sub-Cellular Biochemistry, 89, 47-66. https://doi.org/10.1007/978-981-13-2233-4_2
  • [ Abstract ]
  • [ DOI ]
  The current view on peroxisomes has changed dramatically from being human cell oddities to vital organelles that host several key metabolic pathways. To fulfil over 50 different enzymatic functions, human peroxisomes host either unique peroxisomal proteins or dual-localized proteins. The identification and characterization of the complete peroxisomal proteome in humans is important for diagnosis and treatment of patients with peroxisomal disorders as well as for uncovering novel peroxisomal functions and regulatory modules. Hence, here we compiled a comprehensive list of mammalian peroxisomal and peroxisome-associated proteins by curating results of several quantitative and non-quantitative proteomic studies together with entries in the UniProtKB and Compartments knowledge channel databases. Our analysis gives a holistic view on the mammalian peroxisomal proteome and brings to light potential new peroxisomal and peroxisome-associated proteins. We believe that this dataset, represents a valuable surrogate map of the human peroxisomal proteome.
• 
  Klümper, J., Oeljeklaus, S., Warscheid, B., Erdmann, R., & Schliebs, W. (2018). Using Pull Down Strategies to Analyze the Interactome of Peroxisomal Membrane Proteins in Human Cells. Sub-Cellular Biochemistry, 89, 261-285. https://doi.org/10.1007/978-981-13-2233-4_11
  • [ Abstract ]
  • [ DOI ]
  Different pull-down strategies were successfully applied to gain novel insight into the interactome of human membrane-associated proteins. Here, we compare the outcome, efficiency and potential of pull-down strategies applied to human peroxisomal membrane proteins. Stable membrane-bound protein complexes can be affinity-purified from genetically engineered human cells or subfractions thereof after detergent solubilization, followed by size exclusion chromatography and analysis by mass spectrometry (MS). As exemplified for Protein A-tagged human PEX14, one of the central constituents of the peroxisomal matrix protein import machinery, MS analyses of the affinity-purified complexes revealed an unexpected association of PEX14 with other protein assemblies like the microtubular network or the insertion apparatus for peroxisomal membrane proteins comprising PEX3, PEX16 and PEX19. The latter association was recently supported by using a different pull-down strategy following in vivo proximity labeling with biotin, named BioID, which enabled the identification of various membrane proteins in close proximity of PEX16 in living cells.

• 
  Callegari, S., Oeljeklaus, S., Warscheid, B., Dennerlein, S., Thumm, M., Rehling, P., & Dudek, J. (2017). Phospho-Ubiquitin-PARK2 Complex as a Marker for Mitophagy Defects. Autophagy, 13(1), 201-211. https://doi.org/10.1080/15548627.2016.1254852
  • [ Abstract ]
  • [ DOI ]
  The E3 ubiquitin ligase PARK2 and the mitochondrial protein kinase PINK1 are required for the initiation of mitochondrial damage-induced mitophagy. Together, PARK2 and PINK1 generate a phospho-ubiquitin signal on outer mitochondrial membrane proteins that triggers recruitment of the autophagy machinery. This paper describes the detection of a defined 500-kDa phospho-ubiquitin-rich PARK2 complex that accumulates on mitochondria upon treatment with the membrane uncoupler CCCP. Formation of this complex is dependent on the presence of PINK1 and is absent in mutant forms of PARK2, whereby mitophagy is also arrested. These results signify a functional signaling complex that is essential for the progression of mitophagy. The visualization of the PARK2 signaling complex represents a novel marker for this critical step in mitophagy and can be used to monitor mitophagy progression in PARK2 mutants and to uncover additional upstream factors required for PARK2-mediated mitophagy signaling.
• 
  Fischer, A., Mühlhäuser, W. W. D., Warscheid, B., & Radziwill, G. (2017). Membrane Localization of Acetylated CNK1 Mediates a Positive Feedback on RAF/ERK Signaling. Science Advances, 3(8), e1700475. https://doi.org/10.1126/sciadv.1700475
  • [ Abstract ]
  • [ DOI ]
  Spatiotemporal control is a common mechanism that modulates activity and function of signal transducers in the signaling network. We identified acetylation of CNK1 (connector enhancer of kinase suppressor of Ras-1) as a late step in the activation of CNK1 signaling, accompanied with prolonged stimulation of extracellular signal-regulated kinase (ERK). We identified the acetyltransferase CREB (cyclic adenosine 3',5'-monophosphate response element-binding protein)-binding protein and the deacetylase SIRT2 (sirtuin type 2) as novel binding partners of CNK1, modulating the acetylation state of CNK1. Acetylation of CNK1 at position Lys(414) located in the pleckstrin homology domain drives membrane localization of CNK1 in growth factor-stimulated cells. Inhibition of ERK signaling abolishes CNK1 acetylation. Cosmic database search identified CNK1 mutants at position Arg(426) near the acetylation site in several human tumor types. These mutants show constitutive acetylation and membrane localization. CNK1 mutants substituting Arg(426), the acetylation mimetic mutant CNK1-K414Q, and membrane-anchored CNK1 mutants all interact with the protein kinase CRAF and stimulate ERK-dependent cell proliferation and cell migration. In RAS-transformed cells, CNK1 is acetylated and membrane-bound and drives cell proliferation. Thus, growth factor-stimulated ERK signaling induces CNK1 acetylation, and acetylated CNK1 promotes ERK signaling, demonstrating a novel function of CNK1 as positive feedback regulator of the RAF/MEK (mitogen-activated protein kinase kinase)/ERK pathway. In addition, acetylation of CNK1 is an important step in oncogenic signaling, promoting cell proliferation and migration.
• 
  Blümmel, A.-S., Drepper, F., Knapp, B., Eimer, E., Warscheid, B., Müller, M., & Fröbel, J. (2017). Structural Features of the TatC Membrane Protein That Determine Docking and Insertion of a Twin-Arginine Signal Peptide. The Journal of Biological Chemistry, 292(52), 21320-21329. https://doi.org/10.1074/jbc.M117.812560
  • [ Abstract ]
  • [ DOI ]
  Twin-arginine translocation (Tat) systems transport folded proteins across cellular membranes with the concerted action of mostly three membrane proteins: TatA, TatB, and TatC. Hetero-oligomers of TatB and TatC form circular substrate-receptor complexes with a central binding cavity for twin-arginine-containing signal peptides. After binding of the substrate, energy from an electro-chemical proton gradient is transduced into the recruitment of TatA oligomers and into the actual translocation event. We previously reported that Tat-dependent protein translocation into membrane vesicles of Escherichia coli is blocked by the compound N,N'-dicyclohexylcarbodiimide (DCCD, DCC). We have now identified a highly conserved glutamate residue in the transmembrane region of E. coli TatC, which when modified by DCCD interferes with the deep insertion of a Tat signal peptide into the TatBC receptor complex. Our findings are consistent with a hydrophobic binding cavity formed by TatB and TatC inside the lipid bilayer. Moreover, we found that DCCD mediates discrete intramolecular cross-links of E. coli TatC involving both its N- and C-tails. These results confirm the close proximity of two distant sequence sections of TatC proposed to concertedly function as the primary docking site for twin-arginine signal peptides.
• 
  Morgenstern, M., Stiller, S. B., Lübbert, P., Peikert, C. D., Dannenmaier, S., Drepper, F., Weill, U., Höß, P., Feuerstein, R., Gebert, M., Bohnert, M., van der Laan, M., Schuldiner, M., Schütze, C., Oeljeklaus, S., Pfanner, N., Wiedemann, N., & Warscheid, B. (2017). Definition of a High-Confidence Mitochondrial Proteome at Quantitative Scale. Cell Reports, 19(13), 2836-2852. https://doi.org/10.1016/j.celrep.2017.06.014
  • [ Abstract ]
  • [ DOI ]
  Mitochondria perform central functions in cellular bioenergetics, metabolism, and signaling, and their dysfunction has been linked to numerous diseases. The available studies cover only part of the mitochondrial proteome, and a separation of core mitochondrial proteins from associated fractions has not been achieved. We developed an integrative experimental approach to define the proteome~of yeast mitochondria. We classified {\($>$\) 3,300 proteins of mitochondria and mitochondria-associated fractions and defined 901 high-confidence mitochondrial proteins, expanding the set of mitochondrial proteins by 82. Our analysis includes~protein abundance under fermentable and nonfermentable growth, submitochondrial localization, single-protein~experiments, and subcellular classification of mitochondria-associated fractions. We identified mitochondrial interactors of respiratory chain supercomplexes, ATP synthase, AAA proteases, the mitochondrial contact site and cristae organizing system (MICOS), and the coenzyme Q biosynthesis cluster, as well as mitochondrial proteins with dual cellular localization. The integrative proteome provides a high-confidence~source for the characterization of physiological and pathophysiological functions of mitochondria and their integration into the cellular environment.
• 
  Harsman, A., Oeljeklaus, S., Wenger, C., Huot, J. L., Warscheid, B., & Schneider, A. (2017). Corrigendum: The Non-Canonical Mitochondrial Inner Membrane Presequence Translocase of Trypanosomatids Contains Two Essential Rhomboid-like Proteins. Nature Communications, 8, 16123. https://doi.org/10.1038/ncomms16123
  • [ Abstract ]
  • [ DOI ]
  This corrects the article DOI: 10.1038/ncomms13707.
• 
  Schummer, A., Fischer, S., Oeljeklaus, S., & Warscheid, B. (2017). Study of Peroxisomal Protein Phosphorylation by Functional Proteomics. Methods in Molecular Biology (Clifton, N.J.), 1595, 267-289. https://doi.org/10.1007/978-1-4939-6937-1_26
  • [ Abstract ]
  • [ DOI ]
  Reversible protein phosphorylation is a frequently occurring posttranslational modification mediated by protein kinases and phosphatases that plays an essential role in the regulation of a large number of cellular processes. Evidence is accumulating that protein phosphorylation is also an important mechanism governing processes associated with peroxisome biology. For an improved and detailed understanding of these processes and their regulation it is therefore crucial to study phosphorylation of peroxisome-associated proteins and to determine the phosphorylated amino acid(s). To place peroxisome-related processes into a larger, cellular context, it is further required to identify the kinases and phosphatases catalyzing phosphorylation and dephosphorylation events in peroxisomal proteins. We here provide a strategy for the targeted analysis of peroxisomal phosphoproteins of Saccharomyces cerevisiae combining affinity purification of epitope-tagged peroxisomal proteins with Phos-tag SDS-PAGE and high-resolution mass spectrometry (MS) for the identification and precise localization of in vivo phosphosites. Furthermore, we describe a protocol for an MS-based in vitro kinase assay using recombinant peroxisomal proteins and a selected kinase facilitating the site-resolved analysis of kinase-substrate relationships.
• 
  Niemann, M., Harsman, A., Mani, J., Peikert, C. D., Oeljeklaus, S., Warscheid, B., Wagner, R., & Schneider, A. (2017). tRNAs and Proteins Use the Same Import Channel for Translocation across the Mitochondrial Outer Membrane of Trypanosomes. Proceedings of the National Academy of Sciences of the United States of America, 114(37), E7679-E7687. https://doi.org/10.1073/pnas.1711430114
  • [ Abstract ]
  • [ DOI ]
  Mitochondrial tRNA import is widespread, but the mechanism by which tRNAs are imported remains largely unknown. The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes, and thus imports all tRNAs from the cytosol. Here we show that in T. brucei in vivo import of tRNAs requires four subunits of the mitochondrial outer membrane protein translocase but not the two receptor subunits, one of which is essential for protein import. The latter shows that it is possible to uncouple mitochondrial tRNA import from protein import. Ablation of the intermembrane space domain of the translocase subunit, archaic translocase of the outer membrane (ATOM)14, on the other hand, while not affecting the architecture of the translocase, impedes both protein and tRNA import. A protein import intermediate arrested in the translocation channel prevents both protein and tRNA import. In the presence of tRNA, blocking events of single-channel currents through the pore formed by recombinant ATOM40 were detected in electrophysiological recordings. These results indicate that both types of macromolecules use the same import channel across the outer membrane. However, while tRNA import depends on the core subunits of the protein import translocase, it does not require the protein import receptors, indicating that the two processes are not mechanistically linked.
• 
  Wenger, C., Oeljeklaus, S., Warscheid, B., Schneider, A., & Harsman, A. (2017). A Trypanosomal Orthologue of an Intermembrane Space Chaperone Has a Non-Canonical Function in Biogenesis of the Single Mitochondrial Inner Membrane Protein Translocase. PLoS Pathogens, 13(8), e1006550. https://doi.org/10.1371/journal.ppat.1006550
  • [ Abstract ]
  • [ DOI ]
  Mitochondrial protein import is essential for Trypanosoma brucei across its life cycle and mediated by membrane-embedded heterooligomeric protein complexes, which mainly consist of trypanosomatid-specific subunits. However, trypanosomes contain orthologues of small Tim chaperones that escort hydrophobic proteins across the intermembrane space. Here we have experimentally analyzed three novel trypanosomal small Tim proteins, one of which contains only an incomplete Cx3C motif. RNAi-mediated ablation of TbERV1 shows that their import, as in other organisms, depends on the MIA pathway. Submitochondrial fractionation combined with immunoprecipitation and BN-PAGE reveals two pools of small Tim proteins: a soluble fraction forming 70 kDa complexes, consistent with hexamers and a second fraction that is tightly associated with the single trypanosomal TIM complex. RNAi-mediated ablation of the three proteins leads to a growth arrest and inhibits the formation of the TIM complex. In line with these findings, the changes in the mitochondrial proteome induced by ablation of one small Tim phenocopy the effects observed after ablation of TbTim17. Thus, the trypanosomal small Tims play an unexpected and essential role in the biogenesis of the single TIM complex, which for one of them is not linked to import of TbTim17.
• 
  Ruf, S., Heberle, A. M., Langelaar-Makkinje, M., Gelino, S., Wilkinson, D., Gerbeth, C., Schwarz, J. J., Holzwarth, B., Warscheid, B., Meisinger, C., van Vugt, M. A. T. M., Baumeister, R., Hansen, M., & Thedieck, K. (2017). PLK1 (Polo like Kinase 1) Inhibits MTOR Complex 1 and Promotes Autophagy. Autophagy, 13(3), 486-505. https://doi.org/10.1080/15548627.2016.1263781
  • [ Abstract ]
  • [ DOI ]
  Mechanistic target of rapamycin complex 1 (MTORC1) and polo like kinase 1 (PLK1) are major drivers of cancer cell growth and proliferation, and inhibitors of both protein kinases are currently being investigated in clinical studies. To date, MTORC1's and PLK1's functions are mostly studied separately, and reports on their mutual crosstalk are scarce. Here, we identify PLK1 as a physical MTORC1 interactor in human cancer cells. PLK1 inhibition enhances MTORC1 activity under nutrient sufficiency and in starved cells, and PLK1 directly phosphorylates the MTORC1 component RPTOR/RAPTOR in vitro. PLK1 and MTORC1 reside together at lysosomes, the subcellular site where MTORC1 is active. Consistent with an inhibitory role of PLK1 toward MTORC1, PLK1 overexpression inhibits lysosomal association of the PLK1-MTORC1 complex, whereas PLK1 inhibition promotes lysosomal localization of MTOR. PLK1-MTORC1 binding is enhanced by amino acid starvation, a condition known to increase autophagy. MTORC1 inhibition is an important step in autophagy activation. Consistently, PLK1 inhibition mitigates autophagy in cancer cells both under nutrient starvation and sufficiency, and a role of PLK1 in autophagy is also observed in the invertebrate model organism Caenorhabditis elegans. In summary, PLK1 inhibits MTORC1 and thereby positively contributes to autophagy. Since autophagy is increasingly recognized to contribute to tumor cell survival and growth, we propose that cautious monitoring of MTORC1 and autophagy readouts in clinical trials with PLK1 inhibitors is needed to develop strategies for optimized (combinatorial) cancer therapies targeting MTORC1, PLK1, and autophagy.
• 
  Peikert, C. D., Mani, J., Morgenstern, M., Käser, S., Knapp, B., Wenger, C., Harsman, A., Oeljeklaus, S., Schneider, A., & Warscheid, B. (2017). Charting Organellar Importomes by Quantitative Mass Spectrometry. Nature Communications, 8, 15272. https://doi.org/10.1038/ncomms15272
  • [ Abstract ]
  • [ DOI ]
  Protein import into organelles is essential for all eukaryotes and facilitated by multi-protein translocation machineries. Analysing whether a protein is transported into an organelle is largely restricted to single constituents. This renders knowledge about imported proteins incomplete, limiting our understanding of organellar biogenesis and function. Here we introduce a method that enables charting an organelle's importome. The approach relies on inducible RNAi-mediated knockdown of an essential subunit of a translocase to impair import and quantitative mass spectrometry. To highlight its potential, we established the mitochondrial importome of Trypanosoma brucei, comprising 1,120 proteins including 331 new candidates. Furthermore, the method allows for the identification of proteins with dual or multiple locations and the substrates of distinct protein import pathways. We demonstrate the specificity and versatility of this ImportOmics method by targeting import factors in mitochondria and glycosomes, which demonstrates its potential for globally studying protein import and inventories of organelles.
• 
  Krüger, V., Becker, T., Becker, L., Montilla-Martinez, M., Ellenrieder, L., Vögtle, F.-N., Meyer, H. E., Ryan, M. T., Wiedemann, N., Warscheid, B., Pfanner, N., Wagner, R., & Meisinger, C. (2017). Identification of New Channels by Systematic Analysis of the Mitochondrial Outer Membrane. The Journal of Cell Biology, 216(11), 3485-3495. https://doi.org/10.1083/jcb.201706043
  • [ Abstract ]
  • [ DOI ]
  The mitochondrial outer membrane is essential for communication between mitochondria and the rest of the cell and facilitates the transport of metabolites, ions, and proteins. All mitochondrial outer membrane channels known to date are {\($\beta$\)}-barrel membrane proteins, including the abundant voltage-dependent anion channel and the cation-preferring protein-conducting channels Tom40, Sam50, and Mdm10. We analyzed outer membrane fractions of yeast mitochondria and identified four new channel activities: two anion-preferring channels and two cation-preferring channels. We characterized the cation-preferring channels at the molecular level. The mitochondrial import component Mim1 forms a channel that is predicted to have an {\($\alpha$\)}-helical structure for protein import. The short-chain dehydrogenase-related protein Ayr1 forms an NADPH-regulated channel. We conclude that the mitochondrial outer membrane contains a considerably larger variety of channel-forming proteins than assumed thus far. These findings challenge the traditional view of the outer membrane as an unspecific molecular sieve and indicate a higher degree of selectivity and regulation of metabolite fluxes at the mitochondrial boundary.
• 
  Fischer, A., Weber, W., Warscheid, B., & Radziwill, G. (2017). AKT-dependent Phosphorylation of the SAM Domain Induces Oligomerization and Activation of the Scaffold Protein CNK1. Biochimica Et Biophysica Acta. Molecular Cell Research, 1864(1), 89-100. https://doi.org/10.1016/j.bbamcr.2016.10.009
  • [ Abstract ]
  • [ DOI ]
  Scaffold proteins are hubs for the coordination of intracellular signaling networks. The scaffold protein CNK1 promotes several signal transduction pathway. Here we demonstrate that sterile motif alpha (SAM) domain-dependent oligomerization of CNK1 stimulates CNK1-mediated signaling in growth factor-stimulated cells. We identified Ser22 located within the SAM domain as AKT-dependent phosphorylation site triggering CNK1 oligomerization. Oligomeric CNK1 increased the affinity for active AKT indicating a positive AKT feedback mechanism. A CNK1 mutant lacking the SAM domain and the phosphorylation-defective mutant CNK1(S22A) antagonizes oligomerization and prevents CNK1-driven cell proliferation and matrix metalloproteinase 14 promoter activation. The phosphomimetic mutant CNK1(S22D) constitutively oligomerizes and stimulates CNK1 downstream signaling. Searching the COSMIC database revealed Ser22 as putative target for oncogenic activation of CNK1. Like the phosphomimetic mutant CNK1(S22D), the oncogenic mutant CNK1(S22F) forms clusters in serum-starved cells comparable to clusters of CNK1 in growth factor-stimulated cells. CNK1 clusters induced by activating Ser22 mutants correlate with enhanced cell invasion and binding to and activation of ADP ribosylation factor 1 associated with tumor formation. Mutational analysis indicate that EGF-triggered phosphorylation of Thr8 within the SAM domain prevents AKT binding and antagonizes CNK1-mediated AKT signaling. Our findings reveal SAM domain-dependent oligomerization by AKT as switch for CNK1 activation.
• 
  Naumenko, N., Morgenstern, M., Rucktäschel, R., Warscheid, B., & Rehling, P. (2017). INA Complex Liaises the F(1)F(o)-ATP Synthase Membrane Motor Modules. Nature Communications, 8(1), 1237. https://doi.org/10.1038/s41467-017-01437-z
  • [ Abstract ]
  • [ DOI ]
  The F(1)F(0)-ATP synthase translates a proton flux across the inner mitochondrial membrane into a mechanical rotation, driving anhydride bond formation in the catalytic portion. The complex's membrane-embedded motor forms a proteinaceous channel at the interface between Atp9 ring and Atp6. To prevent unrestricted proton flow dissipating the H(+)-gradient, channel formation is a critical and tightly controlled step during ATP synthase assembly. Here we show that the INA complex (INAC) acts at this decisive step promoting Atp9-ring association with Atp6. INAC binds to newly synthesized mitochondrial-encoded Atp6 and Atp8 in complex with maturation factors. INAC association is retained until the F(1)-portion is built on Atp6/8 and loss of INAC causes accumulation of the free F(1). An independent complex is formed between INAC and the Atp9 ring. We conclude that INAC maintains assembly intermediates of the F(1) F(0)-ATP synthase in a primed state for the terminal assembly step-motor module formation.
• 
  Käser, S., Willemin, M., Schnarwiler, F., Schimanski, B., Poveda-Huertes, D., Oeljeklaus, S., Haenni, B., Zuber, B., Warscheid, B., Meisinger, C., & Schneider, A. (2017). Biogenesis of the Mitochondrial DNA Inheritance Machinery in the Mitochondrial Outer Membrane of Trypanosoma Brucei. PLoS Pathogens, 13(12), e1006808. https://doi.org/10.1371/journal.ppat.1006808
  • [ Abstract ]
  • [ DOI ]
  Mitochondria cannot form de novo but require mechanisms that mediate their inheritance to daughter cells. The parasitic protozoan Trypanosoma brucei has a single mitochondrion with a single-unit genome that is physically connected across the two mitochondrial membranes with the basal body of the flagellum. This connection, termed the tripartite attachment complex (TAC), is essential for the segregation of the replicated mitochondrial genomes prior to cytokinesis. Here we identify a protein complex consisting of three integral mitochondrial outer membrane proteins-TAC60, TAC42 and TAC40-which are essential subunits of the TAC. TAC60 contains separable mitochondrial import and TAC-sorting signals and its biogenesis depends on the main outer membrane protein translocase. TAC40 is a member of the mitochondrial porin family, whereas TAC42 represents a novel class of mitochondrial outer membrane {\($\beta$\)}-barrel proteins. Consequently TAC40 and TAC42 contain C-terminal {\($\beta$\)}-signals. Thus in trypanosomes the highly conserved {\($\beta$\)}-barrel protein assembly machinery plays a major role in the biogenesis of its unique mitochondrial genome segregation system.
• 
  Mokada-Gopal, L., Boeser, A., Lehmann, C. H. K., Drepper, F., Dudziak, D., Warscheid, B., & Voehringer, D. (2017). Identification of Novel STAT6-Regulated Proteins in Mouse B Cells by Comparative Transcriptome and Proteome Analysis. Journal of Immunology (Baltimore, Md. : 1950), 198(9), 3737-3745. https://doi.org/10.4049/jimmunol.1601838
  • [ Abstract ]
  • [ DOI ]
  The transcription factor STAT6 plays a key role in mediating signaling downstream of the receptors for IL-4 and IL-13. In B cells, STAT6 is required for class switch recombination to IgE and for germinal center formation during type 2 immune responses directed against allergens or helminths. In this study, we compared the transcriptomes and proteomes of primary mouse B cells from wild-type and STAT6-deficient mice cultured for 4 d in the presence or absence of IL-4. Microarray analysis revealed that 214 mRNAs were upregulated and 149 were downregulated {\($>$\)3-fold by IL-4 in a STAT6-dependent manner. Across all samples, {\($\sim$\)}5000 proteins were identified by label-free quantitative liquid chromatography/mass spectrometry. A total of 149 proteins was found to be differentially expressed {\($>$\)3-fold between IL-4-stimulated wild-type and STAT6(-/-) B cells (75 upregulated and 74 downregulated). Comparative analysis of the proteome and transcriptome revealed that expression of these proteins was mainly regulated at the transcriptional level, which argues against a major role for posttranscriptional mechanisms that modulate the STAT6-dependent proteome. Nine proteins were selected for confirmation by flow cytometry or Western blot. We show that CD30, CD79b, SLP-76, DEC205, IL-5R{\($\alpha$\)}, STAT5, and Thy1 are induced by IL-4 in a STAT6-dependent manner. In contrast, Syk and Fc receptor-like 1 were downregulated. This dataset provides a framework for further functional analysis of newly identified IL-4-regulated proteins in B cells that may contribute to germinal center formation and IgE switching in type 2 immunity.
• 
  Dolezal, E., Infantino, S., Drepper, F., Börsig, T., Singh, A., Wossning, T., Fiala, G. J., Minguet, S., Warscheid, B., Tarlinton, D. M., Jumaa, H., Medgyesi, D., & Reth, M. (2017). The BTG2-PRMT1 Module Limits Pre-B Cell Expansion by Regulating the CDK4-Cyclin-D3 Complex. Nature Immunology, 18(8), 911-920. https://doi.org/10.1038/ni.3774
  • [ Abstract ]
  • [ DOI ]
  Developing pre-B cells in the bone marrow alternate between proliferation and differentiation phases. We found that protein arginine methyl transferase 1 (PRMT1) and B cell translocation gene 2 (BTG2) are critical components of the pre-B cell differentiation program. The BTG2-PRMT1 module induced a cell-cycle arrest of pre-B cells that was accompanied by re-expression of Rag1 and Rag2 and the onset of immunoglobulin light chain gene rearrangements. We found that PRMT1 methylated cyclin-dependent kinase 4 (CDK4), thereby preventing the formation of a CDK4-Cyclin-D3 complex and cell cycle progression. Moreover, BTG2 in concert with PRMT1 efficiently blocked the proliferation of BCR-ABL1-transformed pre-B cells in vitro and in vivo. Our results identify a key molecular mechanism by which the BTG2-PRMT1 module regulates pre-B cell differentiation and inhibits pre-B cell leukemogenesis.
• 
  Rampelt, H., Bohnert, M., Zerbes, R. M., Horvath, S. E., Warscheid, B., Pfanner, N., & van der Laan, M. (2017). Mic10, a Core Subunit of the Mitochondrial Contact Site and Cristae Organizing System, Interacts with the Dimeric F(1)F(o)-ATP Synthase. Journal of Molecular Biology, 429(8), 1162-1170. https://doi.org/10.1016/j.jmb.2017.03.006
  • [ Abstract ]
  • [ DOI ]
  The mitochondrial contact site and cristae organizing system (MICOS) is crucial for maintaining the architecture of the mitochondrial inner membrane. MICOS is enriched at crista junctions that connect the two inner membrane domains: inner boundary membrane and cristae membrane. MICOS promotes the formation of crista junctions, whereas the oligomeric F(1)F(o)-ATP synthase is crucial for shaping cristae rims, indicating antagonistic functions of these machineries in organizing inner membrane architecture. We report that the MICOS core subunit Mic10, but not Mic60, binds to the F(1)F(o)-ATP synthase. Mic10 selectively associates with the dimeric form of the ATP synthase and supports the formation of ATP synthase oligomers. Our results suggest that Mic10 plays a dual role in mitochondrial inner membrane architecture. In addition to its central function in sculpting crista junctions, a fraction of Mic10 molecules interact with the cristae rim-forming F(1)F(o)-ATP synthase.
• 
  Hoffmann, L., Schummer, A., Reimann, J., Haurat, M. F., Wilson, A. J., Beeby, M., Warscheid, B., & Albers, S.-V. (2017). Expanding the Archaellum Regulatory Network - the Eukaryotic Protein Kinases ArnC and ArnD Influence Motility of Sulfolobus Acidocaldarius. MicrobiologyOpen, 6(1), Article 1. https://doi.org/10.1002/mbo3.414
  • [ Abstract ]
  • [ DOI ]
  Expression of the archaellum, the archaeal-type IV pilus-like rotating motility structure is upregulated under nutrient limitation. This is controlled by a network of regulators, called the archaellum regulatory network (arn). Several of the components of this network in Sulfolobus~acidocaldarius can be phosphorylated, and the deletion of the phosphatase PP2A results in strongly increased motility during starvation, indicating a role for phosphorylation in the regulation of motility. Analysis of the motility of different protein kinase deletion strains revealed that deletion of saci_0965, saci_1181, and saci_1193 resulted in reduced motility, whereas the deletion of saci_1694 resulted in hypermotility. Here ArnC (Saci\_1193) and ArnD (Saci\_1694) are characterized. Purified ArnC and ArnD phosphorylate serine and threonine residues in the C-terminus of the repressor ArnB. arnC is upregulated in starvation medium, whereas arnD is constitutively expressed. However, while differences in the expression and levels of flaB were observed in the {\($\Delta$\)}arnD strain during growth under rich conditions, under nutrient limiting conditions the {\($\Delta$\)}arnC and {\($\Delta$\)}arnD strains showed no large differences in the expression levels of the archaellum or of the studied regulators. This suggests that next to the regulation via the archaellum regulatory network additional regulatory mechanisms of expression and/or activity of the archaellum exist.
• 
  Wróblewska, J. P., Cruz-Zaragoza, L. D., Yuan, W., Schummer, A., Chuartzman, S. G., de Boer, R., Oeljeklaus, S., Schuldiner, M., Zalckvar, E., Warscheid, B., Erdmann, R., & van der Klei, I. J. (2017). Saccharomyces Cerevisiae Cells Lacking Pex3 Contain Membrane Vesicles That Harbor a Subset of Peroxisomal Membrane Proteins. Biochimica Et Biophysica Acta. Molecular Cell Research, 1864(10), 1656-1667. https://doi.org/10.1016/j.bbamcr.2017.05.021
  • [ Abstract ]
  • [ DOI ]
  Pex3 has been proposed to be important for the exit of peroxisomal membrane proteins (PMPs) from the ER, based on the observation that PMPs accumulate at the ER in Saccharomyces cerevisiae pex3 mutant cells. Using a combination of microscopy and biochemical approaches, we show that a subset of the PMPs, including the receptor docking protein Pex14, localizes to membrane vesicles in S. cerevisiae pex3 cells. These vesicles are morphologically distinct from the ER and do not co-sediment with ER markers in cell fractionation experiments. At the vesicles, Pex14 assembles with other peroxins (Pex13, Pex17, and Pex5) to form a complex with a composition similar to the PTS1 import pore in wild-type cells. Fluorescence microscopy studies revealed that also the PTS2 receptor Pex7, the importomer organizing peroxin Pex8, the ubiquitin conjugating enzyme Pex4 with its recruiting PMP Pex22, as well as Pex15 and Pex25 co-localize with Pex14. Other peroxins (including the RING finger complex and Pex27) did not accumulate at these structures, of which Pex11 localized to mitochondria. In line with these observations, proteomic analysis showed that in addition to the docking proteins and Pex5, also Pex7, Pex4/Pex22 and Pex25 were present in Pex14 complexes isolated from pex3 cells. However, formation of the entire importomer was not observed, most likely because Pex8 and the RING proteins were absent in the Pex14 protein complexes. Our data suggest that peroxisomal membrane vesicles can form in the absence of Pex3 and that several PMPs can insert in these vesicles in a Pex3 independent manner.
• 
  Reimann, L., Wiese, H., Leber, Y., Schwäble, A. N., Fricke, A. L., Rohland, A., Knapp, B., Peikert, C. D., Drepper, F., van der Ven, P. F. M., Radziwill, G., Fürst, D. O., & Warscheid, B. (2017). Myofibrillar Z-discs Are a Protein Phosphorylation Hot Spot with Protein Kinase C ({{PKC\($\alpha$\)}}) Modulating Protein Dynamics. Molecular & Cellular Proteomics : MCP, 16(3), 346-367. https://doi.org/10.1074/mcp.M116.065425
  • [ Abstract ]
  • [ DOI ]
  The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast, which were differentiated into myotubes, followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we found significant changes in the relative abundance of 387 proteins in myoblasts versus differentiated myotubes, reflecting the drastic phenotypic conversion of these cells during myogenesis. Interestingly, EPS of differentiated myotubes to induce Z-disc assembly and maturation resulted in increased levels of proteins involved in ATP synthesis, presumably to fulfill the higher energy demand of contracting myotubes. Because an important role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape further warranted in-depth analysis. We therefore established, by global phosphoproteomics of EPS-treated contracting myotubes, a comprehensive site-resolved protein phosphorylation map of the Z-disc and found that it is a phosphorylation hotspot in skeletal myocytes, underscoring its functions in signaling and disease-related processes. In an illustrative fashion, we analyzed the actin-binding multiadaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKC{\($\alpha$\)} phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after photobleaching experiments indicated that this phosphorylation modulates FLNc dynamics. Moreover, FLNc lacking the cleaved Ig-like domain 24 exhibited remarkably fast kinetics and exceedingly high mobility. Our data set provides research community resource for further identification of kinase-mediated changes in myofibrillar protein interactions, kinetics, and mobility that will greatly advance our understanding of Z-disc dynamics and signaling.

• 
  Hannemann, L., Suppanz, I., Ba, Q., MacInnes, K., Drepper, F., Warscheid, B., & Koch, H.-G. (2016). Redox Activation of the Universally Conserved ATPase YchF by Thioredoxin 1. Antioxidants & Redox Signaling, 24(3), 141-156. https://doi.org/10.1089/ars.2015.6272
  • [ Abstract ]
  • [ DOI ]
  AIMS: YchF/Ola1 are unconventional members of the universally conserved GTPase family because they preferentially hydrolyze ATP rather than GTP. These ATPases have been associated with various cellular processes and pathologies, including DNA repair, tumorigenesis, and apoptosis. In particular, a possible role in regulating the oxidative stress response has been suggested for both bacterial and human YchF/Ola1. In this study, we analyzed how YchF responds to oxidative stress and how it potentially regulates the antioxidant response. RESULTS: Our data identify a redox-regulated monomer-dimer equilibrium of YchF as a key event in the functional cycle of YchF. Upon oxidative stress, the oxidation of a conserved and surface-exposed cysteine residue promotes YchF dimerization, which is accompanied by inhibition of the ATPase activity. No dimers were observed in a YchF mutant lacking this cysteine. In vitro, the YchF dimer is dissociated by thioredoxin 1 (TrxA) and this stimulates the ATPase activity. The physiological significance of the YchF-thioredoxin 1 interaction was demonstrated by in vivo cross-linking, which validated this interaction in living cells. This approach also revealed that both the ATPase domain and the helical domain of YchF are in contact with TrxA. INNOVATION: YchF/Ola1 are the first redox-regulated members of the universally conserved GTPase family and are inactivated by oxidation of a conserved cysteine residue within the nucleotide-binding motif. CONCLUSION: Our data provide novel insights into the regulation of the so far ill-defined YchF/Ola1 family of proteins and stipulate their role as negative regulators of the oxidative stress response.
• 
  Renz, C., Oeljeklaus, S., Grinhagens, S., Warscheid, B., Johnsson, N., & Gronemeyer, T. (2016). Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry. PloS One, 11(2), e0148340. https://doi.org/10.1371/journal.pone.0148340
  • [ Abstract ]
  • [ DOI ]
  The septins are a conserved family of GTP-binding proteins that, in the baker's yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified. Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.
• 
  Oeljeklaus, S., Schummer, A., Mastalski, T., Platta, H. W., & Warscheid, B. (2016). Regulation of Peroxisome Dynamics by Phosphorylation. Biochimica Et Biophysica Acta, 1863(5), 1027-1037. https://doi.org/10.1016/j.bbamcr.2015.12.022
  • [ Abstract ]
  • [ DOI ]
  Peroxisomes are highly dynamic organelles that can rapidly change in size, abundance, and protein content in response to alterations in nutritional and other environmental conditions. These dynamic changes in peroxisome features, referred to as peroxisome dynamics, rely on the coordinated action of several processes of peroxisome biogenesis. Revealing the regulatory mechanisms of peroxisome dynamics is an emerging theme in cell biology. These mechanisms are inevitably linked to and synchronized with the biogenesis and degradation of peroxisomes. To date, the key players and basic principles of virtually all steps in the peroxisomal life cycle are known, but regulatory mechanisms remained largely elusive. A number of recent studies put the spotlight on reversible protein phosphorylation for the control of peroxisome dynamics and highlighted peroxisomes as hubs for cellular signal integration and regulation. Here, we will present and discuss the results of several studies performed using yeast and mammalian cells that convey a sense of the impact protein phosphorylation may have on the modulation of peroxisome dynamics by regulating peroxisomal matrix and membrane protein import, proliferation, inheritance, and degradation. We further put forward the idea to make use of current data on phosphorylation sites of peroxisomal and peroxisome-associated proteins reported in advanced large-scale phosphoproteomic studies.
• 
  Stiller, S. B., Höpker, J., Oeljeklaus, S., Schütze, C., Schrempp, S. G., Vent-Schmidt, J., Horvath, S. E., Frazier, A. E., Gebert, N., van der Laan, M., Bohnert, M., Warscheid, B., Pfanner, N., & Wiedemann, N. (2016). Mitochondrial OXA Translocase Plays a Major Role in Biogenesis of Inner-Membrane Proteins. Cell Metabolism, 23(5), 901-908. https://doi.org/10.1016/j.cmet.2016.04.005
  • [ Abstract ]
  • [ DOI ]
  The mitochondrial inner membrane harbors three protein translocases. Presequence translocase and carrier translocase are essential for importing nuclear-encoded proteins. The oxidase assembly (OXA) translocase is required for exporting mitochondrial-encoded proteins; however, different views exist about its relevance for nuclear-encoded proteins. We report that OXA plays a dual role in the biogenesis of nuclear-encoded mitochondrial proteins. First, a systematic analysis of OXA-deficient mitochondria led to~an unexpected expansion of the spectrum of OXA substrates imported via the presequence pathway. Second, biogenesis of numerous metabolite carriers depends on OXA, although they are not imported by~the presequence pathway. We show that OXA is crucial for the biogenesis of the Tim18-Sdh3 module of the carrier translocase. The export translocase OXA is thus required for the import of metabolite carriers by promoting assembly of the carrier translocase. We conclude that OXA is of central importance for the biogenesis of the mitochondrial inner membrane.
• 
  Chan, A., Schummer, A., Fischer, S., Schröter, T., Cruz-Zaragoza, L. D., Bender, J., Drepper, F., Oeljeklaus, S., Kunau, W.-H., Girzalsky, W., Warscheid, B., & Erdmann, R. (2016). Pex17p-Dependent Assembly of Pex14p/Dyn2p-subcomplexes of the Peroxisomal Protein Import Machinery. European Journal of Cell Biology, 95(12), 585-597. https://doi.org/10.1016/j.ejcb.2016.10.004
  • [ Abstract ]
  • [ DOI ]
  Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by peroxisomal targeting sequences (PTS). In the following, the assembled receptor-cargo complex is targeted to the peroxisomal membrane where it docks to the docking-complex as part of the peroxisomal translocation machinery. The docking-complex is composed of Pex13p, Pex14p and in yeast also Pex17p, whose function is still elusive. In order to characterize the function of Pex17p, we compared the composition and size of peroxisomal receptor-docking complexes from wild-type and pex17{\($\Delta$\)} cells. Our data demonstrate that the deficiency of Pex17p affects the stoichiometry of the constituents of an isolated 600kDa complex and that pex17{\($\Delta$\)} cells lack a high molecular weight complex ({\($>$\)}900kDa) of unknown function. We identified the dynein light chain protein Dyn2p as an additional core component of the Pex14p/Pex17p-complex. Both, Pex14p and Pex17p interact directly with Dyn2p, but in vivo, Pex17p turned out to be prerequisite for an association of Dyn2p with Pex14p. Finally, like pex17{\($\Delta$\)} also dyn2{\($\Delta$\)} cells lack the high molecular weight complex. As dyn2{\($\Delta$\)} cells also display reduced peroxisomal function, our data indicate that Dyn2p-dependent formation of the high molecular weight Pex14p-complex is required to maintain peroxisomal function on wild-type level.
• 
  Yilmaz, c S., Drepper, F., Hulstaert, N., v Cerniv c, M. sa, Gevaert, K., Economou, A., Warscheid, B., Martens, L., & Vandermarliere, E. (2016). Xilmass: A New Approach toward the Identification of Cross-Linked Peptides. Analytical Chemistry, 88(20), 9949-9957. https://doi.org/10.1021/acs.analchem.6b01585
  • [ Abstract ]
  • [ DOI ]
  Chemical cross-linking coupled with mass spectrometry plays an important role in unravelling protein interactions, especially weak and transient ones. Moreover, cross-linking complements several structural determination approaches such as cryo-EM. Although several computational approaches are available for the annotation of spectra obtained from cross-linked peptides, there remains room for improvement. Here, we present Xilmass, a novel algorithm to identify cross-linked peptides that introduces two new concepts: (i) the cross-linked peptides are represented in the search database such that the cross-linking sites are explicitly encoded, and (ii) the scoring function derived from the Andromeda algorithm was adapted to score against a theoretical tandem mass spectrometry (MS/MS) spectrum that contains the peaks from all possible fragment ions of a cross-linked peptide pair. The performance of Xilmass was evaluated against the recently published Kojak and the popular pLink algorithms on a calmodulin-plectin complex data set, as well as three additional, published data sets. The results show that Xilmass typically had the highest number of identified distinct cross-linked sites and also the highest number of predicted cross-linked sites.
• 
  Richter-Dennerlein, R., Oeljeklaus, S., Lorenzi, I., Ronsör, C., Bareth, B., Schendzielorz, A. B., Wang, C., Warscheid, B., Rehling, P., & Dennerlein, S. (2016). Mitochondrial Protein Synthesis Adapts to Influx of Nuclear-Encoded Protein. Cell, 167(2), 471-483.e10. https://doi.org/10.1016/j.cell.2016.09.003
  • [ Abstract ]
  • [ DOI ]
  Mitochondrial ribosomes translate membrane integral core subunits of the oxidative phosphorylation system encoded by mtDNA. These translation products associate with nuclear-encoded, imported proteins to form enzyme complexes that produce ATP. Here, we show that human mitochondrial ribosomes display translational plasticity to cope with the supply of imported nuclear-encoded subunits. Ribosomes expressing mitochondrial-encoded COX1 mRNA selectively engage with cytochrome c oxidase assembly factors in the inner membrane. Assembly defects of the cytochrome c oxidase arrest mitochondrial translation in a ribosome nascent chain complex with a partially membrane-inserted COX1 translation product. This complex represents a primed state of the translation product that can be retrieved for assembly. These findings establish a mammalian translational plasticity pathway in mitochondria that enables adaptation of mitochondrial protein synthesis to the influx of nuclear-encoded subunits.
• 
  Dalle Pezze, P., Ruf, S., Sonntag, A. G., Langelaar-Makkinje, M., Hall, P., Heberle, A. M., Razquin Navas, P., van Eunen, K., Tölle, R. C., Schwarz, J. J., Wiese, H., Warscheid, B., Deitersen, J., Stork, B., Fäßler, E., Schäuble, S., Hahn, U., Horvatovich, P., Shanley, D. P., & Thedieck, K. (2016). A Systems Study Reveals Concurrent Activation of AMPK and mTOR by Amino Acids. Nature Communications, 7, 13254. https://doi.org/10.1038/ncomms13254
  • [ Abstract ]
  • [ DOI ]
  Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational-experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca(2+)/calmodulin-dependent protein kinase kinase {\($\beta$\)} (CaMKK{\($\beta$\)}). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes.
• 
  Käser, S., Oeljeklaus, S., T\’y}}{v c, J. rí, Vaughan, S., Warscheid, B., & Schneider, A. (2016). Outer Membrane Protein Functions as Integrator of Protein Import and DNA Inheritance in Mitochondria. Proceedings of the National Academy of Sciences of the United States of America, 113(31), E4467-4475. https://doi.org/10.1073/pnas.1605497113
  • [ Abstract ]
  • [ DOI ]
  Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance.
• 
  Lorenzi, I., Oeljeklaus, S., Ronsör, C., Bareth, B., Warscheid, B., Rehling, P., & Dennerlein, S. (2016). Ribosome-Associated Mba1 Escorts Cox2 from Insertion Machinery to Maturing Assembly Intermediates. Molecular and Cellular Biology, 36(22), 2782-2793. https://doi.org/10.1128/MCB.00361-16
  • [ Abstract ]
  • [ DOI ]
  The three conserved core subunits of the cytochrome c oxidase are encoded by mitochondria in close to all eukaryotes. The Cox2 subunit spans the inner membrane twice, exposing the N and C termini to the intermembrane space. For this, the N terminus is exported cotranslationally by Oxa1 and subsequently undergoes proteolytic maturation in Saccharomyces cerevisiae Little is known about the translocation of the C terminus, but Cox18 has been identified to be a critical protein in this process. Here we find that the scaffold protein Cox20, which promotes processing of Cox2, is in complex with the ribosome receptor Mba1 and translating mitochondrial ribosomes in a Cox2-dependent manner. The Mba1-Cox20 complex accumulates when export of the C terminus of Cox2 is blocked by the loss of the Cox18 protein. While Cox20 engages with Cox18, Mba1 is no longer present at this stage. Our analyses indicate that Cox20 associates with nascent Cox2 and Mba1 to promote Cox2 maturation cotranslationally. We suggest that Mba1 stabilizes the Cox20-ribosome complex and supports the handover of Cox2 to the Cox18 tail export machinery.
• 
  Harsman, A., Oeljeklaus, S., Wenger, C., Huot, J. L., Warscheid, B., & Schneider, A. (2016). The Non-Canonical Mitochondrial Inner Membrane Presequence Translocase of Trypanosomatids Contains Two Essential Rhomboid-like Proteins. Nature Communications, 7, 13707. https://doi.org/10.1038/ncomms13707
  • [ Abstract ]
  • [ DOI ]
  Mitochondrial protein import is essential for all eukaryotes. Here we show that the early diverging eukaryote Trypanosoma brucei has a non-canonical inner membrane (IM) protein translocation machinery. Besides TbTim17, the single member of the Tim17/22/23 family in trypanosomes, the presequence translocase contains nine subunits that co-purify in reciprocal immunoprecipitations and with a presequence-containing substrate that is trapped in the translocation channel. Two of the newly discovered subunits are rhomboid-like proteins, which are essential for growth and mitochondrial protein import. Rhomboid-like proteins were proposed to form the protein translocation pore of the ER-associated degradation system, suggesting that they may contribute to pore formation in the presequence translocase of T. brucei. Pulldown of import-arrested mitochondrial carrier protein shows that the carrier translocase shares eight subunits with the presequence translocase. This indicates that T. brucei may have a single IM translocase that with compositional variations mediates import of presequence-containing and carrier proteins.
• 
  Hübscher, V., Mudholkar, K., Chiabudini, M., Fitzke, E., Wölfle, T., Pfeifer, D., Drepper, F., Warscheid, B., & Rospert, S. (2016). The Hsp70 Homolog Ssb and the 14-3-3 Protein Bmh1 Jointly Regulate Transcription of Glucose Repressed Genes in Saccharomyces Cerevisiae. Nucleic Acids Research, 44(12), 5629-5645. https://doi.org/10.1093/nar/gkw168
  • [ Abstract ]
  • [ DOI ]
  Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of {\($\Delta$\)}reg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation.
• 
  Fischer, A., Warscheid, B., Weber, W., & Radziwill, G. (2016). Optogenetic Clustering of CNK1 Reveals Mechanistic Insights in RAF and AKT Signalling Controlling Cell Fate Decisions. Scientific Reports, 6, 38155. https://doi.org/10.1038/srep38155
  • [ Abstract ]
  • [ DOI ]
  Scaffold proteins such as the multidomain protein CNK1 orchestrate the signalling network by integrating and controlling the underlying pathways. Using an optogenetic approach to stimulate CNK1 uncoupled from upstream effectors, we identified selective clusters of CNK1 that either stimulate RAF-MEK-ERK or AKT signalling depending on the light intensity applied. OptoCNK1 implemented in MCF7 cells induces differentiation at low light intensity stimulating ERK activity whereas stimulation of AKT signalling by higher light intensity promotes cell proliferation. CNK1 clustering in response to increasing EGF concentrations revealed that CNK1 binds to RAF correlating with ERK activation at low EGF dose. At higher EGF dose active AKT binds to CNK1 and phosphorylates and inhibits RAF. Knockdown of CNK1 protects CNK1 from this AKT/RAF crosstalk. In C2 skeletal muscle cells CNK1 expression is induced with the onset of differentiation. Hence, AKT-bound CNK1 counteracts ERK stimulation in differentiated but not in proliferating cells. Ectopically expressed CNK1 facilitates C2 cell differentiation and knockdown of CNK1 impaired the transcriptional network underlying C2 cell differentiation. Thus, CNK1 expression, CNK1 clustering and the thereto related differential signalling processes decide on proliferation and differentiation in a cell type- and cell stage-dependent manner by orchestrating AKT and RAF signalling.

• 
  Dennerlein, S., Oeljeklaus, S., Jans, D., Hellwig, C., Bareth, B., Jakobs, S., Deckers, M., Warscheid, B., & Rehling, P. (2015). MITRAC7 Acts as a COX1-Specific Chaperone and Reveals a Checkpoint during Cytochrome c Oxidase Assembly. Cell Reports, 12(10), 1644-1655. https://doi.org/10.1016/j.celrep.2015.08.009
  • [ Abstract ]
  • [ DOI ]
  Cytochrome c oxidase, the terminal enzyme of the respiratory chain, is assembled from mitochondria- and nuclear-encoded subunits. The MITRAC complex represents the central assembly intermediate during this process as it receives imported subunits and regulates mitochondrial translation of COX1 mRNA. The molecular processes that promote and regulate the progression of assembly downstream of MITRAC are still unknown. Here, we identify MITRAC7 as a constituent of a late form of MITRAC and as a COX1-specific chaperone. MITRAC7 is required for cytochrome c oxidase biogenesis. Surprisingly, loss of MITRAC7 or an increase in its amount causes selective cytochrome c oxidase deficiency in human cells. We demonstrate that increased MITRAC7 levels stabilize and trap COX1 in MITRAC, blocking progression in the assembly process. In contrast, MITRAC7 deficiency leads to turnover of newly synthesized COX1. Accordingly, MITRAC7 affects the biogenesis pathway by stabilizing newly synthesized COX1 in assembly intermediates, concomitantly preventing turnover.
• 
  Stroud, D. A., Maher, M. J., Lindau, C., Vögtle, F.-N., Frazier, A. E., Surgenor, E., Mountford, H., Singh, A. P., Bonas, M., Oeljeklaus, S., Warscheid, B., Meisinger, C., Thorburn, D. R., & Ryan, M. T. (2015). COA6 Is a Mitochondrial Complex IV Assembly Factor Critical for Biogenesis of mtDNA-encoded COX2. Human Molecular Genetics, 24(19), 5404-5415. https://doi.org/10.1093/hmg/ddv265
  • [ Abstract ]
  • [ DOI ]
  Biogenesis of complex IV of the mitochondrial respiratory chain requires assembly factors for subunit maturation, co-factor attachment and stabilization of intermediate assemblies. A pathogenic mutation in COA6, leading to substitution of a conserved tryptophan for a cysteine residue, results in a loss of complex IV activity and cardiomyopathy. Here, we demonstrate that the complex IV defect correlates with a severe loss in complex IV assembly in patient heart but not fibroblasts. Complete loss of COA6 activity using gene editing in HEK293T cells resulted in a profound growth defect due to complex IV deficiency, caused by impaired biogenesis of the copper-bound mitochondrial DNA-encoded subunit COX2 and subsequent accumulation of complex IV assembly intermediates. We show that the pathogenic mutation in COA6 does not affect its import into mitochondria but impairs its maturation and stability. Furthermore, we show that COA6 has the capacity to bind copper and can associate with newly translated COX2 and the mitochondrial copper chaperone SCO1. Our data reveal that COA6 is intricately involved in the copper-dependent biogenesis of COX2.
• 
  Horvath, S. E., Rampelt, H., Oeljeklaus, S., Warscheid, B., van der Laan, M., & Pfanner, N. (2015). Role of Membrane Contact Sites in Protein Import into Mitochondria. Protein Science : A Publication of the Protein Society, 24(3), 277-297. https://doi.org/10.1002/pro.2625
  • [ Abstract ]
  • [ DOI ]
  Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long-standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence-carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture.
• 
  Hünten, S., Kaller, M., Drepper, F., Oeljeklaus, S., Bonfert, T., Erhard, F., Dueck, A., Eichner, N., Friedel, C. C., Meister, G., Zimmer, R., Warscheid, B., & Hermeking, H. (2015). P53-Regulated Networks of Protein, mRNA, miRNA, and lncRNA Expression Revealed by Integrated Pulsed Stable Isotope Labeling With Amino Acids in Cell Culture ({{pSILAC}}) and Next Generation Sequencing ({{NGS}}) Analyses. Molecular & Cellular Proteomics : MCP, 14(10), 2609-2629. https://doi.org/10.1074/mcp.M115.050237
  • [ Abstract ]
  • [ DOI ]
  We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics (pulsed stable isotope labeling with amino acids in cell culture/pSILAC) in the colorectal cancer cell line SW480. This was combined with mRNA and noncoding RNA expression analyses by next generation sequencing (RNA-, miR-Seq). Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated proteins (542 up, 569 down), mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down) and lncRNAs (270 up, 123 down). Changes in protein and mRNA expression levels showed a positive correlation (r = 0.50, p {\($<$\) 0.0001). In total, we detected 133 direct p53 target genes that were differentially expressed and displayed p53 occupancy in the vicinity of their promoter. More transcriptionally induced genes displayed occupied p53 binding sites (4.3\% mRNAs, 7.2\% miRNAs, 6.3\% lncRNAs, 5.9\% proteins) than repressed genes (2.4\% mRNAs, 3.2\% miRNAs, 0.8\% lncRNAs, 1.9\% proteins), suggesting indirect mechanisms of repression. Around 50% of the down-regulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3'-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed up-regulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibits proliferation in SW480 cells. Furthermore, KLF12, HMGB1 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486-5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of KLF12, HMGB1 and CIT was detected in advanced stages of cancer. In conclusion, the integration of multiple omics methods allowed the comprehensive identification of direct and indirect effectors of p53 that provide new insights and leads into the mechanisms of p53-mediated tumor suppression.
• 
  Song, J.-G., Kostan, J., Drepper, F., Knapp, B., de Almeida Ribeiro, E. J., Konarev, P. V., Grishkovskaya, I., Wiche, G., Gregor, M., Svergun, D. I., Warscheid, B., & Djinovi’c}}-Carugo, K. (2015). Structural Insights into Ca2+-Calmodulin Regulation of Plectin 1a-Integrin {\($B$\)4 Interaction in Hemidesmosomes. Structure (London, England : 1993), 23(3), 558-570. https://doi.org/10.1016/j.str.2015.01.011
  • [ Abstract ]
  • [ DOI ]
  The mechanical stability of epithelial cells, which protect organisms from harmful external factors, is maintained by hemidesmosomes via the interaction between plectin 1a (P1a) and integrin {\($\alpha$\)}6{\($\beta$\)}4. Binding of calcium-calmodulin (Ca(2+)-CaM) to P1a together with phosphorylation of integrin {\($\beta$\)}4 disrupts this complex, resulting in disassembly of hemidesmosomes. We present structures of the P1a actin binding domain either in complex with the N-ter lobe of Ca(2+)-CaM or with the first pair of integrin {\($\beta$\)}4 fibronectin domains. Ca(2+)-CaM binds to the N-ter isoform-specific tail of P1a in a unique manner, via its N-ter lobe in an extended conformation. Structural, cell biology, and biochemical studies suggest the following model: binding of Ca(2+)-CaM to an intrinsically disordered N-ter segment of plectin converts it to an {\($\alpha$\)} helix, which repositions calmodulin to displace integrin {\($\beta$\)}4 by steric repulsion. This model could serve as a blueprint for studies aimed at understanding how Ca(2+)-CaM or EF-hand motifs regulate F-actin-based cytoskeleton.
• 
  Gemmecker, S., Schaub, P., Koschmieder, J., Brausemann, A., Drepper, F., Rodriguez-Franco, M., Ghisla, S., Warscheid, B., Einsle, O., & Beyer, P. (2015). Phytoene Desaturase from Oryza Sativa: Oligomeric Assembly, Membrane Association and Preliminary 3D-Analysis. PloS One, 10(7), e0131717. https://doi.org/10.1371/journal.pone.0131717
  • [ Abstract ]
  • [ DOI ]
  Recombinant phytoene desaturase (PDS-His6) from rice was purified to near-homogeneity and shown to be enzymatically active in a biphasic, liposome-based assay system. The protein contains FAD as the sole protein-bound redox-cofactor. Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase. The herbicidal PDS-inhibitor norflurazon is capable of arresting the reaction by stabilizing the intermediary FAD(red), while an excess of the quinone acceptor relieves this blockage, indicating competition. The enzyme requires its homo-oligomeric association for activity. The sum of data collected through gel permeation chromatography, non-denaturing polyacrylamide electrophoresis, chemical cross-linking, mass spectrometry and electron microscopy techniques indicate that the high-order oligomers formed in solution are the basis for an active preparation. Of these, a tetramer consisting of dimers represents the active unit. This is corroborated by our preliminary X-ray structural analysis that also revealed similarities of the protein fold with the sequence-inhomologous bacterial phytoene desaturase CRTI and other oxidoreductases of the GR2-family of flavoproteins. This points to an evolutionary relatedness of CRTI and PDS yielding different carotene desaturation sequences based on homologous protein folds.
• 
  Mani, J., Desy, S., Niemann, M., Chanfon, A., Oeljeklaus, S., Pusnik, M., Schmidt, O., Gerbeth, C., Meisinger, C., Warscheid, B., & Schneider, A. (2015). Mitochondrial Protein Import Receptors in Kinetoplastids Reveal Convergent Evolution over Large Phylogenetic Distances. Nature Communications, 6, 6646. https://doi.org/10.1038/ncomms7646
  • [ Abstract ]
  • [ DOI ]
  Mitochondrial protein import is essential for all eukaryotes and mediated by hetero-oligomeric protein translocases thought to be conserved within all eukaryotes. We have identified and analysed the function and architecture of the non-conventional outer membrane (OM) protein translocase in the early diverging eukaryote Trypanosoma brucei. It consists of six subunits that show no obvious homology to translocase components of other species. Two subunits are import receptors that have a unique topology and unique protein domains and thus evolved independently of the prototype receptors Tom20 and Tom70. Our study suggests that protein import receptors were recruited to the core of the OM translocase after the divergence of the major eukaryotic supergroups. Moreover, it links the evolutionary history of mitochondrial protein import receptors to the origin of the eukaryotic supergroups.
• 
  Höhr, A. I. C., Straub, S. P., Warscheid, B., Becker, T., & Wiedemann, N. (2015). Assembly of {\($\beta$\)}-Barrel Proteins in the Mitochondrial Outer Membrane. Biochimica Et Biophysica Acta, 1853(1), 74-88. https://doi.org/10.1016/j.bbamcr.2014.10.006
  • [ Abstract ]
  • [ DOI ]
  Mitochondria evolved through endosymbiosis of a Gram-negative progenitor with a host cell to generate eukaryotes. Therefore, the outer membrane of mitochondria and Gram-negative bacteria contain pore proteins with {\($\beta$\)}-barrel topology. After synthesis in the cytosol, {\($\beta$\)}-barrel precursor proteins are first transported into the mitochondrial intermembrane space. Folding and membrane integration of {\($\beta$\)}-barrel proteins depend on the mitochondrial sorting and assembly machinery (SAM) located in the outer membrane, which is related to the {\($\beta$\)}-barrel assembly machinery (BAM) in bacteria. The SAM complex recognizes {\($\beta$\)}-barrel proteins by a {\($\beta$\)}-signal in the C-terminal {\($\beta$\)}-strand that is required to initiate {\($\beta$\)}-barrel protein insertion into the outer membrane. In addition, the SAM complex is crucial to form membrane contacts with the inner mitochondrial membrane by interacting with the mitochondrial contact site and cristae organizing system (MICOS) and shares a subunit with the endoplasmic reticulum-mitochondria encounter structure (ERMES) that links the outer mitochondrial membrane to the endoplasmic reticulum (ER).
• 
  Fischer, A., Brummer, T., Warscheid, B., & Radziwill, G. (2015). Differential Tyrosine Phosphorylation Controls the Function of CNK1 as a Molecular Switch in Signal Transduction. Biochimica Et Biophysica Acta, 1853(11 Pt A), 2847-2855. https://doi.org/10.1016/j.bbamcr.2015.08.014
  • [ Abstract ]
  • [ DOI ]
  Scaffold proteins are multidomain proteins without enzymatic function that play a central role in coordinating signaling processes. The scaffold protein CNK1 interacts with pathway-specific signaling proteins and thereby regulates these respective pathways. Here, we revealed tyrosine phosphorylation as a critical regulation mechanism to control the function of CNK1. We identified Tyr 26 as a PDGF-induced and, additionally, Tyr 519 and Tyr 665 as SRC-induced tyrosine phosphorylation sites. Phosphomimetic mutants indicate that phosphorylation of Tyr 519 recruits CNK1 to the nucleus and additional phosphorylation of Tyr 26 enables CNK1 to promote SRE-dependent gene expression. Contrary, mutants preventing tyrosine phosphorylation promote matrix metalloproteinase MMP14 promoter activity. CNK1-driven cell proliferation partially depends on its tyrosine phosphorylation. Upon PDGF stimulation, CNK1 is recruited to the plasma membrane mediated by SRC. Knock down of CNK1 prevents PDGF-induced SRE-dependent gene expression, MMP14 promoter activity and cell proliferation. Thus, tyrosine phosphorylation is an important mechanism to control the subcellular localization of CNK1 and its distinct biological functions.
• 
  Hagen, S., Drepper, F., Fischer, S., Fodor, K., Passon, D., Platta, H. W., Zenn, M., Schliebs, W., Girzalsky, W., Wilmanns, M., Warscheid, B., & Erdmann, R. (2015). Structural Insights into Cargo Recognition by the Yeast PTS1 Receptor. The Journal of Biological Chemistry, 290(44), 26610-26626. https://doi.org/10.1074/jbc.M115.657973
  • [ Abstract ]
  • [ DOI ]
  The peroxisomal matrix protein import is facilitated by cycling import receptors that shuttle between the cytosol and the peroxisomal membrane. The import receptor Pex5p mediates the import of proteins harboring a peroxisomal targeting signal of type I (PTS1). Purified recombinant Pex5p forms a dimeric complex with the PTS1-protein Pcs60p in vitro with a KD of 0.19 {\($\mu$\)}m. To analyze the structural basis for receptor-cargo recognition, the PTS1 and adjacent amino acids of Pcs60p were systematically scanned for Pex5p binding by an in vitro site-directed photo-cross-linking approach. The cross-linked binding regions of the receptor were subsequently identified by high resolution mass spectrometry. Most cross-links were found with TPR6, TPR7, as well as the 7C-loop of Pex5p. Surface plasmon resonance analysis revealed a bivalent interaction mode for Pex5p and Pcs60p. Interestingly, Pcs60p lacking its C-terminal tripeptide sequence was efficiently cross-linked to the same regions of Pex5p. The KD value of the interaction of truncated Pcs60p and Pex5p was in the range of 7.7 {\($\mu$\)}m. Isothermal titration calorimetry and surface plasmon resonance measurements revealed a monovalent binding mode for the interaction of Pex5p and Pcs60p lacking the PTS1. Our data indicate that Pcs60p contains a second contact site for its receptor Pex5p, beyond the C-terminal tripeptide. The physiological relevance of the ancillary binding region was supported by in vivo import studies. The bivalent binding mode might be explained by a two-step concept as follows: first, cargo recognition and initial tethering by the PTS1-receptor Pex5p; second, lock-in of receptor and cargo.
• 
  Böttinger, L., Oeljeklaus, S., Guiard, B., Rospert, S., Warscheid, B., & Becker, T. (2015). Mitochondrial Heat Shock Protein ({{Hsp}}) 70 and Hsp10 Cooperate in the Formation of Hsp60 Complexes. The Journal of Biological Chemistry, 290(18), 11611-11622. https://doi.org/10.1074/jbc.M115.642017
  • [ Abstract ]
  • [ DOI ]
  Mitochondrial Hsp70 (mtHsp70) mediates essential functions for mitochondrial biogenesis, like import and folding of proteins. In these processes, the chaperone cooperates with cochaperones, the presequence translocase, and other chaperone systems. The chaperonin Hsp60, together with its cofactor Hsp10, catalyzes folding of a subset of mtHsp70 client proteins. Hsp60 forms heptameric ring structures that provide a cavity for protein folding. How the Hsp60 rings are assembled is poorly understood. In a comprehensive interaction study, we found that mtHsp70 associates with Hsp60 and Hsp10. Surprisingly, mtHsp70 interacts with Hsp10 independently of Hsp60. The mtHsp70-Hsp10 complex binds to the unassembled Hsp60 precursor to promote its assembly into mature Hsp60 complexes. We conclude that coupling to Hsp10 recruits mtHsp70 to mediate the biogenesis of the heptameric Hsp60 rings.
• 
  Sachelaru, I., Petriman, N. A., Kudva, R., Kuhn, P., Welte, T., Knapp, B., Drepper, F., Warscheid, B., & Koch, H.-G. (2015). YidC Occupies the Lateral Gate of the SecYEG Translocon and Is Sequentially Displaced by a Nascent Membrane Protein. The Journal of Biological Chemistry, 290(23), 14492. https://doi.org/10.1074/jbc.A112.446583
  • [ DOI ]
• 
  Kuhn, P., Draycheva, A., Vogt, A., Petriman, N.-A., Sturm, L., Drepper, F., Warscheid, B., Wintermeyer, W., & Koch, H.-G. (2015). Ribosome Binding Induces Repositioning of the Signal Recognition Particle Receptor on the Translocon. The Journal of Cell Biology, 211(1), 91-104. https://doi.org/10.1083/jcb.201502103
  • [ Abstract ]
  • [ DOI ]
  Cotranslational protein targeting delivers proteins to the bacterial cytoplasmic membrane or to the eukaryotic endoplasmic reticulum membrane. The signal recognition particle (SRP) binds to signal sequences emerging from the ribosomal tunnel and targets the ribosome-nascent-chain complex (RNC) to the SRP receptor, termed FtsY in bacteria. FtsY interacts with the fifth cytosolic loop of SecY in the SecYEG translocon, but the functional role of the interaction is unclear. By using photo-cross-linking and fluorescence resonance energy transfer measurements, we show that FtsY-SecY complex formation is guanosine triphosphate independent but requires a phospholipid environment. Binding of an SRP-RNC complex exposing a hydrophobic transmembrane segment induces a rearrangement of the SecY-FtsY complex, which allows the subsequent contact between SecY and ribosomal protein uL23. These results suggest that direct RNC transfer to the translocon is guided by the interaction between SRP and translocon-bound FtsY in a quaternary targeting complex.
• 
  Schwarz, J. J., Wiese, H., Tölle, R. C., Zarei, M., Dengjel, J., Warscheid, B., & Thedieck, K. (2015). Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 ({{mTORC1}}) Substrate. Molecular & Cellular Proteomics : MCP, 14(8), 2042-2055. https://doi.org/10.1074/mcp.M114.045807
  • [ Abstract ]
  • [ DOI ]
  The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and investigated in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation.
• 
  Chojnacka, M., Gornicka, A., Oeljeklaus, S., Warscheid, B., & Chacinska, A. (2015). Cox17 Protein Is an Auxiliary Factor Involved in the Control of the Mitochondrial Contact Site and Cristae Organizing System. The Journal of Biological Chemistry, 290(24), 15304-15312. https://doi.org/10.1074/jbc.M115.645069
  • [ Abstract ]
  • [ DOI ]
  The mitochondrial contact site and cristae organizing system (MICOS) is a recently discovered protein complex that is crucial for establishing and maintaining the proper inner membrane architecture and contacts with the outer membrane of mitochondria. The ways in which the MICOS complex is assembled and its integrity is regulated remain elusive. Here, we report a direct link between Cox17, a protein involved in the assembly of cytochrome c oxidase, and the MICOS complex. Cox17 interacts with Mic60, thereby modulating MICOS complex integrity. This interaction does not involve Sco1, a partner of Cox17 in transferring copper ions to cytochrome c oxidase. However, the Cox17-MICOS interaction is regulated by copper ions. We propose that Cox17 is a newly identified factor involved in maintaining the architecture of the MICOS complex.
• 
  Wiese, H., Gelis, L., Wiese, S., Reichenbach, C., Jovancevic, N., Osterloh, M., Meyer, H. E., Neuhaus, E. M., Hatt, H. H., Radziwill, G., & Warscheid, B. (2015). Quantitative Phosphoproteomics Reveals the Protein Tyrosine Kinase Pyk2 as a Central Effector of Olfactory Receptor Signaling in Prostate Cancer Cells. Biochimica Et Biophysica Acta, 1854(6), 632-640. https://doi.org/10.1016/j.bbapap.2014.09.002
  • [ Abstract ]
  • [ DOI ]
  The prostate-specific G-protein-coupled receptor 1 (PSGR1) is an olfactory receptor specifically expressed in the prostate gland. PSGR1 expression is elevated both in benign prostatic hyperplasia tissue and in prostate cancer. Stimulation of PSGR1 by the odorant {\($\beta$\)}-ionone leads to an increase in the intracellular Ca(2+) concentration, activation of mitogen-activated protein (MAP) kinases and a decrease in prostate cancer cell proliferation. To further extend our knowledge about PSGR1 signaling in prostate cancer cells, we performed a quantitative phosphoproteomics study using stable isotope labeling by amino acids in cell culture and mass spectrometry. We report 51 differentially regulated phosphorylation sites in 24 proteins with functions in cytoskeletal remodeling, signaling and ion transport. Activation of PSGR1 evoked an increase in intracellular pH mediated by the sodium/hydrogen exchanger NHE1. Furthermore, we report the protein tyrosine kinase Pyk2 as a central effector of PSGR1 signaling cascades in LNCaP cells. Our data show that phosphorylation of p38 MAP kinase is triggered by Pyk2. In addition, we confirmed dephosphorylation of the tumor suppressor protein N-myc downstream regulated gene 1 (NDRG1) at Ser330 downstream of Pyk2. Since NDRG1 impacts oncogenic signaling pathways interfering with tumor progression, we suggest that the Pyk2-NDRG1 axis is possibly involved in conveying the anti-proliferative effect of {\($\beta$\)}-ionone in prostate cancer cells. This article is part of a Special Issue entitled: Medical Proteomics.
• 
  Wrobel, L., Topf, U., Bragoszewski, P., Wiese, S., Sztolsztener, M. E., Oeljeklaus, S., Varabyova, A., Lirski, M., Chroscicki, P., Mroczek, S., Januszewicz, E., Dziembowski, A., Koblowska, M., Warscheid, B., & Chacinska, A. (2015). Mistargeted Mitochondrial Proteins Activate a Proteostatic Response in the Cytosol. Nature, 524(7566), 485-488. https://doi.org/10.1038/nature14951
  • [ Abstract ]
  • [ DOI ]
  Most of the mitochondrial proteome originates from nuclear genes and is transported into the mitochondria after synthesis in the cytosol. Complex machineries which maintain the specificity of protein import and sorting include the TIM23 translocase responsible for the transfer of precursor proteins into the matrix, and the mitochondrial intermembrane space import and assembly (MIA) machinery required for the biogenesis of intermembrane space proteins. Dysfunction of mitochondrial protein sorting pathways results in diminishing specific substrate proteins, followed by systemic pathology of the organelle and organismal death. The cellular responses caused by accumulation of mitochondrial precursor proteins in the cytosol are mainly unknown. Here we present a comprehensive picture of the changes in the cellular transcriptome and proteome in response to a mitochondrial import defect and precursor over-accumulation stress. Pathways were identified that protect the cell against mitochondrial biogenesis defects by inhibiting protein synthesis and by activation of the proteasome, a major machine for cellular protein clearance. Proteasomal activity is modulated in proportion to the quantity of mislocalized mitochondrial precursor proteins in the cytosol. We propose that this type of unfolded protein response activated by mistargeting of proteins (UPRam) is beneficial for the cells. UPRam provides a means for buffering the consequences of physiological slowdown in mitochondrial protein import and for counteracting pathologies that are caused or contributed by mitochondrial dysfunction.

• 
  Oeljeklaus, S., Schummer, A., Suppanz, I., & Warscheid, B. (2014). SILAC Labeling of Yeast for the Study of Membrane Protein Complexes. Methods in Molecular Biology (Clifton, N.J.), 1188, 23-46. https://doi.org/10.1007/978-1-4939-1142-4_3
  • [ Abstract ]
  • [ DOI ]
  Despite their simplicity compared to multicellular organisms, single-celled yeasts such as the baker's yeast Saccharomyces cerevisiae are widely recognized as model organisms for the study of eukaryotic cell biology. To gain deeper insights into the molecular mechanisms underlying cellular processes, it is of utmost interest to establish the interactome of distinct proteins and to thoroughly analyze the composition of individual protein complexes and their dynamics. Combining affinity purification of epitope-tagged proteins with high-resolution mass spectrometry and quantitative proteomics strategies, in particular stable isotope labeling by amino acids in cell culture (SILAC), represents an unbiased and powerful approach for a most accurate characterization of protein complexes. In this chapter, we provide detailed protocols for the generation of yeast strains (S. cerevisiae) amenable to SILAC-labeling, for epitope tagging of a protein of interest for affinity purification, and for the SILAC-based characterization of membrane protein complexes including the identification of stable core components and transient interaction partners.
• 
  Kaller, M., Oeljeklaus, S., Warscheid, B., & Hermeking, H. (2014). Identification of microRNA Targets by Pulsed SILAC. Methods in Molecular Biology (Clifton, N.J.), 1188, 327-349. https://doi.org/10.1007/978-1-4939-1142-4_23
  • [ Abstract ]
  • [ DOI ]
  Pulsed stable isotope labeling by amino acids in cell culture (pulsed SILAC or pSILAC) allows to monitor and quantify the de novo synthesis of proteins in an unbiased fashion on a proteome-wide scale. The high applicability of this metabolic labeling technique has been demonstrated for the identification of posttranscriptional changes in gene expression on the proteome level, in particular those caused by microRNAs. The application of pSILAC allows the selective quantification of newly synthesized proteins and thus the detection of differences in protein translation. This is of particular interest in the case of microRNA-mediated regulations, which characteristically cause rather modest decreases in protein amounts that may be difficult to detect by other proteomic methods. Here, we describe a detailed protocol for using pSILAC to track miRNA-mediated changes in protein expression, using the p53-induced miR-34a microRNA as a prototypic example of microRNA-mediated regulations.
• 
  Mehnert, C. S., Rampelt, H., Gebert, M., Oeljeklaus, S., Schrempp, S. G., Kochbeck, L., Guiard, B., Warscheid, B., & van der Laan, M. (2014). The Mitochondrial ADP/ATP Carrier Associates with the Inner Membrane Presequence Translocase in a Stoichiometric Manner. The Journal of Biological Chemistry, 289(39), 27352-27362. https://doi.org/10.1074/jbc.M114.556498
  • [ Abstract ]
  • [ DOI ]
  The majority of mitochondrial proteins are synthesized with amino-terminal signal sequences. The presequence translocase of the inner membrane (TIM23 complex) mediates the import of these preproteins. The essential TIM23 core complex closely cooperates with partner protein complexes like the presequence translocase-associated import motor and the respiratory chain. The inner mitochondrial membrane also contains a large number of metabolite carriers, but their association with preprotein translocases has been controversial. We performed a comprehensive analysis of the TIM23 interactome based on stable isotope labeling with amino acids in cell culture. Subsequent biochemical studies on identified partner proteins showed that the mitochondrial ADP/ATP carrier associates with the membrane-embedded core of the TIM23 complex in a stoichiometric manner, revealing an unexpected connection of mitochondrial protein biogenesis to metabolite transport. Our data indicate that direct TIM23-AAC coupling may support preprotein import into mitochondria when respiratory activity is low.
• 
  Wiese, H., Kuhlmann, K., Wiese, S., Stoepel, N. S., Pawlas, M., Meyer, H. E., Stephan, C., Eisenacher, M., Drepper, F., & Warscheid, B. (2014). Comparison of Alternative MS/MS and Bioinformatics Approaches for Confident Phosphorylation Site Localization. Journal of Proteome Research, 13(2), 1128-1137. https://doi.org/10.1021/pr400402s
  • [ Abstract ]
  • [ DOI ]
  Over the past years, phosphoproteomics has advanced to a prime tool in signaling research. Since then, an enormous amount of information about in vivo protein phosphorylation events has been collected providing a treasure trove for gaining a better understanding of the molecular processes involved in cell signaling. Yet, we still face the problem of how to achieve correct modification site localization. Here we use alternative fragmentation and different bioinformatics approaches for the identification and confident localization of phosphorylation sites. Phosphopeptide-enriched fractions were analyzed by multistage activation, collision-induced dissociation and electron transfer dissociation (ETD), yielding complementary phosphopeptide identifications. We further found that MASCOT, OMSSA and Andromeda each identified a distinct set of phosphopeptides allowing the number of site assignments to be increased. The postsearch engine SLoMo provided confident phosphorylation site localization, whereas different versions of PTM-Score integrated in MaxQuant differed in performance. Based on high-resolution ETD and higher collisional dissociation (HCD) data sets from a large synthetic peptide and phosphopeptide reference library reported by Marx et al. [Nat. Biotechnol. 2013, 31 (6), 557-564], we show that an Andromeda/PTM-Score probability of 1 is required to provide an false localization rate (FLR) of 1% for HCD data, while 0.55 is sufficient for high-resolution ETD spectra. Additional analyses of HCD data demonstrated that for phosphotyrosine peptides and phosphopeptides containing two potential phosphorylation sites, PTM-Score probability cutoff values of {\($<$\)1 can be applied to ensure an FLR of 1%. Proper adjustment of localization probability cutoffs allowed us to significantly increase the number of confident sites with an FLR of {\($<$\)1%.Our findings underscore the need for the systematic assessment of FLRs for different score values to report confident modification site localization.
• 
  Schnarwiler, F., Niemann, M., Doiron, N., Harsman, A., Käser, S., Mani, J., Chanfon, A., Dewar, C. E., Oeljeklaus, S., Jackson, C. B., Pusnik, M., Schmidt, O., Meisinger, C., Hiller, S., Warscheid, B., Schnaufer, A. C., Ochsenreiter, T., & Schneider, A. (2014). Trypanosomal TAC40 Constitutes a Novel Subclass of Mitochondrial {\($\beta$\)}-Barrel Proteins Specialized in Mitochondrial Genome Inheritance. Proceedings of the National Academy of Sciences of the United States of America, 111(21), 7624-7629. https://doi.org/10.1073/pnas.1404854111
  • [ Abstract ]
  • [ DOI ]
  Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a {\($\beta$\)}-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum-mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a {\($\beta$\)}-barrel protein of the mitochondrial porin family that mediates a DNA-cytoskeleton linkage that is essential for mitochondrial DNA inheritance.
• 
  Lytovchenko, O., Naumenko, N., Oeljeklaus, S., Schmidt, B., von der Malsburg, K., Deckers, M., Warscheid, B., van der Laan, M., & Rehling, P. (2014). The INA Complex Facilitates Assembly of the Peripheral Stalk of the Mitochondrial F1Fo-ATP Synthase. The EMBO Journal, 33(15), 1624-1638. https://doi.org/10.15252/embj.201488076
  • [ Abstract ]
  • [ DOI ]
  Mitochondrial F1Fo-ATP synthase generates the bulk of cellular ATP. This molecular machine assembles from nuclear- and mitochondria-encoded subunits. Whereas chaperones for formation of the matrix-exposed hexameric F1-ATPase core domain have been identified, insight into how the nuclear-encoded F1-domain assembles with the membrane-embedded Fo-region is lacking. Here we identified the INA complex (INAC) in the inner membrane of mitochondria as an assembly factor involved in this process. Ina22 and Ina17 are INAC constituents that physically associate with the F1-module and peripheral stalk, but not with the assembled F1Fo-ATP synthase. Our analyses show that loss of Ina22 and Ina17 specifically impairs formation of the peripheral stalk that connects the catalytic F1-module to the membrane embedded Fo-domain. We conclude that INAC represents a matrix-exposed inner membrane protein complex that facilitates peripheral stalk assembly and thus promotes a key step in the biogenesis of mitochondrial F1Fo-ATP synthase.
• 
  Kuhlmann, K., Tschapek, A., Wiese, H., Eisenacher, M., Meyer, H. E., Hatt, H. H., Oeljeklaus, S., & Warscheid, B. (2014). The Membrane Proteome of Sensory Cilia to the Depth of Olfactory Receptors. Molecular & Cellular Proteomics : MCP, 13(7), 1828-1843. https://doi.org/10.1074/mcp.M113.035378
  • [ Abstract ]
  • [ DOI ]
  In the nasal cavity, the nonmotile cilium of olfactory sensory neurons (OSNs) constitutes the chemosensory interface between the ambient environment and the brain. The unique sensory organelle facilitates odor detection for which it includes all necessary components of initial and downstream olfactory signal transduction. In addition to its function in olfaction, a more universal role in modulating different signaling pathways is implicated, for example, in neurogenesis, apoptosis, and neural regeneration. To further extend our knowledge about this multifunctional signaling organelle, it is of high importance to establish a most detailed proteome map of the ciliary membrane compartment down to the level of transmembrane receptors. We detached cilia from mouse olfactory epithelia via Ca(2+)/K(+) shock followed by the enrichment of ciliary membrane proteins at alkaline pH, and we identified a total of 4,403 proteins by gel-based and gel-free methods in conjunction with high resolution LC/MS. This study is the first to report the detection of 62 native olfactory receptor proteins and to provide evidence for their heterogeneous expression at the protein level. Quantitative data evaluation revealed four ciliary membrane-associated candidate proteins (the annexins ANXA1, ANXA2, ANXA5, and S100A5) with a suggested function in the regulation of olfactory signal transduction, and their presence in ciliary structures was confirmed by immunohistochemistry. Moreover, we corroborated the ciliary localization of the potassium-dependent Na(+)/Ca(2+) exchanger (NCKX) 4 and the plasma membrane Ca(2+)-ATPase 1 (PMCA1) involved in olfactory signal termination, and we detected for the first time NCKX2 in olfactory cilia. Through comparison with transcriptome data specific for mature, ciliated OSNs, we finally delineated the membrane ciliome of OSNs. The membrane proteome of olfactory cilia established here is the most complete today, thus allowing us to pave new avenues for the study of diverse molecular functions and signaling pathways in and out of olfactory cilia and thus to advance our understanding of the biology of sensory organelles in general.

• 
  Böttinger, L., Guiard, B., Oeljeklaus, S., Kulawiak, B., Zufall, N., Wiedemann, N., Warscheid, B., van der Laan, M., & Becker, T. (2013). A Complex of Cox4 and Mitochondrial Hsp70 Plays an Important Role in the Assembly of the Cytochrome c Oxidase. Molecular Biology of the Cell, 24(17), 2609-2619. https://doi.org/10.1091/mbc.E13-02-0106
  • [ Abstract ]
  • [ DOI ]
  The formation of the mature cytochrome c oxidase (complex IV) involves the association of nuclear- and mitochondria-encoded subunits. The assembly of nuclear-encoded subunits like cytochrome c oxidase subunit 4 (Cox4) into the mature complex is poorly understood. Cox4 is crucial for the stability of complex IV. To find specific biogenesis factors, we analyze interaction partners of Cox4 by affinity purification and mass spectroscopy. Surprisingly, we identify a complex of Cox4, the mitochondrial Hsp70 (mtHsp70), and its nucleotide-exchange factor mitochondrial GrpE (Mge1). We generate a yeast mutant of mtHsp70 specifically impaired in the formation of this novel mtHsp70-Mge1-Cox4 complex. Strikingly, the assembly of Cox4 is strongly decreased in these mutant mitochondria. Because Cox4 is a key factor for the biogenesis of complex IV, we conclude that the mtHsp70-Mge1-Cox4 complex plays an important role in the formation of cytochrome c oxidase. Cox4 arrests at this chaperone complex in the absence of mature complex IV. Thus the mtHsp70-Cox4 complex likely serves as a novel delivery system to channel Cox4 into the assembly line when needed.
• 
  Gronemeyer, T., Wiese, S., Ofman, R., Bunse, C., Pawlas, M., Hayen, H., Eisenacher, M., Stephan, C., Meyer, H. E., Waterham, H. R., Erdmann, R., Wanders, R. J., & Warscheid, B. (2013). The Proteome of Human Liver Peroxisomes: Identification of Five New Peroxisomal Constituents by a Label-Free Quantitative Proteomics Survey. PloS One, 8(2), e57395. https://doi.org/10.1371/journal.pone.0057395
  • [ Abstract ]
  • [ DOI ]
  The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or {\($\beta$\)}-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD(+) becomes regenerated during fatty acid {\($\beta$\)}-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.
• 
  Jank, T., Bogdanovi’c}}, X., Wirth, C., Haaf, E., Spoerner, M., Böhmer, K. E., Steinemann, M., Orth, J. H. C., Kalbitzer, H. R., Warscheid, B., Hunte, C., & Aktories, K. (2013). A Bacterial Toxin Catalyzing Tyrosine Glycosylation of Rho and Deamidation of Gq and Gi Proteins. Nature Structural & Molecular Biology, 20(11), 1273-1280. https://doi.org/10.1038/nsmb.2688
  • [ Abstract ]
  • [ DOI ]
  Entomopathogenic Photorhabdus asymbiotica is an emerging pathogen in humans. Here, we identified a P. asymbiotica protein toxin (PaTox), which contains a glycosyltransferase and a deamidase domain. PaTox mono-O-glycosylates Y32 (or Y34) of eukaryotic Rho GTPases by using UDP-N-acetylglucosamine (UDP-GlcNAc). Tyrosine glycosylation inhibits Rho activation and prevents interaction with downstream effectors, resulting in actin disassembly, inhibition of phagocytosis and toxicity toward insects and mammalian cells. The crystal structure of the PaTox glycosyltransferase domain in complex with UDP-GlcNAc determined at 1.8-AA} resolution represents a canonical GT-A fold and is the smallest glycosyltransferase toxin known. (1)H-NMR analysis identifies PaTox as a retaining glycosyltransferase. The glutamine-deamidase domain of PaTox blocks GTP hydrolysis of heterotrimeric G{\($\alpha$\)}q/11 and G{\($\alpha$\)}i proteins, thereby activating RhoA. Thus, PaTox hijacks host GTPase signaling in a bidirectional manner by deamidation-induced activation and glycosylation-induced inactivation of GTPases.
• 
  Ieva, R., Heißwolf, A. K., Gebert, M., Vögtle, F.-N., Wollweber, F., Mehnert, C. S., Oeljeklaus, S., Warscheid, B., Meisinger, C., van der Laan, M., & Pfanner, N. (2013). Mitochondrial Inner Membrane Protease Promotes Assembly of Presequence Translocase by Removing a Carboxy-Terminal Targeting Sequence. Nature Communications, 4, 2853. https://doi.org/10.1038/ncomms3853
  • [ Abstract ]
  • [ DOI ]
  The presequence translocase of the inner mitochondrial membrane (TIM23 complex) is essential for importing cleavable preproteins into mitochondria. The preproteins contain amino-terminal targeting sequences that are removed by the mitochondrial processing peptidase (MPP). Some preproteins carry bipartite presequences that are cleaved twice, by MPP and the inner membrane protease (IMP). Here, we report that the TIM23 complex is altered in mitochondria lacking the IMP subunit Imp1 although none of the TIM23 components contains a bipartite presequence. We show that the TIM23 subunit Mgr2 is processed by IMP, but not by MPP. The cytosolic precursor of Mgr2 contains a carboxy-terminal sequence that promotes targeting to mitochondria, but impairs stable assembly and function of the mature TIM23 complex. IMP removes the carboxy-terminal targeting sequence and thus promotes proper assembly of the TIM23 complex. Our results reveal carboxy-terminal processing as a new mechanism in the biogenesis of the mitochondrial inner membrane.
• 
  David, C., Koch, J., Oeljeklaus, S., Laernsack, A., Melchior, S., Wiese, S., Schummer, A., Erdmann, R., Warscheid, B., & Brocard, C. (2013). A Combined Approach of Quantitative Interaction Proteomics and Live-Cell Imaging Reveals a Regulatory Role for Endoplasmic Reticulum ({{ER}}) Reticulon Homology Proteins in Peroxisome Biogenesis. Molecular & Cellular Proteomics : MCP, 12(9), 2408-2425. https://doi.org/10.1074/mcp.M112.017830
  • [ Abstract ]
  • [ DOI ]
  Peroxisome biogenesis initiates at the endoplasmic reticulum (ER) and maturation allows for the formation of metabolically active organelles. Yet, peroxisomes can also multiply by growth and division. Several proteins, called peroxins, are known to participate in these processes but little is known about their organization to orchestrate peroxisome proliferation. Here, we demonstrate that regulation of peroxisome proliferation relies on the integrity of the tubular ER network. Using a dual track SILAC-based quantitative interaction proteomics approach, we established a comprehensive network of stable as well as transient interactions of the peroxin Pex30p, an integral membrane protein. Through association with merely ER resident proteins, in particular with proteins containing a reticulon homology domain, and with other peroxins, Pex30p designates peroxisome contact sites at ER subdomains. We show that Pex30p traffics through the ER and segregates in punctae to which peroxisomes specifically append, and we ascertain its transient interaction with all subunits of the COPI coatomer complex suggesting the involvement of a vesicle-mediated transport. We establish that the membrane protein Pex30p facilitates the connection of peroxisomes to the ER. Taken together, our data indicate that Pex30p-containing protein complexes act as focal points from which peroxisomes can form and that the tubular ER architecture organized by the reticulon homology proteins Rtn1p, Rtn2p and Yop1p controls this process.
• 
  Gronemeyer, T., Wiese, S., Grinhagens, S., Schollenberger, L., Satyagraha, A., Huber, L. A., Meyer, H. E., Warscheid, B., & Just, W. W. (2013). Localization of Rab Proteins to Peroxisomes: A Proteomics and Immunofluorescence Study. FEBS Letters, 587(4), 328-338. https://doi.org/10.1016/j.febslet.2012.12.025
  • [ Abstract ]
  • [ DOI ]
  A proteomics screen was initiated to identify Rab proteins regulating transport to and away from peroxisomes. Mass spectrometry-based protein correlation profiling of rat liver organelles and immunofluorescence analysis of the peroxisome candidate Rab proteins revealed Rab6, Rab10, Rab14 and Rab18 to associate with the peroxisomal membrane. While Rab14 localized to peroxisomes predominantly in its dominant-active form, other Rab proteins associated with peroxisomes in both their GTP- and GDP-bound state. In summary, our data suggest that Rab6, Rab10, Rab14 and Rab18 associate with the peroxisomal compartment and similar as previously shown for Rab8, Rab18 in its GDP-bound state favors peroxisome proliferation.
• 
  Qiu, J., Wenz, L.-S., Zerbes, R. M., Oeljeklaus, S., Bohnert, M., Stroud, D. A., Wirth, C., Ellenrieder, L., Thornton, N., Kutik, S., Wiese, S., Schulze-Specking, A., Zufall, N., Chacinska, A., Guiard, B., Hunte, C., Warscheid, B., van der Laan, M., Pfanner, N., … Becker, T. (2013). Coupling of Mitochondrial Import and Export Translocases by Receptor-Mediated Supercomplex Formation. Cell, 154(3), 596-608. https://doi.org/10.1016/j.cell.2013.06.033
  • [ Abstract ]
  • [ DOI ]
  The mitochondrial outer membrane harbors two protein translocases that are essential for cell viability: the translocase of the outer mitochondrial membrane (TOM) and the sorting and assembly machinery (SAM). The precursors of {\($\beta$\)}-barrel proteins use both translocases-TOM for import to the intermembrane space and SAM for export into the outer membrane. It is unknown if the translocases cooperate and where the {\($\beta$\)}-barrel of newly imported proteins is formed. We established a position-specific assay for monitoring {\($\beta$\)}-barrel formation in vivo and in organello and demonstrated that the {\($\beta$\)}-barrel was formed and membrane inserted while the precursor was bound to SAM. {\($\beta$\)}-barrel formation was inhibited by SAM mutants and, unexpectedly, by mutants of the central import receptor, Tom22. We show that the cytosolic domain of Tom22 links TOM and SAM into a supercomplex, facilitating precursor transfer on the intermembrane space side. Our study reveals receptor-mediated coupling of import and export translocases as a means of precursor channeling.
• 
  Lindemann, C., Lupilova, N., Müller, A., Warscheid, B., Meyer, H. E., Kuhlmann, K., Eisenacher, M., & Leichert, L. I. (2013). Redox Proteomics Uncovers Peroxynitrite-Sensitive Proteins That Help Escherichia Coli to Overcome Nitrosative Stress. The Journal of Biological Chemistry, 288(27), 19698-19714. https://doi.org/10.1074/jbc.M113.457556
  • [ Abstract ]
  • [ DOI ]
  Peroxynitrite is a highly reactive chemical species with antibacterial properties that are synthesized in immune cells. In a proteomic approach, we identified specific target proteins of peroxynitrite-induced modifications in Escherichia coli. Although peroxynitrite caused a fairly indiscriminate nitration of tyrosine residues, reversible modifications of protein thiols were highly specific. We used a quantitative redox proteomic method based on isotope-coded affinity tag chemistry and identified four proteins consistently thiol-modified in cells treated with peroxynitrite as follows: AsnB, FrmA, MaeB, and RidA. All four were required for peroxynitrite stress tolerance in vivo. Three of the identified proteins were modified at highly conserved cysteines, and MaeB and FrmA are known to be directly involved in the oxidative and nitrosative stress response in E. coli. In in vitro studies, we could show that the activity of RidA, a recently discovered enamine/imine deaminase, is regulated in a specific manner by the modification of its single conserved cysteine. Mutation of this cysteine 107 to serine generated a constitutively active protein that was not susceptible to peroxynitrite.
• 
  Cristodero, M., Mani, J., Oeljeklaus, S., Aeberhard, L., Hashimi, H., Ramrath, D. J. F., Lukev s, J., Warscheid, B., & Schneider, A. (2013). Mitochondrial Translation Factors of Trypanosoma Brucei: Elongation Factor-Tu Has a Unique Subdomain That Is Essential for Its Function. Molecular Microbiology, 90(4), 744-755. https://doi.org/10.1111/mmi.12397
  • [ Abstract ]
  • [ DOI ]
  Mitochondrial translation in the parasitic protozoan Trypanosoma brucei relies on imported eukaryotic-type tRNAs as well as on bacterial-type ribosomes that have the shortest known rRNAs. Here we have identified the mitochondrial translation elongation factors EF-Tu, EF-Ts, EF-G1 and release factor RF1 of trypanosomatids and show that their ablation impairs growth and oxidative phosphorylation. In vivo labelling experiments and a SILAC-based analysis of the global proteomic changes induced by EF-Tu RNAi directly link EF-Tu to mitochondrial translation. Moreover, EF-Tu RNAi reveals downregulation of many nuclear encoded subunits of cytochrome oxidase as well as of components of the bc1-complex, whereas most cytosolic ribosomal proteins were upregulated. Interestingly, T.,brucei>EF-Tu has a 30-amino-acid-long, highly charged subdomain, which is unique to trypanosomatids. A combination of RNAi and complementation experiments shows that this subdomain is essential for EF-Tu function, but that it can be replaced by a similar sequence found in eukaryotic EF-1a, the cytosolic counterpart of EF-Tu. A recent cryo-electron microscopy study revealed that trypanosomatid mitochondrial ribosomes have a unique intersubunit space that likely harbours the EF-Tu binding site. These findings suggest that the trypanosomatid-specific EF-Tu subdomain serves as an adaption for binding to these unusual mitochondrial ribosomes.
• 
  Sachelaru, I., Petriman, N. A., Kudva, R., Kuhn, P., Welte, T., Knapp, B., Drepper, F., Warscheid, B., & Koch, H.-G. (2013). YidC Occupies the Lateral Gate of the SecYEG Translocon and Is Sequentially Displaced by a Nascent Membrane Protein. The Journal of Biological Chemistry, 288(23), 16295-16307. https://doi.org/10.1074/jbc.M112.446583
  • [ Abstract ]
  • [ DOI ]
  Most membrane proteins are co-translationally inserted into the lipid bilayer via the universally conserved SecY complex and they access the lipid phase presumably via a lateral gate in SecY. In bacteria, the lipid transfer of membrane proteins from the SecY channel is assisted by the SecY-associated protein YidC, but details on the SecY-YidC interaction are unknown. By employing an in vivo and in vitro site-directed cross-linking approach, we have mapped the SecY-YidC interface and found YidC in contact with all four transmembrane domains of the lateral gate. This interaction did not require the SecDFYajC complex and was not influenced by SecA binding to SecY. In contrast, ribosomes dissociated the YidC contacts to lateral gate helices 2b and 8. The major contact between YidC and the lateral gate was lost in the presence of ribosome nascent chains and new SecY-YidC contacts appeared. These data demonstrate that the SecY-YidC interaction is influenced by nascent-membrane-induced lateral gate movements.
• 
  Niemann, M., Wiese, S., Mani, J., Chanfon, A., Jackson, C., Meisinger, C., Warscheid, B., & Schneider, A. (2013). Mitochondrial Outer Membrane Proteome of Trypanosoma Brucei Reveals Novel Factors Required to Maintain Mitochondrial Morphology. Molecular & Cellular Proteomics : MCP, 12(2), 515-528. https://doi.org/10.1074/mcp.M112.023093
  • [ Abstract ]
  • [ DOI ]
  Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, two-thirds of which have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins, 33 of which are specific to trypanosomatids, remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and for the first time identified factors that control mitochondrial shape in T. brucei.

• 
  Piechura, H., Oeljeklaus, S., & Warscheid, B. (2012). SILAC for the Study of Mammalian Cell Lines and Yeast Protein Complexes. Methods in Molecular Biology (Clifton, N.J.), 893, 201-221. https://doi.org/10.1007/978-1-61779-885-6_14
  • [ Abstract ]
  • [ DOI ]
  Through crucial advancements in quantitative mass spectrometry (MS), proteomics has evolved from taking mere "snapshots" of proteomes to thoroughly studying dynamic changes in entire proteomes and characterizing intricate protein-protein interaction or signaling networks. Thus, quantitative MS-based proteomics offers the unique potential to place proteins into their functional context and, moreover, to improve our understanding of the molecular processes involved in the development, survival, or pathology of cells and organisms. Among the vast variety of techniques developed for the accurate quantification of proteins via MS, stable isotope labeling by amino acids in cell culture (SILAC) arguably represents the most elegant method. In this chapter, we provide a detailed protocol for the establishment of SILAC for mammalian cell culture systems. In addition, to exemplify the high versatility of SILAC for addressing different biological questions, we describe the successful "pairing" of SILAC with conventional affinity purification (AP)-MS approaches allowing for accurately characterizing protein complexes.
• 
  Gebert, M., Schrempp, S. G., Mehnert, C. S., Heißwolf, A. K., Oeljeklaus, S., Ieva, R., Bohnert, M., von der Malsburg, K., Wiese, S., Kleinschroth, T., Hunte, C., Meyer, H. E., Haferkamp, I., Guiard, B., Warscheid, B., Pfanner, N., & van der Laan, M. (2012). Mgr2 Promotes Coupling of the Mitochondrial Presequence Translocase to Partner Complexes. The Journal of Cell Biology, 197(5), 595-604. https://doi.org/10.1083/jcb.201110047
  • [ Abstract ]
  • [ DOI ]
  Many mitochondrial proteins are synthesized with N-terminal presequences in the cytosol. The presequence translocase of the inner mitochondrial membrane (TIM23) translocates preproteins into and across the membrane and associates with the matrix-localized import motor. The TIM23 complex consists of three core components and Tim21, which interacts with the translocase of the outer membrane (TOM) and the respiratory chain. We have identified a new subunit of the TIM23 complex, the inner membrane protein Mgr2. Mitochondria lacking Mgr2 were deficient in the Tim21-containing sorting form of the TIM23 complex. Mgr2 was required for binding of Tim21 to TIM23(CORE), revealing a binding chain of TIM23(CORE)-Mgr2/Tim21-respiratory chain. Mgr2-deficient yeast cells were defective in growth at elevated temperature, and the mitochondria were impaired in TOM-TIM23 coupling and the import of presequence-carrying preproteins. We conclude that Mgr2 is a coupling factor of the presequence translocase crucial for cell growth at elevated temperature and for efficient protein import.
• 
  Welte, T., Kudva, R., Kuhn, P., Sturm, L., Braig, D., Müller, M., Warscheid, B., Drepper, F., & Koch, H.-G. (2012). Promiscuous Targeting of Polytopic Membrane Proteins to SecYEG or YidC by the Escherichia Coli Signal Recognition Particle. Molecular Biology of the Cell, 23(3), 464-479. https://doi.org/10.1091/mbc.E11-07-0590
  • [ Abstract ]
  • [ DOI ]
  Protein insertion into the bacterial inner membrane is facilitated by SecYEG or YidC. Although SecYEG most likely constitutes the major integration site, small membrane proteins have been shown to integrate via YidC. We show that YidC can also integrate multispanning membrane proteins such as mannitol permease or TatC, which had been considered to be exclusively integrated by SecYEG. Only SecA-dependent multispanning membrane proteins strictly require SecYEG for integration, which suggests that SecA can only interact with the SecYEG translocon, but not with the YidC insertase. Targeting of multispanning membrane proteins to YidC is mediated by signal recognition particle (SRP), and we show by site-directed cross-linking that the C-terminus of YidC is in contact with SRP, the SRP receptor, and ribosomal proteins. These findings indicate that SRP recognizes membrane proteins independent of the downstream integration site and that many membrane proteins can probably use either SecYEG or YidC for integration. Because protein synthesis is much slower than protein transport, the use of YidC as an additional integration site for multispanning membrane proteins may prevent a situation in which the majority of SecYEG complexes are occupied by translating ribosomes during cotranslational insertion, impeding the translocation of secretory proteins.
• 
  Eisenacher, M., Kohl, M., Wiese, S., Hebeler, R., Meyer, H. E., Warscheid, B., & Stephan, C. (2012). Find Pairs: The Module for Protein Quantification of the PeakQuant Software Suite. Omics : A Journal of Integrative Biology, 16(9), 457-467. https://doi.org/10.1089/omi.2011.0140
  • [ Abstract ]
  • [ DOI ]
  Accurate quantification of proteins is one of the major tasks in current proteomics research. To address this issue, a wide range of stable isotope labeling techniques have been developed, allowing one to quantitatively study thousands of proteins by means of mass spectrometry. In this article, the FindPairs module of the PeakQuant software suite is detailed. It facilitates the automatic determination of protein abundance ratios based on the automated analysis of stable isotope-coded mass spectrometric data. Furthermore, it implements statistical methods to determine outliers due to biological as well as technical variance of proteome data obtained in replicate experiments. This provides an important means to evaluate the significance in obtained protein expression data. For demonstrating the high applicability of FindPairs, we focused on the quantitative analysis of proteome data acquired in (14)N/(15)N labeling experiments. We further provide a comprehensive overview of the features of the FindPairs software, and compare these with existing quantification packages. The software presented here supports a wide range of proteomics applications, allowing one to quantitatively assess data derived from different stable isotope labeling approaches, such as (14)N/(15)N labeling, SILAC, and iTRAQ. The software is publicly available at http://www.medizinisches-proteom-center.de/software and free for academic use.
• 
  Bohnert, M., Wenz, L.-S., Zerbes, R. M., Horvath, S. E., Stroud, D. A., von der Malsburg, K., Müller, J. M., Oeljeklaus, S., Perschil, I., Warscheid, B., Chacinska, A., Veenhuis, M., van der Klei, I. J., Daum, G., Wiedemann, N., Becker, T., Pfanner, N., & van der Laan, M. (2012). Role of Mitochondrial Inner Membrane Organizing System in Protein Biogenesis of the Mitochondrial Outer Membrane. Molecular Biology of the Cell, 23(20), 3948-3956. https://doi.org/10.1091/mbc.E12-04-0295
  • [ Abstract ]
  • [ DOI ]
  Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport-associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of {\($\beta$\)}-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import {\($\beta$\)}-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of {\($\beta$\)}-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane {\($\beta$\)}-barrel proteins.
• 
  Wenk, M., Ba, Q., Erichsen, V., MacInnes, K., Wiese, H., Warscheid, B., & Koch, H.-G. (2012). A Universally Conserved ATPase Regulates the Oxidative Stress Response in Escherichia Coli. The Journal of Biological Chemistry, 287(52), 43585-43598. https://doi.org/10.1074/jbc.M112.413070
  • [ Abstract ]
  • [ DOI ]
  YchF is an evolutionarily conserved ATPase of unknown function. In humans, the YchF homologue hOla1 appears to influence cell proliferation and was found to be up-regulated in many tumors. A possible involvement in regulating the oxidative stress response was also suggested, but details on the underlying mechanism are lacking. For gaining insight into YchF function, we used Escherichia coli as a model organism and found that YchF overexpression resulted in H(2)O(2) hypersensitivity. This was not caused by transcriptional or translational down-regulation of H(2)O(2)-scavenging enzymes. Instead, we observed YchF-dependent inhibition of catalase activity and a direct interaction with the major E. coli catalase KatG. KatG inhibition was dependent on the ATPase activity of YchF and was regulated by post-translational modifications, most likely including an H(2)O(2)-dependent dephosphorylation. We furthermore showed that YchF expression is repressed by the transcription factor OxyR and further post-translationally modified in response to H(2)O(2). In summary, our data show that YchF functions as a novel negative regulator of the oxidative stress response in E. coli. Considering the available data on hOla1, YchF/Ola1 most likely execute similar functions in bacteria and humans, and their up-regulation inhibits the ability of the cells to scavenge damaging reactive oxygen species.
• 
  Zerbes, R. M., Bohnert, M., Stroud, D. A., von der Malsburg, K., Kram, A., Oeljeklaus, S., Warscheid, B., Becker, T., Wiedemann, N., Veenhuis, M., van der Klei, I. J., Pfanner, N., & van der Laan, M. (2012). Role of MINOS in Mitochondrial Membrane Architecture: Cristae Morphology and Outer Membrane Interactions Differentially Depend on Mitofilin Domains. Journal of Molecular Biology, 422(2), 183-191. https://doi.org/10.1016/j.jmb.2012.05.004
  • [ Abstract ]
  • [ DOI ]
  The mitochondrial inner membrane contains a large protein complex crucial for membrane architecture, the mitochondrial inner membrane organizing system (MINOS). MINOS is required for keeping cristae membranes attached to the inner boundary membrane via crista junctions and interacts with protein complexes of the mitochondrial outer membrane. To study if outer membrane interactions and maintenance of cristae morphology are directly coupled, we generated mutant forms of mitofilin/Fcj1 (formation of crista junction protein 1), a core component of MINOS. Mitofilin consists of a transmembrane anchor in the inner membrane and intermembrane space domains, including a coiled-coil domain and a conserved C-terminal domain. Deletion of the C-terminal domain disrupted the MINOS complex and led to release of cristae membranes from the inner boundary membrane, whereas the interaction of mitofilin with the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM) were enhanced. Deletion of the coiled-coil domain also disturbed the MINOS complex and cristae morphology; however, the interactions of mitofilin with TOM and SAM were differentially affected. Finally, deletion of both intermembrane space domains disturbed MINOS integrity as well as interactions with TOM and SAM. Thus, the intermembrane space domains of mitofilin play distinct roles in interactions with outer membrane complexes and maintenance of MINOS and cristae morphology, demonstrating that MINOS contacts to TOM and SAM are not sufficient for the maintenance of inner membrane architecture.
• 
  Pusnik, M., Mani, J., Schmidt, O., Niemann, M., Oeljeklaus, S., Schnarwiler, F., Warscheid, B., Lithgow, T., Meisinger, C., & Schneider, A. (2012). An Essential Novel Component of the Noncanonical Mitochondrial Outer Membrane Protein Import System of Trypanosomatids. Molecular Biology of the Cell, 23(17), 3420-3428. https://doi.org/10.1091/mbc.E12-02-0107
  • [ Abstract ]
  • [ DOI ]
  The mitochondrial outer membrane protein Tom40 is the general entry gate for imported proteins in essentially all eukaryotes. Trypanosomatids lack Tom40, however, and use instead a protein termed the archaic translocase of the outer mitochondrial membrane (ATOM). Here we report the discovery of pATOM36, a novel essential component of the trypanosomal outer membrane protein import system that interacts with ATOM. pATOM36 is not related to known Tom proteins from other organisms and mediates the import of matrix proteins. However, there is a group of precursor proteins whose import is independent of pATOM36. Domain-swapping experiments indicate that the N-terminal presequence-containing domain of the substrate proteins at least in part determines the dependence on pATOM36. Secondary structure profiling suggests that pATOM36 is composed largely of {\($\alpha$\)}-helices and its assembly into the outer membrane is independent of the sorting and assembly machinery complex. Taken together, these results show that pATOM36 is a novel component associated with the ATOM complex that promotes the import of a subpopulation of proteins into the mitochondrial matrix.
• 
  Mick, D. U., Dennerlein, S., Wiese, H., Reinhold, R., Pacheu-Grau, D., Lorenzi, I., Sasarman, F., Weraarpachai, W., Shoubridge, E. A., Warscheid, B., & Rehling, P. (2012). MITRAC Links Mitochondrial Protein Translocation to Respiratory-Chain Assembly and Translational Regulation. Cell, 151(7), 1528-1541. https://doi.org/10.1016/j.cell.2012.11.053
  • [ Abstract ]
  • [ DOI ]
  Mitochondrial respiratory-chain complexes assemble from subunits of dual genetic origin assisted by specialized assembly factors. Whereas core subunits are translated on mitochondrial ribosomes, others are imported after cytosolic translation. How imported subunits are ushered to assembly intermediates containing mitochondria-encoded subunits is unresolved. Here, we report a comprehensive dissection of early cytochrome c oxidase assembly intermediates containing proteins required for normal mitochondrial translation and reveal assembly factors promoting biogenesis of human respiratory-chain complexes. We find that TIM21, a subunit of the inner-membrane presequence translocase, is also present in the major assembly intermediates containing newly mitochondria-synthesized and imported respiratory-chain subunits, which we term MITRAC complexes. Human TIM21 is dispensable for protein import but required for integration of early-assembling, presequence-containing subunits into respiratory-chain intermediates. We establish an unexpected molecular link between the TIM23 transport machinery and assembly of respiratory-chain complexes that regulate mitochondrial protein synthesis in response to their assembly state.
• 
  Puetz, S., Schroeter, M. M., Piechura, H., Reimann, L., Hunger, M. S., Lubomirov, L. T., Metzler, D., Warscheid, B., & Pfitzer, G. (2012). New Insights into Myosin Phosphorylation during Cyclic Nucleotide-Mediated Smooth Muscle Relaxation. Journal of Muscle Research and Cell Motility, 33(6), 471-483. https://doi.org/10.1007/s10974-012-9306-9
  • [ Abstract ]
  • [ DOI ]
  Nitrovasodilators and agonists, via an increase in intracellular cyclic nucleotide levels, can induce smooth muscle relaxation without a concomitant decrease in phosphorylation of the regulatory light chains (RLC) of myosin. However, since cyclic nucleotide-induced relaxation is associated with a decrease in intracellular [Ca(2+)], and hence, a decreased activity of MLCK, we tested the hypothesis that the site responsible for the elevated RLC phosphorylation is not Ser19. Smooth muscle strips from gastric fundus were isometrically contracted with ET-1 which induced an increase in monophosphorylation from 9~textpm}~1~% under resting conditions (PSS) to 36~textpm}~1~% determined with 2D-PAGE. Electric field stimulation induced a rapid, largely NO-mediated relaxation with a half time of 8~s, which was associated with an initial decline in RLC phosphorylation to 18~% within 2~s and a rebound to 34~% after 30~s whereas relaxation was sustained. In contrast, phosphorylation of RLC at Ser19 probed with phosphospecific antibodies declined in parallel with force. LC/MS and western blot analysis with phosphospecific antibodies against monophosphorylated Thr18 indicate that Thr18 is significantly monophosphorylated during sustained relaxation. We therefore suggest that (i) monophosphorylation of Thr18 rather than Ser19 is responsible for the phosphorylation rebound during sustained EFS-induced relaxation of mouse gastric fundus, and (ii) that relaxation can be ascribed to dephosphorylation of Ser19, the site considered to be responsible for regulation of smooth muscle tone.
• 
  Oeljeklaus, S., Reinartz, B. S., Wolf, J., Wiese, S., Tonillo, J., Podwojski, K., Kuhlmann, K., Stephan, C., Meyer, H. E., Schliebs, W., Brocard, C., Erdmann, R., & Warscheid, B. (2012). Identification of Core Components and Transient Interactors of the Peroxisomal Importomer by Dual-Track Stable Isotope Labeling with Amino Acids in Cell Culture Analysis. Journal of Proteome Research, 11(4), 2567-2580. https://doi.org/10.1021/pr3000333
  • [ Abstract ]
  • [ DOI ]
  The importomer complex plays an essential role in the biogenesis of peroxisomes by mediating the translocation of matrix proteins across the organellar membrane. A central part of this highly dynamic import machinery is the docking complex consisting of Pex14p, Pex13p, and Pex17p that is linked to the RING finger complex (Pex2p, Pex10p, Pex12p) via Pex8p. To gain detailed knowledge on the molecular players governing peroxisomal matrix protein import and, thus, the integrity and functionality of peroxisomes, we aimed at a most comprehensive investigation of stable and transient interaction partners of Pex14p, the central component of the importomer. To this end, we performed a thorough quantitative proteomics study based on epitope tagging of Pex14p combined with dual-track stable isotope labeling with amino acids in cell culture-mass spectrometry (SILAC-MS) analysis of affinity-purified Pex14p complexes and statistics. The results led to the establishment of the so far most extensive Pex14p interactome, comprising 9 core and further 12 transient components. We confirmed virtually all known Pex14p interaction partners including the core constituents of the importomer as well as Pex5p, Pex11p, Pex15p, and Dyn2p. More importantly, we identified new transient interaction partners (Pex25p, Hrr25p, Esl2p, prohibitin) that provide a valuable resource for future investigations on the functionality, dynamics, and regulation of the peroxisomal importomer.
• 
  Vukotic, M., Oeljeklaus, S., Wiese, S., Vögtle, F. N., Meisinger, C., Meyer, H. E., Zieseniss, A., Katschinski, D. M., Jans, D. C., Jakobs, S., Warscheid, B., Rehling, P., & Deckers, M. (2012). Rcf1 Mediates Cytochrome Oxidase Assembly and Respirasome Formation, Revealing Heterogeneity of the Enzyme Complex. Cell Metabolism, 15(3), 336-347. https://doi.org/10.1016/j.cmet.2012.01.016
  • [ Abstract ]
  • [ DOI ]
  The terminal enzyme of the mitochondrial respiratory chain, cytochrome oxidase, transfers electrons to molecular oxygen, generating water. Within the inner~mitochondrial membrane, cytochrome oxidase assembles into supercomplexes, together with other respiratory chain complexes, forming so-called respirasomes. Little is known about how these higher oligomeric structures are attained. Here we report on Rcf1 and Rcf2 as cytochrome oxidase subunits in S.~cerevisiae. While Rcf2 is specific to yeast, Rcf1 is a conserved subunit with two human orthologs, RCF1a and RCF1b. Rcf1 is required for growth in hypoxia and complex assembly of subunits Cox13 and Rcf2, as well as for the oligomerization of~a subclass of cytochrome oxidase complexes into respirasomes. Our analyses reveal that the cytochrome oxidase of mitochondria displays intrinsic heterogeneity with regard to its subunit composition and that distinct forms of respirasomes can be formed by complex variants.

• 
  Stroud, D. A., Oeljeklaus, S., Wiese, S., Bohnert, M., Lewandrowski, U., Sickmann, A., Guiard, B., van der Laan, M., Warscheid, B., & Wiedemann, N. (2011). Composition and Topology of the Endoplasmic Reticulum-Mitochondria Encounter Structure. Journal of Molecular Biology, 413(4), 743-750. https://doi.org/10.1016/j.jmb.2011.09.012
  • [ Abstract ]
  • [ DOI ]
  Eukaryotic cells contain multiple organelles, which are functionally and structurally interconnected. The endoplasmic reticulum-mitochondria encounter structure (ERMES) forms a junction between mitochondria and the endoplasmic reticulum (ER). Four ERMES proteins are known in yeast, the ER-anchored protein Mmm1 and three mitochondria-associated proteins, Mdm10, Mdm12 and Mdm34, with functions related to mitochondrial morphology and protein biogenesis. We mapped the glycosylation sites of ERMES and demonstrate that three asparagine residues in the N-terminal domain of Mmm1 are glycosylated. While the glycosylation is dispensable, the cytosolic C-terminal domain of Mmm1 that connects to the Mdm proteins is required for Mmm1 function. To analyze the composition of ERMES, we determined the subunits by quantitative mass spectrometry. We identified the calcium-binding GTPase Gem1 as a new ERMES subunit, revealing that ERMES is composed of five genuine subunits. Taken together, ERMES represents a platform that integrates components with functions in formation of ER-mitochondria junctions, maintenance of mitochondrial morphology, protein biogenesis and calcium binding.
• 
  von der Malsburg, K., Müller, J. M., Bohnert, M., Oeljeklaus, S., Kwiatkowska, P., Becker, T., Loniewska-Lwowska, A., Wiese, S., Rao, S., Milenkovic, D., Hutu, D. P., Zerbes, R. M., Schulze-Specking, A., Meyer, H. E., Martinou, J.-C., Rospert, S., Rehling, P., Meisinger, C., Veenhuis, M., … van der Laan, M. (2011). Dual Role of Mitofilin in Mitochondrial Membrane Organization and Protein Biogenesis. Developmental Cell, 21(4), 694-707. https://doi.org/10.1016/j.devcel.2011.08.026
  • [ Abstract ]
  • [ DOI ]
  The mitochondrial inner membrane consists of two domains, inner boundary membrane and cristae membrane that are connected by crista junctions. Mitofilin/Fcj1 was reported to be involved in formation of crista junctions, however, different views exist on its function and possible partner proteins. We report that mitofilin plays a dual role. Mitofilin is part of a large inner membrane complex, and we identify five partner proteins as constituents of the mitochondrial inner membrane organizing system (MINOS) that is required for keeping cristae membranes connected to the inner boundary membrane. Additionally, mitofilin is coupled to the outer membrane and promotes protein import via the mitochondrial intermembrane space assembly pathway. Our findings indicate that mitofilin is a central component~of MINOS and functions as a multifunctional regulator of mitochondrial architecture and protein biogenesis.
• 
  Kaller, M., Liffers, S.-T., Oeljeklaus, S., Kuhlmann, K., Röh, S., Hoffmann, R., Warscheid, B., & Hermeking, H. (2011). Genome-Wide Characterization of miR-34a Induced Changes in Protein and mRNA Expression by a Combined Pulsed SILAC and Microarray Analysis. Molecular & Cellular Proteomics : MCP, 10(8), M111.010462. https://doi.org/10.1074/mcp.M111.010462
  • [ Abstract ]
  • [ DOI ]
  The gene encoding the miR-34a microRNA is a transcriptional target of the p53 tumor suppressor protein and subject to epigenetic inactivation in colorectal cancer and numerous other tumor types. Here, we combined pulsed SILAC (pSILAC) and microarray analyses to identify miR-34a-induced changes in protein and mRNA expression. pSILAC allowed to quantify the de novo protein synthesis of 1206 proteins after activation of a conditional miR-34a allele in a colorectal cancer cell line. {\($\sim$\)}19% of the detected proteins were differentially regulated, with 113 proteins being down- and 115 up-regulated. The proteins with a miR-34a seed-matching-sequence in the 3'-untranslated region (UTR) of the corresponding mRNA showed a clear bias toward translational repression. Proteins involved in DNA replication, e.g. the MCM proteins, and cell proliferation, were over-represented among indirectly down-regulated proteins lacking a miR-34a seed-match. The decrease in de novo protein synthesis of direct miR-34a targets correlated with reduced levels of the corresponding mRNA in most cases, indicating an interdependence of both types of regulation. In addition, 43 mRNAs encoding proteins not detected by pSILAC were down-regulated after miR-34a expression and contained miR-34a seed-matches. The direct regulation of selected miR-34a target-mRNAs was confirmed using reporter assays. Via down-regulation of the proteins encoded by these mRNAs miR-34a presumably inhibits glycolysis (LDHA), WNT-signaling (LEF1), invasion/migration (AXL) and lipid metabolism (ACSL1, ACSL4). Furthermore, miR-34a may activate p53 by inhibiting its acetylation (MTA2, HDAC1) and degradation (YY1). In summary, miR-34a presumably participates in multiple tumor suppressive pathways by directly and indirectly suppressing the expression of numerous, critical proteins.
• 
  Bharti, P., Schliebs, W., Schievelbusch, T., Neuhaus, A., David, C., Kock, K., Herrmann, C., Meyer, H. E., Wiese, S., Warscheid, B., Theiss, C., & Erdmann, R. (2011). PEX14 Is Required for Microtubule-Based Peroxisome Motility in Human Cells. Journal of Cell Science, 124(Pt 10), 1759-1768. https://doi.org/10.1242/jcs.079368
  • [ Abstract ]
  • [ DOI ]
  We have established a procedure for isolating native peroxisomal membrane protein complexes from cultured human cells. Protein-A-tagged peroxin 14 (PEX14), a central component of the peroxisomal protein translocation machinery was genomically expressed in Flp-In-293 cells and purified from digitonin-solubilized membranes. Size-exclusion chromatography revealed the existence of distinct multimeric PEX14 assemblies at the peroxisomal membrane. Using mass spectrometric analysis, almost all known human peroxins involved in protein import were identified as constituents of the PEX14 complexes. Unexpectedly, tubulin was discovered to be the major PEX14-associated protein, and direct binding of the proteins was demonstrated. Accordingly, peroxisomal remnants in PEX14-deficient cells have lost their ability to move along microtubules. In vivo and in vitro analyses indicate that the physical binding to tubulin is mediated by the conserved N-terminal domain of PEX14. Thus, human PEX14 is a multi-tasking protein that not only facilitates peroxisomal protein import but is also required for peroxisome motility by serving as membrane anchor for microtubules.
• 
  Gebert, N., Gebert, M., Oeljeklaus, S., von der Malsburg, K., Stroud, D. A., Kulawiak, B., Wirth, C., Zahedi, R. P., Dolezal, P., Wiese, S., Simon, O., Schulze-Specking, A., Truscott, K. N., Sickmann, A., Rehling, P., Guiard, B., Hunte, C., Warscheid, B., van der Laan, M., … Wiedemann, N. (2011). Dual Function of Sdh3 in the Respiratory Chain and TIM22 Protein Translocase of the Mitochondrial Inner Membrane. Molecular Cell, 44(5), 811-818. https://doi.org/10.1016/j.molcel.2011.09.025
  • [ Abstract ]
  • [ DOI ]
  The mitochondrial inner membrane harbors the complexes of the respiratory chain and translocase complexes for precursor proteins. We have identified a further subunit of the carrier translocase (TIM22 complex) that surprisingly is identical to subunit 3 of respiratory complex II, succinate dehydrogenase (Sdh3). The membrane-integral protein Sdh3 plays specific functions in electron transfer in complex II. We show by genetic and biochemical approaches that Sdh3 also plays specific functions in the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex. We conclude that the assembly of Sdh3 with different partner proteins, Sdh4 and Tim18, recruits it to two different mitochondrial membrane complexes with functions in bioenergetics and protein biogenesis, respectively.
• 
  Pusnik, M., Schmidt, O., Perry, A. J., Oeljeklaus, S., Niemann, M., Warscheid, B., Lithgow, T., Meisinger, C., & Schneider, A. (2011). Mitochondrial Preprotein Translocase of Trypanosomatids Has a Bacterial Origin. Current Biology : CB, 21(20), 1738-1743. https://doi.org/10.1016/j.cub.2011.08.060
  • [ Abstract ]
  • [ DOI ]
  Mitochondria are found in all eukaryotic cells and derive from a bacterial endosymbiont [1, 2]. The evolution of a protein import system was a prerequisite for the conversion of the endosymbiont into a true organelle. Tom40, the essential component of the protein translocase of the outer membrane, is conserved in mitochondria of almost all eukaryotes but lacks bacterial orthologs [3-6]. It serves as the gateway through which all mitochondrial proteins are imported. The parasitic protozoa Trypanosoma brucei and its relatives do not have a Tom40-like protein, which raises the question of how proteins are imported by their mitochondria [7, 8]. Using a combination of bioinformatics and in vivo and in vitro studies, we have discovered that T. brucei likely employs a different import channel, termed ATOM (archaic translocase of the outer mitochondrial membrane). ATOM mediates the import of nuclear-encoded proteins into mitochondria and is essential for viability of trypanosomes. It is not related to Tom40 but is instead an ortholog of a subgroup of the Omp85 protein superfamily that is involved in membrane translocation and insertion of bacterial outer membrane proteins [9]. This suggests that the protein import channel in trypanosomes is a relic of an archaic protein transport system that was operational in the ancestor of all eukaryotes.
• 
  Debelyy, M. O., Platta, H. W., Saffian, D., Hensel, A., Thoms, S., Meyer, H. E., Warscheid, B., Girzalsky, W., & Erdmann, R. (2011). Ubp15p, a Ubiquitin Hydrolase Associated with the Peroxisomal Export Machinery. The Journal of Biological Chemistry, 286(32), 28223-28234. https://doi.org/10.1074/jbc.M111.238600
  • [ Abstract ]
  • [ DOI ]
  Peroxisomal matrix protein import is facilitated by cycling receptors shuttling between the cytosol and the peroxisomal membrane. One crucial step in this cycle is the ATP-dependent release of the receptors from the peroxisomal membrane. This step is facilitated by the peroxisomal AAA (ATPases associated with various cellular activities) proteins Pex1p and Pex6p with ubiquitination of the receptor being the main signal for its export. Here we report that the AAA complex contains dislocase as well as deubiquitinating activity. Ubp15p, a ubiquitin hydrolase, was identified as a novel constituent of the complex. Ubp15p partially localizes to peroxisomes and is capable of cleaving off ubiquitin moieties from the type I peroxisomal targeting sequence (PTS1) receptor Pex5p. Furthermore, Ubp15p-deficient cells are characterized by a stress-related PTS1 import defect. The results merge into a picture in which removal of ubiquitin from the PTS1 receptor Pex5p is a specific event and might represent a vital step in receptor recycling.
• 
  Kohl, M., Wiese, S., & Warscheid, B. (2011). Cytoscape: Software for Visualization and Analysis of Biological Networks. Methods in Molecular Biology (Clifton, N.J.), 696, 291-303. https://doi.org/10.1007/978-1-60761-987-1_18
  • [ Abstract ]
  • [ DOI ]
  Substantial progress has been made in the field of "omics" research (e.g., Genomics, Transcriptomics, Proteomics, and Metabolomics), leading to a vast amount of biological data. In order to represent large biological data sets in an easily interpretable manner, this information is frequently visualized as graphs, i.e., a set of nodes and edges. Nodes are representations of biological molecules and edges connect the nodes depicting some kind of relationship. Obviously, there is a high demand for computer-based assistance for both visualization and analysis of biological data, which are often heterogeneous and retrieved from different sources. This chapter focuses on software tools that assist in visual exploration and analysis of biological networks. Global requirements for such programs are discussed. Utilization of visualization software is exemplified using the widely used Cytoscape tool. Additional information about the use of Cytoscape is provided in the Notes section. Furthermore, special features of alternative software tools are highlighted in order to assist researchers in the choice of an adequate program for their specific requirements.
• 
  Kuhn, P., Weiche, B., Sturm, L., Sommer, E., Drepper, F., Warscheid, B., Sourjik, V., & Koch, H.-G. (2011). The Bacterial SRP Receptor, SecA and the Ribosome Use Overlapping Binding Sites on the SecY Translocon. Traffic (Copenhagen, Denmark), 12(5), 563-578. https://doi.org/10.1111/j.1600-0854.2011.01167.x
  • [ Abstract ]
  • [ DOI ]
  Signal recognition particle (SRP)-dependent protein targeting is a universally conserved process that delivers proteins to the bacterial cytoplasmic membrane or to the endoplasmic reticulum membrane in eukaryotes. Crucial during targeting is the transfer of the ribosome-nascent chain complex (RNC) from SRP to the Sec translocon. In eukaryotes, this step is co-ordinated by the SR{\($\beta$\)} subunit of the SRP receptor (SR), which probably senses a vacant translocon by direct interaction with the translocon. Bacteria lack the SR{\($\beta$\)} subunit and how they co-ordinate RNC transfer is unknown. By site-directed cross-linking and fluorescence resonance energy transfer (FRET) analyses, we show that FtsY, the bacterial SR{\($\alpha$\)} homologue, binds to the exposed C4/C5 loops of SecY, the central component of the bacterial Sec translocon. The same loops serve also as binding sites for SecA and the ribosome. The FtsY-SecY interaction involves at least the A domain of FtsY, which attributes an important function to this so far ill-defined domain. Binding of FtsY to SecY residues, which are also used by SecA and the ribosome, probably allows FtsY to sense an available translocon and to align the incoming SRP-RNC with the protein conducting channel. Thus, the Escherichia coli FtsY encompasses the functions of both the eukaryotic SR{\($\alpha$\)} and SR{\($\beta$\)} subunits in one single protein.

• 
  Schollenberger, L., Gronemeyer, T., Huber, C. M., Lay, D., Wiese, S., Meyer, H. E., Warscheid, B., Saffrich, R., Peränen, J., Gorgas, K., & Just, W. W. (2010). RhoA Regulates Peroxisome Association to Microtubules and the Actin Cytoskeleton. PloS One, 5(11), e13886. https://doi.org/10.1371/journal.pone.0013886
  • [ Abstract ]
  • [ DOI ]
  The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.
• 
  El-Mohsnawy, E., Kopczak, M. J., Schlodder, E., Nowaczyk, M., Meyer, H. E., Warscheid, B., Karapetyan, N. V., & Rögner, M. (2010). Structure and Function of Intact Photosystem 1 Monomers from the Cyanobacterium Thermosynechococcus Elongatus. Biochemistry, 49(23), 4740-4751. https://doi.org/10.1021/bi901807p
  • [ Abstract ]
  • [ DOI ]
  Until now, the functional and structural characterization of monomeric photosystem 1 (PS1) complexes from Thermosynechococcus elongatus has been hampered by the lack of a fully intact PS1 preparation; for this reason, the three-dimensional crystal structure at 2.5 A resolution was determined with the trimeric PS1 complex [Jordan, P., et al. (2001) Nature 411 (6840), 909-917]. Here we show the possibility of isolating from this cyanobacterium the intact monomeric PS1 complex which preserves all subunits and the photochemical activity of the isolated trimeric complex. Moreover, the equilibrium between these complexes in the thylakoid membrane can be shifted by a high-salt treatment in favor of monomeric PS1 which can be quantitatively extracted below the phase transition temperature. Both monomers and trimers exhibit identical posttranslational modifications of their subunits and the same reaction centers but differ in the long-wavelength antenna chlorophylls. Their chlorophyll/P700 ratio (108 for the monomer and 112 for the trimer) is slightly higher than in the crystal structure, confirming mild preparation conditions. Interaction of antenna chlorophylls of the monomers within the trimer leads to a larger amount of long-wavelength chlorophylls, resulting in a higher photochemical activity of the trimers under red or far-red illumination. The dynamic equilibrium between monomers and trimers in the thylakoid membrane may indicate a transient monomer population in the course of biogenesis and could also be the basis for short-term adaptation of the cell to changing environmental conditions.
• 
  Mick, D. U., Vukotic, M., Piechura, H., Meyer, H. E., Warscheid, B., Deckers, M., & Rehling, P. (2010). Coa3 and Cox14 Are Essential for Negative Feedback Regulation of COX1 Translation in Mitochondria. The Journal of Cell Biology, 191(1), 141-154. https://doi.org/10.1083/jcb.201007026
  • [ Abstract ]
  • [ DOI ]
  Regulation of eukaryotic cytochrome oxidase assembly occurs at the level of Cox1 translation, its central mitochondria-encoded subunit. Translation of COX1 messenger RNA is coupled to complex assembly in a negative feedback loop: the translational activator Mss51 is thought to be sequestered to assembly intermediates, rendering it incompetent to promote translation. In this study, we identify Coa3 (cytochrome oxidase assembly factor 3; Yjl062w-A), a novel regulator of mitochondrial COX1 translation and cytochrome oxidase assembly. We show that Coa3 and Cox14 form assembly intermediates with newly synthesized Cox1 and are required for Mss51 association with these complexes. Mss51 exists in equilibrium between a latent, translational resting, and a committed, translation-effective, state that are represented as distinct complexes. Coa3 and Cox14 promote formation of the latent state and thus down-regulate COX1 expression. Consequently, lack of Coa3 or Cox14 function traps Mss51 in the committed state and promotes Cox1 synthesis. Our data indicate that Coa1 binding to sequestered Mss51 in complex with Cox14, Coa3, and Cox1 is essential for full inactivation.
• 
  Managadze, D., Würtz, C., Wiese, S., Meyer, H. E., Niehaus, G., Erdmann, R., Warscheid, B., & Rottensteiner, H. (2010). A Proteomic Approach towards the Identification of the Matrix Protein Content of the Two Types of Microbodies in Neurospora Crassa. Proteomics, 10(18), 3222-3234. https://doi.org/10.1002/pmic.201000095
  • [ Abstract ]
  • [ DOI ]
  Microbodies (peroxisomes) comprise a class of organelles with a similar biogenesis but remarkable biochemical heterogeneity. Here, we purified the two distinct microbody family members of filamentous fungi, glyoxysomes and Woronin bodies, from Neurospora crassa and analyzed their protein content by HPLC/ESI-MS/MS. In the purified Woronin bodies, we unambiguously identified only hexagonal 1 (HEX1), suggesting that the matrix is probably exclusively filled with the HEX1 hexagonal crystal. The proteomic analysis of highly purified glyoxysomes allowed the identification of 191 proteins. Among them were 16 proteins with a peroxisomal targeting signal type 1 (PTS1) and three with a PTS2. The collection also contained the previously described N. crassa glyoxysomal matrix proteins FOX2 and ICL1 that lack a typical PTS. Three PTS1 proteins were identified that likely represent the long sought glyoxysomal acyl-CoA dehydrogenases of filamentous fungi. Two of them were demonstrated by subcellular localization studies to be indeed glyoxysomal. Furthermore, two PTS proteins were identified that are suggested to be involved in the detoxification of nitroalkanes. Since the glyoxysomal localization was experimentally demonstrated for one of these enzymes, a new biochemical reaction is expected to be associated with microbody function.
• 
  Managadze, D., Würtz, C., Wiese, S., Schneider, M., Girzalsky, W., Meyer, H. E., Erdmann, R., Warscheid, B., & Rottensteiner, H. (2010). Identification of PEX33, a Novel Component of the Peroxisomal Docking Complex in the Filamentous Fungus Neurospora Crassa. European Journal of Cell Biology, 89(12), 955-964. https://doi.org/10.1016/j.ejcb.2010.07.003
  • [ Abstract ]
  • [ DOI ]
  The docking complex of peroxisomal matrix protein import is composed of PEX13 and PEX14 in all species analyzed so far, whereas only yeast appears to possess an additional component, PEX17. In this report we isolated PEX14 complexes of Neurospora crassa. Among the complex constituents, one protein designated as PEX33 possessed homology to PEX14 but only in a short N-terminal domain. The PEX14/PEX33 interaction was verified by means of two-hybrid analysis. Moreover, PEX33 was shown to interact with itself and the PTS1-receptor PEX5. Localization studies demonstrated that PEX33 constitutes a glyoxysomal protein. Growth tests of the pex33 deletion strain revealed a defect of this strain in the biogenesis of glyoxysomes and Woronin bodies. As the function of PEX33 was not redundant to that of PEX14, it is a genuine novel peroxin. Based on our experimental data, the function of PEX33 seems to resemble that of yeast PEX17 despite clear structural differences.
• 
  Klein-Scory, S., Kübler, S., Diehl, H., Eilert-Micus, C., Reinacher-Schick, A., Stühler, K., Warscheid, B., Meyer, H. E., Schmiegel, W., & Schwarte-Waldhoff, I. (2010). Immunoscreening of the Extracellular Proteome of Colorectal Cancer Cells. BMC Cancer, 10, 70. https://doi.org/10.1186/1471-2407-10-70
  • [ Abstract ]
  • [ DOI ]
  BACKGROUND: The release of proteins from tumors can trigger an immune response in cancer patients involving T lymphocytes and B lymphocytes, which results in the generation of antibodies to tumor-derived proteins. Many studies aim to use humoral immune responses, namely autoantibody profiles, directly, as clinical biomarkers. Alternatively, the antibody immune response as an amplification system for tumor associated alterations may be used to indicate putative protein biomarkers with high sensitivity. Aiming at the latter approach we here have implemented an autoantibody profiling strategy which particularly focuses on proteins released by tumor cells in vitro: the so-called secretome. METHODS: For immunoscreening, the extracellular proteome of five colorectal cancer cell lines was resolved on 2D gels, immobilized on PVDF membranes and used for serological screening with individual sera from 21 colorectal cancer patients and 24 healthy controls. All of the signals from each blot were assigned to a master map, and autoantigen candidates were defined based of the pattern of immunoreactivities. The corresponding proteins were isolated from preparative gels, identified by MALDI-MS and/or by nano-HPLC/ESI-MS/MS and exemplarily confirmed by duplex Western blotting combining the human serum samples with antibodies directed against the protein(s) of interest. RESULTS: From 281 secretome proteins stained with autoantibodies in total we first defined the "background patterns" of frequently immunoreactive extracellular proteins in healthy and diseased people. An assignment of these proteins, among them many nominally intracellular proteins, to the subset of exosomal proteins within the secretomes revealed a large overlap. On this basis we defined and consequently confirmed novel biomarker candidates such as the extreme C-terminus of the extracellular matrix protein agrin within the set of cancer-enriched immunoreactivities. CONCLUSIONS: Our findings suggest, first, that autoantibody responses may be due, in large part, to cross-presentation of antigens to the immune system via exosomes, membrane vesicles released by tumor cells and constituting a significant fraction of the secretome. In addition, this immunosecretomics approach has revealed novel biomarker candidates, some of them secretome-specific, and thus serves as a promising complementary tool to the frequently reported immunoproteomic studies for biomarker discovery.
• 
  Resemann, A., Wunderlich, D., Rothbauer, U., Warscheid, B., Leonhardt, H., Fuchser, J., Kuhlmann, K., & Suckau, D. (2010). Top-down de Novo Protein Sequencing of a 13.6 kDa Camelid Single Heavy Chain Antibody by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight/Time-of-Flight Mass Spectrometry. Analytical Chemistry, 82(8), 3283-3292. https://doi.org/10.1021/ac1000515
  • [ Abstract ]
  • [ DOI ]
  The primary structure of a 13.6 kDa single heavy chain camelid antibody (V(H)H) was determined by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) top-down sequence analysis. The majority of the sequence was obtained by mass spectrometric de novo sequencing, with the N-terminal 14 amino acid residues being determined using T(3)-sequencing and database interrogation. The determined sequence was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of a tryptic digest, which also provided high-energy collisionally induced dissociation (CID) data permitting the clear assignment of 3 of the 14 isobaric Leu/Ile residues. Five of the 11 Leu/Ile ambiguities could be resolved by homology comparisons with known V(H)H sequences. The monoisotopic molecular weight of the V(H)H was determined by ultrahigh-resolution orthogonal electrospray (ESI)-TOF analysis and found to be 13 610.6066 Da, in excellent agreement with the established sequence. To our knowledge, this is the first time that the entire primary structure of a protein with a molecular weight {\($>$\)13 kDa has been established by mass spectrometric top-down sequencing.
• 
  Meiser, C. K., Piechura, H., Werner, T., Dittmeyer-Schäfer, S., Meyer, H. E., Warscheid, B., Schaub, G. A., & Balczun, C. (2010). Kazal-Type Inhibitors in the Stomach of Panstrongylus Megistus (Triatominae, {{Reduviidae}}). Insect Biochemistry and Molecular Biology, 40(4), 345-353. https://doi.org/10.1016/j.ibmb.2010.02.011
  • [ Abstract ]
  • [ DOI ]
  Triatomines inhibit the clotting of ingested blood in the stomach (anterior midgut). After verifying this phenomenon in Panstrongylus megistus using coagulation assays, a full-length cDNA encoding a Kazal-like inhibitor was amplified by PCR. The open reading frame encodes a putative precursor protein of 412 amino acid residues, which was named PmStKaz and contains seven Kazal-like domains forming four Kazal-type inhibitors. A single domain inhibitor and three double-domain inhibitors possess sequence identities of up to 91% to the respective domains of Kazal-type inhibitors from other triatomines. The gene is expressed in the stomach (anterior midgut) but not in the small intestine (posterior midgut), salivary glands or haemocytes. After hydrophobic interaction chromatography of the stomach contents, four fractions (numbers 1-4) inhibited the activity of trypsin, fraction 2 that of subtilisin A, fractions 1, 3 and 4 that of plasmin, and fractions 3 and 4 that of thrombin. After ion exchange chromatography, MALDI-TOF-MS analysis of the intact proteins in fractions 3 and 4 showed diverse masses correlating to PmStKaz IV-V and PmStKaz II-III, respectively. Both proteins seem to be present in several isoforms with variant amino- and carboxy-terminal ends. In reverse zymography of the proteins of the stomach contents after separation by isoelectric focusing and non-reducing SDS-PAGE, much higher concentrations of isoforms of PmStKaz II-III and IV-V were evident than of PmStKaz I and VI-VII.
• 
  Zboralski, D., Warscheid, B., Klein-Scory, S., Malas, M. B., Becker, H., Böckmann, M., Meyer, H. E., Schmiegel, W., Simon-Assmann, P., & Schwarte-Waldhoff, I. (2010). Uncoupled Responses of Smad4-deficient Cancer Cells to TNFalpha Result in Secretion of Monomeric Laminin-Gamma2. Molecular Cancer, 9(1), 65. https://doi.org/10.1186/1476-4598-9-65
  • [ Abstract ]
  • [ DOI ]
  BACKGROUND: Functional loss of the tumor suppressor Smad4 is involved in pancreatic and colorectal carcinogenesis and has been associated with the acquisition of invasiveness. We have previously demonstrated that the heterotrimeric basement membrane protein laminin-332 is a Smad4 target. Namely, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-332; its loss is thus implicated in the reduced or discontinuous deposition of the heterotrimeric basement membrane molecule as evident in carcinomas. Uncoupled expression of laminin genes, on the other hand, namely overexpression of the laminin-gamma2 chain is an impressive marker at invasive edges of carcinomas where tumor cells are maximally exposed to signals from stromal cell types like macrophages. As Smad4 is characterized as an integrator of multiple extracellular stimuli in a strongly contextual manner, we asked if loss of Smad4 may also be involved in uncoupled expression of laminin genes in response to altered environmental stimuli. Here, we address Smad4 dependent effects of the prominent inflammatory cytokine TNFalpha on tumor cells. RESULTS: Smad4-reconstituted colon carcinoma cells like adenoma cells respond to TNFalpha with an increased expression of all three chains encoding laminin-332; coincubation with TGFbeta and TNFalpha leads to synergistic induction and to the secretion of large amounts of the heterotrimer. In contrast, in Smad4-deficient cells TNFalpha can induce expression of the gamma2 and beta3 but not the alpha3 chain. Surprisingly, this uncoupled induction of laminin-332 chains in Smad4-negative cells rather than causing intracellular accumulation is followed by the release of gamma2 into the medium, either in a monomeric form or in complexes with as yet unknown proteins. Soluble gamma2 is associated with increased cell migration. CONCLUSIONS: Loss of Smad4 may lead to uncoupled induction of laminin-gamma2 in response to TNFalpha and may therefore represent one of the mechanisms which underlie accumulation of laminin-gamma2 at the invasive margin of a tumor. The finding, that gamma2 is secreted from tumor cells in significant amounts and is associated with increased cell migration may pave the way for further investigation to better understand its functional relevance for tumor progression.
• 
  Rasche, S., Toetter, B., Adler, J., Tschapek, A., Doerner, J. F., Kurtenbach, S., Hatt, H., Meyer, H., Warscheid, B., & Neuhaus, E. M. (2010). Tmem16b Is Specifically Expressed in the Cilia of Olfactory Sensory Neurons. Chemical Senses, 35(3), 239-245. https://doi.org/10.1093/chemse/bjq007
  • [ Abstract ]
  • [ DOI ]
  Calcium-activated chloride channels (CaCCs) are involved in many physiological processes, including sensory signal transduction, but only little is known to date about their structure and function. We performed a proteome analysis of the olfactory epithelium (OE) membrane proteome and identified so far uncharacterized membrane proteins as candidate channels. One of the most abundant membrane proteins in olfactory sensory neurons (OSNs) was Tmem16b, a member of a recently identified family of CaCCs. In addition to former studies performed on Tmem16b, we show here that Tmem16b expression is highly specific for the OE, in contrast to the closely related Tmem16a, which shows a broad expression pattern in secretory epithelial cells. Native Tmem16b is localized in the cilia of the OSNs, which is in agreement with previous electrophysiological recordings.

• 
  Grunau, S., Schliebs, W., Linnepe, R., Neufeld, C., Cizmowski, C., Reinartz, B., Meyer, H. E., Warscheid, B., Girzalsky, W., & Erdmann, R. (2009). Peroxisomal Targeting of PTS2 Pre-Import Complexes in the Yeast Saccharomyces Cerevisiae. Traffic (Copenhagen, Denmark), 10(4), 451-460. https://doi.org/10.1111/j.1600-0854.2008.00876.x
  • [ Abstract ]
  • [ DOI ]
  Posttranslational matrix protein import into peroxisomes uses either one of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. Unlike the PTS1 receptor Pex5p, the PTS2 receptor Pex7p is necessary but not sufficient to target cargo proteins into the peroxisomal matrix and requires coreceptors. Saccharomyces cerevisiae possesses two coreceptors, Pex18p and Pex21p, with a redundant but not a clearly defined function. To gain further insight into the early events of this import pathway, PTS2 pre-import complexes of S. cerevisiae were isolated and characterized by determination of size and protein composition in wild-type and different mutant strains. Mass spectrometric analysis of the cytosolic PTS2 pre-import complex indicates that Fox3p is the only abundant PTS2 protein under oleate growth conditions. Our data strongly suggest that the formation of the ternary cytosolic PTS2 pre-import complex occurs hierarchically. First, Pex7p recognizes cargo proteins through its PTS2 in the cytosol. In a second step, the coreceptor binds to this complex, and finally, this ternary 150 kDa pre-import complex docks at the peroxisomal membrane, where both the PTS1 and the PTS2 import pathways converge. Gel filtration analysis of membrane-bound subcomplexes suggests that Pex13p provides the initial binding partner at the peroxisomal membrane, whereas Pex14p assembles with Pex18p in high-molecular-weight complexes after or during dissociation of the PTS2 receptor.
• 
  Oeljeklaus, S., Meyer, H. E., & Warscheid, B. (2009). Advancements in Plant Proteomics Using Quantitative Mass Spectrometry. Journal of Proteomics, 72(3), 545-554. https://doi.org/10.1016/j.jprot.2008.11.008
  • [ Abstract ]
  • [ DOI ]
  Due to innovative advancements in quantitative MS technologies, proteomics has evolved from taking mere "snapshots" of distinct proteomes in a defined state to monitoring, for instance, changes in abundance, location and/or posttranslational modification(s) of proteins under various conditions, thereby facilitating the functional characterization of proteins in large scale experiments. In plant biology, MS-based quantitative proteomics strategies utilizing stable isotope labeling or label-free methods for protein quantification have only recently been started to find increasing application to comparative and functional proteomics analyses. This review summarizes latest trends and applications in MS-based quantitative plant proteomics and provides insight into different technologies available. In addition, the studies presented here illustrate the enormous potential of quantitative MS for the analysis of important functional aspects with the emphasis on organellar and phosphoproteomics as well as dynamics and turnover of proteins in plants.
• 
  Poschmann, G., Sitek, B., Sipos, B., Ulrich, A., Wiese, S., Stephan, C., Warscheid, B., Klöppel, G., Vander Borght, A., Ramaekers, F. C. S., Meyer, H. E., & Stühler, K. (2009). Identification of Proteomic Differences between Squamous Cell Carcinoma of the Lung and Bronchial Epithelium. Molecular & Cellular Proteomics : MCP, 8(5), 1105-1116. https://doi.org/10.1074/mcp.M800422-MCP200
  • [ Abstract ]
  • [ DOI ]
  Proteins that exhibit different expression levels in normal and malignant lung cells are good candidate biomarkers to improve early diagnosis and intervention. We used a quantitative approach and compared the proteome of microdissected cells from normal human bronchial epithelium and squamous cell carcinoma tumors of histopathological grades G2 and G3. DIGE analysis and subsequent MS-based protein identification revealed that 32 non-redundant proteins were differentially regulated between the respective tissue types. These proteins are mainly involved in energy pathways, cell growth or maintenance mechanisms, protein metabolism, and the regulation of DNA and RNA metabolism. The expression of some of these proteins was analyzed by immunohistochemistry using tissue microarrays containing tissue specimen of 55 patients, including normal bronchial epithelium, squamous cell carcinomas, adenocarcinomas, and large cell carcinomas. The results of the immunohistochemical studies correlated with the proteome study data and revealed that particularly HSP47 and a group of cytokeratins (i.e. cytokeratins 6a, 16, and 17) are significantly co-regulated in squamous cell carcinoma. Furthermore cytokeratin 17 showed significantly higher abundance in G2 grade compared with G3 grade squamous cell carcinomas in both the gel-based and the immunohistochemical analysis. Therefore this protein might be used as a marker for stratification between different tumor grades.
• 
  Mölleken, C., Sitek, B., Henkel, C., Poschmann, G., Sipos, B., Wiese, S., Warscheid, B., Broelsch, C., Reiser, M., Friedman, S. L., Tornoe, I., Schlosser, A., Klöppel, G., Schmiegel, W., Meyer, H. E., Holmskov, U., & Stühler, K. (2009). Detection of Novel Biomarkers of Liver Cirrhosis by Proteomic Analysis. Hepatology (Baltimore, Md.), 49(4), 1257-1266. https://doi.org/10.1002/hep.22764
  • [ Abstract ]
  • [ DOI ]
  Hepatic cirrhosis is a life-threatening disease arising from different chronic liver disorders. One major cause for hepatic cirrhosis is chronic hepatitis C. Chronic hepatitis C is characterized by a highly variable clinical course, with at least 20% developing liver cirrhosis within 40 years. Only liver biopsy allows a reliable evaluation of the course of hepatitis C by grading inflammation and staging fibrosis, and thus serum biomarkers for hepatic fibrosis with high sensitivity and specificity are needed. To identify new candidate biomarkers for hepatic fibrosis, we performed a proteomic approach of microdissected cirrhotic septa and liver parenchyma cells. In cirrhotic septa, we detected an increasing expression of cell structure associated proteins, including actin, prolyl 4-hydroxylase, tropomyosin, calponin, transgelin, and human microfibril-associated protein 4 (MFAP-4). Tropomyosin, calponin, and transgelin reflect a contribution of activated stellate cells/myofibroblasts to chronic liver injury. The expression of tropomyosin, transgelin, and MFAP-4, an extracellular matrix associated protein, were further evaluated by immunohistochemistry. Tropomyosin and MFAP-4 demonstrated high serum levels in patients with hepatic cirrhosis of different causes. CONCLUSION: A quantitative analysis of MFAP-4 serum levels in a large number of patients showed MFAP-4 as novel candidate biomarker with high diagnostic accuracy for prediction of nondiseased liver versus cirrhosis [area under receiver operating characteristic curve (AUC) = 0.97, P {\($<$\) 0.0001] as well as stage 0 versus stage 4 fibrosis (AUC = 0.84, P {\($<$\) 0.0001), and stages 0 to 3 versus stage 4 fibrosis (AUC = 0.76, P {\($<$\) 0.0001).
• 
  Oeljeklaus, S., Meyer, H. E., & Warscheid, B. (2009). New Dimensions in the Study of Protein Complexes Using Quantitative Mass Spectrometry. FEBS Letters, 583(11), 1674-1683. https://doi.org/10.1016/j.febslet.2009.04.018
  • [ Abstract ]
  • [ DOI ]
  Mass spectrometry combined with affinity purification techniques has evolved as a prime tool for the in-depth study of distinct protein complexes and protein-protein interactions. It fueled proteome-wide studies leading to the establishment of intricate cellular protein interaction networks. Recent innovative advancements in quantitative protein mass spectrometry act as driving force for the design of ingenious strategies in interaction proteomics facilitating the acquisition of interaction data with improved accuracy and, most intriguingly, the elucidation of functional aspects by monitoring transient interactions as well as dynamic changes in composition, stoichiometry, localization and post-translational modification of protein complexes under various conditions.

• 
  Grou, C. P., Carvalho, A. F., Pinto, M. P., Wiese, S., Piechura, H., Meyer, H. E., Warscheid, B., Sá-Miranda, C., & Azevedo, J. E. (2008). Members of the E2D ({{UbcH5}}) Family Mediate the Ubiquitination of the Conserved Cysteine of Pex5p, the Peroxisomal Import Receptor. The Journal of Biological Chemistry, 283(21), 14190-14197. https://doi.org/10.1074/jbc.M800402200
  • [ Abstract ]
  • [ DOI ]
  According to current models of peroxisomal biogenesis, newly synthesized peroxisomal matrix proteins are transported into the organelle by Pex5p. Pex5p recognizes these proteins in the cytosol, mediates their membrane translocation, and is exported back into the cytosol in an ATP-dependent manner. We have previously shown that export of Pex5p is preceded by (and requires) monoubiquitination of a conserved cysteine residue present at its N terminus. In yeasts, and probably also in plants, ubiquitination of Pex5p is mediated by a specialized ubiquitin-conjugating enzyme, Pex4p. In mammals, the identity of this enzyme has remained unknown for many years. Here, we provide evidence suggesting that E2D1/2/3 (UbcH5a/b/c) are the mammalian functional counterparts of yeast/plant Pex4p. The mechanistic implications of these findings are discussed.
• 
  Barbour, J., Neuhaus, E. M., Piechura, H., Stoepel, N., Mashukova, A., Brunert, D., Sitek, B., Stühler, K., Meyer, H. E., Hatt, H., & Warscheid, B. (2008). New Insight into Stimulus-Induced Plasticity of the Olfactory Epithelium in Mus Musculus by Quantitative Proteomics. Journal of Proteome Research, 7(4), 1594-1605. https://doi.org/10.1021/pr7005796
  • [ Abstract ]
  • [ DOI ]
  The olfactory system is exposed to a plethora of chemical compounds throughout an organism's lifespan. Anticipation of stimuli and construction of appropriate neural filters present a significant challenge. This may be addressed via modulation of the protein composition of the sensory epithelium in response to environmental conditions. To reveal the mechanisms governing these changes, we employed a comprehensive quantitative proteomics strategy. Two groups of juvenile mice were treated with either pulsed or continuous application of octanal. After 20 days of treatment, we performed a behavioral study and conducted electrophysiological recordings from the olfactory epithelium (OE). Both treated groups demonstrated peripheral desensitization to octanal; however, only the 'continuous' group exhibited habituation. To obtain novel insight into the molecular mechanisms underpinning the peripheral desensitization to octanal, the OE proteomes of octanal-treated mice versus control were quantitatively analyzed using two-dimensional difference gel electrophoresis. We identified several significantly regulated proteins that were functionally classified as calcium-binding proteins, cytoskeletal proteins, and lipocalins. The calcium-binding proteins and cytoskeletal proteins were up-regulated in the 'pulsed' group, whereas in the 'continuous' group, four lipocalins were significantly down-regulated. Uniquely, the lipocalin odorant-binding protein Ia was drastically down-regulated in both groups. The identified proteins reflect changes throughout the entire OE, corresponding to changes in neuronal, non-neuronal, and pericellular processes. We report the regulation of several promising candidates for the investigation of odorant-induced changes of the OE. Among these proteins are different lipocalins, which seem to play a crucial role in the regulation of the sensitivity of the olfactory system.
• 
  Tribl, F., Lohaus, C., Dombert, T., Langenfeld, E., Piechura, H., Warscheid, B., Meyer, H. E., & Marcus, K. (2008). Towards Multidimensional Liquid Chromatography Separation of Proteins Using Fluorescence and Isotope-Coded Protein Labelling for Quantitative Proteomics. Proteomics, 8(6), 1204-1211. https://doi.org/10.1002/pmic.200700736
  • [ Abstract ]
  • [ DOI ]
  HPLC has emerged as a valuable tool for separating proteins. To address the analysis of complex proteomes and quantitative changes of proteins therein, we developed a multidimensional LC (MDLC)-based approach followed by large gel 1-D SDS-PAGE. Here we present a novel strategy that allows for simultaneously identifying and quantifying differentially regulated proteins following three separation and fractionation steps. This MDLC platform integrates advantages of dual protein labelling using both fluorescence and isotope-coded tags for subsequent detection and quantification of abundance ratios of proteins by MS.
• 
  Hebeler, R., Oeljeklaus, S., Reidegeld, K. A., Eisenacher, M., Stephan, C., Sitek, B., Stühler, K., Meyer, H. E., Sturre, M. J. G., Dijkwel, P. P., & Warscheid, B. (2008). Study of Early Leaf Senescence in Arabidopsis Thaliana by Quantitative Proteomics Using Reciprocal 14N/15N Labeling and Difference Gel Electrophoresis. Molecular & Cellular Proteomics : MCP, 7(1), 108-120. https://doi.org/10.1074/mcp.M700340-MCP200
  • [ Abstract ]
  • [ DOI ]
  Leaf senescence represents the final stage of leaf development and is associated with fundamental changes on the level of the proteome. For the quantitative analysis of changes in protein abundance related to early leaf senescence, we designed an elaborate double and reverse labeling strategy simultaneously employing fluorescent two-dimensional DIGE as well as metabolic (15)N labeling followed by MS. Reciprocal (14)N/(15)N labeling of entire Arabidopsis thaliana plants showed that full incorporation of (15)N into the proteins of the plant did not cause any adverse effects on development and protein expression. A direct comparison of DIGE and (15)N labeling combined with MS showed that results obtained by both quantification methods correlated well for proteins showing low to moderate regulation factors. Nano HPLC/ESI-MS/MS analysis of 21 protein spots that consistently exhibited abundance differences in nine biological replicates based on both DIGE and MS resulted in the identification of 13 distinct proteins and protein subunits that showed significant regulation in Arabidopsis mutant plants displaying advanced leaf senescence. Ribulose 1,5-bisphosphate carboxylase/oxygenase large and three of its four small subunits were found to be down-regulated, which reflects the degradation of the photosynthetic machinery during leaf senescence. Among the proteins showing higher abundance in mutant plants were several members of the glutathione S-transferase family class phi and quinone reductase. Up-regulation of these proteins fits well into the context of leaf senescence since they are generally involved in the protection of plant cells against reactive oxygen species which are increasingly generated by lipid degradation during leaf senescence. With the exception of one glutathione S-transferase isoform, none of these proteins has been linked to leaf senescence before.

• 
  Mick, D. U., Wagner, K., van der Laan, M., Frazier, A. E., Perschil, I., Pawlas, M., Meyer, H. E., Warscheid, B., & Rehling, P. (2007). Shy1 Couples Cox1 Translational Regulation to Cytochrome c Oxidase Assembly. The EMBO Journal, 26(20), 4347-4358. https://doi.org/10.1038/sj.emboj.7601862
  • [ Abstract ]
  • [ DOI ]
  Cytochrome c oxidase (complex IV) of the respiratory chain is assembled from nuclear and mitochondrially-encoded subunits. Defects in the assembly process lead to severe human disorders such as Leigh syndrome. Shy1 is an assembly factor for complex IV in Saccharomyces cerevisiae and mutations of its human homolog, SURF1, are the most frequent cause for Leigh syndrome. We report that Shy1 promotes complex IV biogenesis through association with different protein modules; Shy1 interacts with Mss51 and Cox14, translational regulators of Cox1. Additionally, Shy1 associates with the subcomplexes of complex IV that are potential assembly intermediates. Formation of these subcomplexes depends on Coa1 (YIL157c), a novel assembly factor that cooperates with Shy1. Moreover, partially assembled forms of complex IV bound to Shy1 and Cox14 can associate with the bc1 complex to form transitional supercomplexes. We suggest that Shy1 links Cox1 translational regulation to complex IV assembly and supercomplex formation.
• 
  Wiese, S., Gronemeyer, T., Ofman, R., Kunze, M., Grou, C. P., Almeida, J. A., Eisenacher, M., Stephan, C., Hayen, H., Schollenberger, L., Korosec, T., Waterham, H. R., Schliebs, W., Erdmann, R., Berger, J., Meyer, H. E., Just, W., Azevedo, J. E., Wanders, R. J. A., & Warscheid, B. (2007). Proteomics Characterization of Mouse Kidney Peroxisomes by Tandem Mass Spectrometry and Protein Correlation Profiling. Molecular & Cellular Proteomics : MCP, 6(12), 2045-2057. https://doi.org/10.1074/mcp.M700169-MCP200
  • [ Abstract ]
  • [ DOI ]
  The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by alpha- and beta-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate approximately 50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.
• 
  Wiese, S., Reidegeld, K. A., Meyer, H. E., & Warscheid, B. (2007). Protein Labeling by iTRAQ: A New Tool for Quantitative Mass Spectrometry in Proteome Research. Proteomics, 7(3), 340-350. https://doi.org/10.1002/pmic.200600422
  • [ Abstract ]
  • [ DOI ]
  A novel, MS-based approach for the relative quantification of proteins, relying on the derivatization of primary amino groups in intact proteins using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled proteins do not differ in mass; accordingly, their corresponding proteolytic peptides appear as single peaks in MS scans. Because quantitative information is provided by isotope-encoded reporter ions that can only be observed in MS/MS spectra, we analyzed the fragmentation behavior of ESI and MALDI ions of peptides generated from iTRAQ-labeled proteins using a TOF/TOF and/or a QTOF instrument. We observed efficient liberation of reporter ions for singly protonated peptides at low-energy collision conditions. In contrast, increased collision energies were required to liberate the iTRAQ label from lysine side chains of doubly charged peptides and, thus, to observe reporter ions suitable for relative quantification of proteins with high accuracy. We then developed a quantitative strategy that comprises labeling of intact proteins by iTRAQ followed by gel electrophoresis and peptide MS/MS analyses. As proof of principle, mixtures of five different proteins in various concentration ratios were quantified, demonstrating the general applicability of the approach presented here to quantitative MS-based proteomics.

• 
  Frazier, A. E., Taylor, R. D., Mick, D. U., Warscheid, B., Stoepel, N., Meyer, H. E., Ryan, M. T., Guiard, B., & Rehling, P. (2006). Mdm38 Interacts with Ribosomes and Is a Component of the Mitochondrial Protein Export Machinery. The Journal of Cell Biology, 172(4), 553-564. https://doi.org/10.1083/jcb.200505060
  • [ Abstract ]
  • [ DOI ]
  Saccharomyces cerevisiae Mdm38 and Ylh47 are homologues of human Letm1, a protein implicated in Wolf-Hirschhorn syndrome. We analyzed the function of Mdm38 and Ylh47 in yeast mitochondria to gain insight into the role of Letm1. We find that mdm38Delta mitochondria have reduced amounts of certain mitochondrially encoded proteins and low levels of complex III and IV and accumulate unassembled Atp6 of complex V of the respiratory chain. Mdm38 is especially required for efficient transport of Atp6 and cytochrome b across the inner membrane, whereas Ylh47 plays a minor role in this process. Both Mdm38 and Ylh47 form stable complexes with mitochondrial ribosomes, similar to what has been reported for Oxa1, a central component of the mitochondrial export machinery. Our results indicate that Mdm38 functions as a component of an Oxa1-independent insertion machinery in the inner membrane and that Mdm38 plays a critical role in the biogenesis of the respiratory chain by coupling ribosome function to protein transport across the inner membrane.
• 
  Nowaczyk, M. M., Hebeler, R., Schlodder, E., Meyer, H. E., Warscheid, B., & Rögner, M. (2006). Psb27, a Cyanobacterial Lipoprotein, Is Involved in the Repair Cycle of Photosystem II. The Plant Cell, 18(11), 3121-3131. https://doi.org/10.1105/tpc.106.042671
  • [ Abstract ]
  • [ DOI ]
  Photosystem II (PSII) performs one of the key reactions on our planet: the light-driven oxidation of water. This fundamental but very complex process requires PSII to act in a highly coordinated fashion. Despite detailed structural information on the fully assembled PSII complex, the dynamic aspects of formation, processing, turnover, and degradation of PSII with at least 19 subunits and various cofactors are still not fully understood. Transient complexes are especially difficult to characterize due to low abundance, potential heterogeneity, and instability. Here, we show that Psb27 is involved in the assembly of the water-splitting site of PSII and in the turnover of the complex. Psb27 is a bacterial lipoprotein with a specific lipid modification as shown by matrix-assisted laser-desorption ionization time of flight mass spectrometry. The combination of HPLC purification of four different PSII subcomplexes and (15)N pulse label experiments revealed that lipoprotein Psb27 is part of a preassembled PSII subcomplex that represents a distinct intermediate in the repair cycle of PSII.

• 
  Warscheid, B., & Fenselau, C. (2004). A Targeted Proteomics Approach to the Rapid Identification of Bacterial Cell Mixtures by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Proteomics, 4(10), 2877-2892. https://doi.org/10.1002/pmic.200400911
  • [ Abstract ]
  • [ DOI ]
  A proteomic approach to the rapid identification of bacteria is presented, which relies on the solubilization of a limited number of proteins from intact cells combined with on-probe tryptic digestion. Within 20 min, complete cleavage products of a limited set of bacterial proteins with molecular masses of about 4-125 kDa were obtained by on-probe digestion with immobilized trypsin. Bacterial peptides suitable for unimolecular decomposition analysis were generated within 5 min, and the sequence information obtained allowed identification of abundant proteins, and accordingly, their bacterial sources via searches in the NCBI database. Analysis of fragmentation products was also shown to allow for identification of bacterial peptides identical in mass but differing slightly in amino acid sequence by manual data analysis. In this work, Bacillus subtilis 168, B. globigii, B. sphaericus 14577, B. cereus T, and B. anthracis Sterne were examined, and various cold shock proteins were identified in all species. In addition, DNA-binding, 60 kDa-heat shock, surface-related and other stress-protective proteins were identified in the bacterial cell digests, and species-specific tryptic peptides could be generated from each of the Bacillus species studied. Bacterial peptides could be analyzed with greater sensitivity and mass accuracy than the parent proteins. The applicability of this targeted proteomics approach to the rapid identification of Bacillus species was further established by analyzing binary cell mixtures.
• 
  Whiteaker, J. R., Warscheid, B., Pribil, P., Hathout, Y., & Fenselau, C. (2004). Complete Sequences of Small Acid-Soluble Proteins from Bacillus Globigii. Journal of Mass Spectrometry : JMS, 39(10), 1113-1121. https://doi.org/10.1002/jms.668
  • [ Abstract ]
  • [ DOI ]
  Three abundant small acid-soluble proteins (SASPs) from spores of Bacillus globigii were sequenced using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with post-source decay and nanoelectrospray collision-induced dissociation tandem mass spectrometry. The proteins were extracted from spores with 1 M HCl. Scanning electron micrographs of spores before and after acid extraction show that the spores retain their overall structure but have a shriveled texture following the acid treatment. Extracted SASPs were purified by high-performance liquid chromatography and molecular masses of the SASPs were identified at 7068 (SASP-1), 7332 (SASP-2), and 8889 (gamma-SASP). De novo peptide sequencing was used to determine the protein sequences. The correct ordering of peptide sequences was aided by mapping overlapping enzymatic digests and by comparison with homologous SASPs from Bacillus stearothermophilus. B. globigii is used in many field tests as a surrogate for B. anthracis. Thus complete SASP sequences from B. globigii will facilitate the development of methods for rapid identification of bacteria based on mass spectrometry and the examination of taxonomic relationships between Bacillus species.

• 
  Warscheid, B., Kückelmann, U., & Hoffmann, T. (2003). Direct Quantitative Analysis of Organic Compounds in the Gas and Particle Phase Using a Modified Atmospheric Pressure Chemical Ionization Source in Combination with Ion Trap Mass Spectrometry. Analytical Chemistry, 75(6), 1410-1417. https://doi.org/10.1021/ac025788d
  • [ Abstract ]
  • [ DOI ]
  A slightly modified atmospheric pressure chemical ionization source is employed for direct quantitative analysis of volatile or semivolatile organic compounds in air. The method described here is based on the direct introduction of an analyte in the gas or particle phase, or both, into the ion source of a commercial ion trap mass spectrometer. For quantitation, a standard solution is directly transferred into the vaporizer unit of the ion source via a deactivated fused-silica capillary by using the sheath liquid syringe pump, which is part of the mass spectrometer. The standard addition procedure is conducted by varying the pump rate of a diluted solution of the standard compound in methanol/water. A N2 sheath gas flow is applied for optimal vaporization and mixing with the analyte gas stream. By performing detailed reagent ion monitoring experiments, it is shown that the relative signal intensity of [M + H]+ ions is dependent on the relative humidity of the analyte gas stream as well as the composition and concentration of CI reagent ions. The method is validated by a comparison of the standard addition results with a calibration test gas of known concentration. To demonstrate the potential of atmospheric pressure chemical ionization mass spectrometry as a quantitative analytical technique for on-line investigations, a tropospherically relevant reaction is carried out in a 493-L reaction chamber at atmospheric pressure and 296 K in synthetic air at 50% relative humidity. Finally, the applicability of the technique to rapidly differentiate between analytes in the gas and particle phase is demonstrated.
• 
  English, R. D., Warscheid, B., Fenselau, C., & Cotter, R. J. (2003). Bacillus Spore Identification via Proteolytic Peptide Mapping with a Miniaturized MALDI TOF Mass Spectrometer. Analytical Chemistry, 75(24), 6886-6893. https://doi.org/10.1021/ac034624+
  • [ Abstract ]
  • [ DOI ]
  An approach is tested here as a rapid screening method for Bacillus spore species employing bacterial peptide analysis with a miniaturized MALDI TOF mass spectrometer. A limited set of tryptic peptides was generated in situ following selective solubilization of the small, acid-soluble protein family (SASP) from spore samples on the MALDI sample holder. To facilitate species identification, a compact database was created comprising masses of the tryptic cleavage products generated in silico from all Bacillus and Clostridium SASPs whose sequences are available in public databases. Experimental measurements were matched against the custom-made database, and a published statistical model was then used to evaluate the probability of false identifications.
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  Warscheid, B., & Fenselau, C. (2003). Characterization of Bacillus Spore Species and Their Mixtures Using Postsource Decay with a Curved-Field Reflectron. Analytical Chemistry, 75(20), 5618-5627. https://doi.org/10.1021/ac034200f
  • [ Abstract ]
  • [ DOI ]
  A strategy is proposed for the rapid identification of Bacillus spores, which relies on the selective release of a family of proteins, referred to as small, acid-soluble spore proteins (SASPs). In this work, SASPs were selectively solubilized from Bacillus spores on the MALDI sample plate by using 10% TFA. Proteolytic digests of SASPs generated in situ from spores of B. subtilis 168, B. globigii, B. thuringiensis subs. Kurstaki HD-1, B. cereus T, and the nonpathogenic strain B. anthracis Sterne were prepared in 5-25 min by using trypsin immobilized on Agarose beads and subsequently analyzed by MALDI-TOFMS using a curved-field reflectron. Protein identification was obtained by partial sequencing of distinctive tryptic peptides from Bacillus spores via post-source decay analysis combined with genome-based database searches by Mascot Sequence Query. Various unique SASPs were identified, allowing the characterization of Bacillus species by obtaining sequence-specific information on single peptides. The applicability of this approach for the rapid identification of Bacillus species was further established by analyzing spore mixtures.
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  Warscheid, B., Jackson, K., Sutton, C., & Fenselau, C. (2003). MALDI Analysis of Bacilli in Spore Mixtures by Applying a Quadrupole Ion Trap Time-of-Flight Tandem Mass Spectrometer. Analytical Chemistry, 75(20), 5608-5617. https://doi.org/10.1021/ac0344081
  • [ Abstract ]
  • [ DOI ]
  A novel ion trap time-of-flight hybrid mass spectrometer (qIT-TOF MS) has been applied for peptide sequencing in proteolytic digests generated from spore mixtures of Bacilli. The method of on-probe solubilization and in situ proteolytic digestion of small, acid-soluble spore proteins has been recently developed in our laboratory, and microorganism identification in less than 20 min was accomplished. In this study, tryptic peptides were generated in situ from complex spore mixtures of B. subtilis 168, B. globigii, B. thuringiensis subs. Kurstaki, and B. cereus T, respectively. MALDI analysis of bacterial peptides generated was performed with an average mass resolving power of 6200 and a mass accuracy of up to 10 ppm using a trap-TOF tandem configuration. Precursor ions of interest were usually selected and stored in the quadrupole ion trap with their complete isotope distribution by choosing a window of +/- 2 Da. Sequence-specific information on isolated protonated peptides was gained via tandem MS experiments with an average mass resolving power of 4450 for product ion analysis, and protein and bacterial sources were identified by database searching.

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  Warscheid, B., & Hoffmann, T. (2002). Direct Analysis of Highly Oxidised Organic Aerosol Constituents by On-Line Ion Trap Mass Spectrometry in the Negative-Ion Mode. Rapid Communications in Mass Spectrometry : RCM, 16(6), 496-504. https://doi.org/10.1002/rcm.602
  • [ Abstract ]
  • [ DOI ]
  On-line ion trap mass spectrometry (ITMS) enables the characterisation of constituents of biogenic secondary organic aerosols in complex organic reaction mixtures. This real-time analysis is achieved by directly introducing the airborne particles into the ion source of the mass spectrometer. Negative-ion chemical ionisation at atmospheric pressure (APCI(-)) was used as the ionisation method of choice. The aerosols were generated from the gas-phase ozonolysis of two C10H16-terpenes (alpha-pinene and limonene), and investigated by performing on-line APCI(-)-ITMS(n). Highly oxidised compounds were tentatively identified as important particle-phase products. Based on recent investigations of low-energy collision-induced dissociation pathways of a wide range of deprotonated multifunctional carboxylic acid species derived from monoterpene precursors (Warscheid B, Hoffmann T. Rapid Commun. Mass Spectrom. 2001; 15: 2259), the formation of structurally different C10H16O5 and C10H16O6 species, such as acidic esters from alpha-pinene and aldo-hydroxycarboxylic acids from limonene, is proposed.