Publications

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  Coordination of cytochrome bc1 complex assembly at MICOS. Zerbes, Ralf M; Colina-Tenorio, Lilia; Bohnert, Maria; von der Malsburg, Karina; Peikert, Christian D; Mehnert, Carola S; Perschil, Inge; Klar, Rhena F U; de Boer, Rinse; Kram, Anita; van der Klei, Ida; Oeljeklaus, Silke; Warscheid, Bettina; Rampelt, Heike; van der Laan, Martin. In EMBO Reports. Springer Science and Business Media LLC, 2024.
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  Identification of TFG- and Autophagy-Regulated Proteins and Glycerophospholipids in B Cells. Steinmetz, Tobit D.; Thomas, Jana; Reimann, Lena; Himmelreich, Ann-Kathrin; Schulz, Sebastian R.; Golombek, Florian; Castiglione, Kathrin; Jäck, Hans-Martin; Brodesser, Susanne; Warscheid, Bettina; Mielenz, Dirk. In Journal of proteome research, 23(5), pp. 1615–1633. United States, 2024.
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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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  Mass spectrometry-based proteomics to study mutants and interactomes of mitochondrial translocation proteins. Oeljeklaus, Silke; Sharma, Lakshita; Bender, Julian; Warscheid, Bettina. In Mitochondrial Translocases Part B, pp. 101–152. Elsevier, 2024.
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  Identification of TMEM126A as OXA1L-interacting Protein Reveals Cotranslational Quality Control in Mitochondria. Poerschke, Sabine; Oeljeklaus, Silke; Cruz-Zaragoza, Luis Daniel; Schenzielorz, Alexander; Dahal, Drishan; Hillen, Hauke Sven; Das, Hirak; Kremer, Laura Sophie; Valpadashi, Anusha; Breuer, Mirjam; Sattmann, Johannes; Richter-Dennerlein, Ricarda; Warscheid, Bettina; Dennerlein, Sven; Rehling, Peter. In Molecular cell, 84(2), pp. 345–358.e5. United States, 2024.
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  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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  Intermembrane Space-Localized TbTim15 Is an Essential Subunit of the Single Mitochondrial Inner Membrane Protein Translocase of Trypanosomes. von Känel, Corinne; Oeljeklaus, Silke; Wenger, Christoph; Stettler, Philip; Harsman, Anke; Warscheid, Bettina; Schneider, André. In Molecular microbiology. England, 2024.
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  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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  Protein phosphatase-1 regulates the binding of filamin C to FILIP1 in cultured skeletal muscle cells under mechanical stress. Kokot, Thomas; Zimmermann, Johannes P.; Schwäble, Anja N.; Reimann, Lena; Herr, Anna L.; Höfflin, Nico; Köhn, Maja; Warscheid, Bettina. In Scientific Reports, 14(1), p. 27348. 2024.
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  The actin-binding protein filamin c (FLNc) is a key mediator in the response of skeletal muscle cells to mechanical stress. In addition to its function as a structural scaffold, FLNc acts as a signaling adaptor which is phosphorylated at S2234 in its mechanosensitive domain 20 (d20) through AKT. Here, we discovered a strong dephosphorylation of FLNc-pS2234 in cultured skeletal myotubes under acute mechanical stress, despite high AKT activity. We found that all three protein phosphatase 1 (PP1) isoforms are part of the FLNc d18-21 interactome. Enzymatic assays demonstrate that PP1 efficiently dephosphorylates FLNc-pS2234 and in vitro and in cells upon PP1 activation using specific modulators. FLNc-pS2234 dephosphorylation promotes the interaction with FILIP1, a mediator for filamin degradation. Altogether, we present a model in which dephosphorylation of FLNc d20 by the dominant action of PP1c prevails over AKT activity to promote the binding of the filamin degradation-inducing factor FILIP1 during acute mechanical stress.
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  Ageing-Dependent Thiol Oxidation Reveals Early Oxidation of Proteins with Core Proteostasis Functions. Jonak, Katarzyna; Suppanz, Ida; Bender, Julian; Chacinska, Agnieszka; Warscheid, Bettina; Topf, Ulrike. In Life science alliance, 7(5). United States, 2024.
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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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  Pam16 and Pam18 were repurposed during Trypanosoma brucei evolution to regulate the replication of mitochondrial DNA. von Känel, Corinne; Stettler, Philip; Esposito, Carmela; Berger, Stephan; Amodeo, Simona; Oeljeklaus, Silke; Calderaro, Salvatore; Durante, Ignacio M.; Rašková, Vendula; Warscheid, Bettina; Schneider, André. In PLOS Biology, 22(8), pp. 1–33. Public Library of Science, 2024.
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  Protein import and genome replication are essential processes for mitochondrial biogenesis and propagation. The J-domain proteins Pam16 and Pam18 regulate the presequence translocase of the mitochondrial inner membrane. In the protozoan Trypanosoma brucei, their counterparts are TbPam16 and TbPam18, which are essential for the procyclic form (PCF) of the parasite, though not involved in mitochondrial protein import. Here, we show that during evolution, the 2 proteins have been repurposed to regulate the replication of maxicircles within the intricate kDNA network, the most complex mitochondrial genome known. TbPam18 and TbPam16 have inactive J-domains suggesting a function independent of heat shock proteins. However, their single transmembrane domain is essential for function. Pulldown of TbPam16 identifies a putative client protein, termed MaRF11, the depletion of which causes the selective loss of maxicircles, akin to the effects observed for TbPam18 and TbPam16. Moreover, depletion of the mitochondrial proteasome results in increased levels of MaRF11. Thus, we have discovered a protein complex comprising TbPam18, TbPam16, and MaRF11, that controls maxicircle replication. We propose a working model in which the matrix protein MaRF11 functions downstream of the 2 integral inner membrane proteins TbPam18 and TbPam16. Moreover, we suggest that the levels of MaRF11 are controlled by the mitochondrial proteasome.
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  Identification of phosphatases that dephosphorylate the co-chaperone BAG3. Kokot, Thomas; Zimmermann, Johannes P; Chand, Yamini; Krier, Fabrice; Reimann, Lena; Scheinost, Laura; Höfflin, Nico; Esch, Alessandra; Höhfeld, Jörg; Warscheid, Bettina; Köhn, Maja. In Life Science Alliance, 8(2), p. e202402734. Life Science Alliance, LLC, 2024.
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  PEX39 facilitates the peroxisomal import of PTS2 proteins. Chen, Walter W.; Rodrigues, Tony A.; Wendscheck, Daniel; Pedrosa, Ana G.; Yang, Chendong; Francisco, Tânia; Möcklinghoff, Till; Zografakis, Alexandros; Nunes-Silva, Bernardo; Avraham, Reut Ester; Silva, Ana R.; Ferreira, Maria J.; Das, Hirak; Bender, Julian; Oeljeklaus, Silke; Sondhi, Varun; Schuldiner, Maya; Zalckvar, Einat; Hofmann, Kay; Waterham, Hans R.; DeBerardinis, Ralph J.; Azevedo, Jorge E.; Warscheid, Bettina. Cold Spring Harbor Laboratory, 2024.
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  A Msp1-containing Complex Removes Orphaned Proteins in the Mitochondrial Outer Membrane of T. Brucei. Gerber, Markus; Suppanz, Ida; Oeljeklaus, Silke; Niemann, Moritz; Käser, Sandro; Warscheid, Bettina; Schneider, André; Dewar, Caroline E. In Life science alliance, 6(11). United States, 2023.
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  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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  COX17 Acetylation via MOF-KANSL Complex Promotes Mitochondrial Integrity and Function. Guhathakurta, Sukanya; Erdogdu, Niyazi Umut; Hoffmann, Juliane J.; Grzadzielewska, Iga; Schendzielorz, Alexander; Seyfferth, Janine; Maa}rtensson, Christoph U.; Corrado, Mauro; Karoutas, Adam; Warscheid, Bettina; Pfanner, Nikolaus; Becker, Thomas; Akhtar, Asifa. In Nature metabolism, 5(11), pp. 1931–1952. Germany, 2023.
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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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  Reprogramming of the Transcriptome after Heat Stress Mediates Heat Hormesis in Caenorhabditis Elegans. Xu, Fan; Li, Ruoyao; von Gromoff, Erika D.; Drepper, Friedel; Knapp, Bettina; Warscheid, Bettina; Baumeister, Ralf; Qi, Wenjing. In Nature communications, 14(1), p. 4176. England, 2023.
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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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  Conformational Regulation and Target-Myristoyl Switch of Calcineurin B Homologous Protein 3. Becker, Florian; Fuchs, Simon; Refisch, Lukas; Drepper, Friedel; Bildl, Wolfgang; Schulte, Uwe; Liang, Shuo; Heinicke, Jonas Immanuel; Hansen, Sierra C.; Kreutz, Clemens; Warscheid, Bettina; Fakler, Bernd; Mymrikov, Evgeny V.; Hunte, Carola. In eLife, 12. England, 2023.
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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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  Immunoproteasome-Specific Subunit PSMB9 Induction Is Required to Regulate Cellular Proteostasis upon Mitochondrial Dysfunction. Kim, Minji; Serwa, Remigiusz A.; Samluk, Lukasz; Suppanz, Ida; Kodroń, Agata; Stk epkowski, Tomasz M.; Elancheliyan, Praveenraj; Tsegaye, Biniyam; Oeljeklaus, Silke; Wasilewski, Michal; Warscheid, Bettina; Chacinska, Agnieszka. In Nature communications, 14(1), p. 4092. England, 2023.
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  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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  The Mba1 Homologue of Trypanosoma Brucei Is Involved in the Biogenesis of Oxidative Phosphorylation Complexes. Wenger, Christoph; Harsman, Anke; Niemann, Moritz; Oeljeklaus, Silke; von Känel, Corinne; Calderaro, Salvatore; Warscheid, Bettina; Schneider, André. In Molecular microbiology, 119(5), pp. 537–550. England, 2023.
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  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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  Phosphoproteomics Profiling Defines a Target Landscape of the Basophilic Protein Kinases AKT, S6K, and RSK in Skeletal Myotubes. Fricke, Anna L.; Mühlhäuser, Wignand W. D.; Reimann, Lena; Zimmermann, Johannes P.; Reichenbach, Christa; Knapp, Bettina; Peikert, Christian D.; Heberle, Alexander M.; Faessler, Erik; Schäuble, Sascha; Hahn, Udo; Thedieck, Kathrin; Radziwill, Gerald; Warscheid, Bettina. In Journal of proteome research, 22(3), pp. 768–789. United States, 2023.
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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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  Phosphorylation of the Receptor Protein Pex5p Modulates Import of Proteins into Peroxisomes. Fischer, Sven; Bürgi, Jérôme; Gabay-Maskit, Shiran; Maier, Renate; Mastalski, Thomas; Yifrach, Eden; Obarska-Kosinska, Agnieszka; Rudowitz, Markus; Erdmann, Ralf; Platta, Harald W.; Wilmanns, Matthias; Schuldiner, Maya; Zalckvar, Einat; Oeljeklaus, Silke; Drepper, Friedel; Warscheid, Bettina. In Biological chemistry, 404(2-3), pp. 135–155. Germany, 2023.
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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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  Analysis of Yeast Peroxisomes via Spatial Proteomics. Das, Hirak; Zografakis, Alexandros; Oeljeklaus, Silke; Warscheid, Bettina. In Methods in molecular biology (Clifton, N.J.), 2643, pp. 13–31. United States, 2023.
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  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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  Characterization of a Highly Diverged Mitochondrial ATP Synthase F(o) Subunit in Trypanosoma Brucei. Dewar, Caroline E.; Oeljeklaus, Silke; Wenger, Christoph; Warscheid, Bettina; Schneider, André. In The Journal of biological chemistry, 298(4), p. 101829. United States, 2022.
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  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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  Regulating Peroxisome-ER Contacts via the ACBD5-VAPB Tether by FFAT Motif Phosphorylation and {{GSK3\($\beta$\)}}. Kors, Suzan; Hacker, Christian; Bolton, Chloe; Maier, Renate; Reimann, Lena; Kitchener, Emily J. A.; Warscheid, Bettina; Costello, Joseph L.; Schrader, Michael. In The Journal of cell biology, 221(3). United States, 2022.
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  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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  A Newly Identified Secreted Larval Antigen Elicits Basophil-Dependent Protective Immunity against N. Brasiliensis Infection. Thuma, Natalie; Döhler, Daniela; Mielenz, Dirk; Sticht, Heinrich; Radtke, Daniel; Reimann, Lena; Warscheid, Bettina; Voehringer, David. In Frontiers in immunology, 13, p. 979491. Switzerland, 2022.
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  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.
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  The Endoplasmic Reticulum Membrane Protein Complex Localizes to the Mitochondrial - Endoplasmic Reticulum Interface and Its Subunits Modulate Phospholipid Biosynthesis in Trypanosoma Brucei. Iyer, Advaitha; Niemann, Moritz; Serricchio, Mauro; Dewar, Caroline E.; Oeljeklaus, Silke; Farine, Luce; Warscheid, Bettina; Schneider, André; Bütikofer, Peter. In PLoS pathogens, 18(5), p. e1009717. United States, 2022.
  • [ Abstract ]
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  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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  MaxQuant and MSstats in Galaxy Enable Reproducible Cloud-Based Analysis of Quantitative Proteomics Experiments for Everyone. Pinter, Niko; Glätzer, Damian; Fahrner, Matthias; Fröhlich, Klemens; Johnson, James; Grüning, Björn Andreas; Warscheid, Bettina; Drepper, Friedel; Schilling, Oliver; Föll, Melanie Christine. In Journal of proteome research, 21(6), pp. 1558–1565. United States, 2022.
  • [ 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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  Mistargeting of Aggregation Prone Mitochondrial Proteins Activates a Nucleus-Mediated Posttranscriptional Quality Control Pathway in Trypanosomes. Dewar, Caroline E.; Oeljeklaus, Silke; Mani, Jan; Mühlhäuser, Wignand W. D.; von Känel, Corinne; Zimmermann, Johannes; Ochsenreiter, Torsten; Warscheid, Bettina; Schneider, André. In Nature communications, 13(1), p. 3084. England, 2022.
  • [ 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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  P166 Links Membrane and Intramitochondrial Modules of the Trypanosomal Tripartite Attachment Complex. Schimanski, Bernd; Aeschlimann, Salome; Stettler, Philip; Käser, Sandro; Gomez-Fabra Gala, Maria; Bender, Julian; Warscheid, Bettina; Vögtle, F.-Nora; Schneider, André. In PLoS pathogens, 18(6), p. e1010207. United States, 2022.
  • [ 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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  Author Correction: Mitochondrial Proteins: From Biogenesis to Functional Networks. Pfanner, Nikolaus; Warscheid, Bettina; Wiedemann, Nils. In Nature reviews. Molecular cell biology, 22(5), p. 367. England, 2021.
  • [ DOI ]
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  2nSILAC for Quantitative Proteomics of Prototrophic Baker’s Yeast. Dannenmaier, Stefan; Oeljeklaus, Silke; Warscheid, Bettina. In Methods in molecular biology (Clifton, N.J.), Vol. 2228, pp. 253–270. United States, 2021.
  • [ 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.
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  The Universally Conserved ATPase YchF Regulates Translation of Leaderless mRNA in Response to Stress Conditions. Landwehr, Victoria; Milanov, Martin; Angebauer, Larissa; Hong, Jiang; Jüngert, Gabriela; Hiersemenzel, Anna; Siebler, Ariane; Schmit, Fränk; Öztürk, Yavuz; Dannenmaier, Stefan; Drepper, Friedel; Warscheid, Bettina; Koch, Hans-Georg. In Frontiers in molecular biosciences, 8, p. 643696. Switzerland, 2021.
  • [ 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.
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  Subcellular Proteomics. Christopher, Josie A.; Stadler, Charlotte; Martin, Claire E.; Morgenstern, Marcel; Pan, Yanbo; Betsinger, Cora N.; Rattray, David G.; Mahdessian, Diana; Gingras, Anne-Claude; Warscheid, Bettina; Lehtiö, Janne; Cristea, Ileana M.; Foster, Leonard J.; Emili, Andrew; Lilley, Kathryn S. In Nature reviews. Methods primers, 1, p. 32. England, 2021.
  • [ 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.
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  Molecular Basis of F-actin Regulation and Sarcomere Assembly via Myotilin. Kostan, Julius; Pavv siv c, Miha; Puv z, Vid; Schwarz, Thomas C.; Drepper, Friedel; Molt, Sibylle; Graewert, Melissa Ann; Schreiner, Claudia; Sajko, Sara; van der Ven, Peter F. M.; Onipe, Adekunle; Svergun, Dmitri I.; Warscheid, Bettina; Konrat, Robert; Fürst, Dieter O.; Lenarv civ c, Brigita; Djinovi’c}}-Carugo, Kristina. In PLoS biology, 19(4), p. e3001148. United States, 2021.
  • [ 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.
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  Order from Disorder in the Sarcomere: FATZ Forms a Fuzzy but Tight Complex and Phase-Separated Condensates with {\($\alpha$\)}-Actinin. Sponga, Antonio; Arolas, Joan L.; Schwarz, Thomas C.; Jeffries, Cy M.; Rodriguez Chamorro, Ariadna; Kostan, Julius; Ghisleni, Andrea; Drepper, Friedel; Polyansky, Anton; De Almeida Ribeiro, Euripedes; Pedron, Miriam; Zawadzka-Kazimierczuk, Anna; Mlynek, Georg; Peterbauer, Thomas; Doto, Pierantonio; Schreiner, Claudia; Hollerl, Eneda; Mateos, Borja; Geist, Leonhard; Faulkner, Georgine; Kozminski, Wiktor; Svergun, Dmitri I.; Warscheid, Bettina; Zagrovic, Bojan; Gautel, Mathias; Konrat, Robert; Djinovi’c}}-Carugo, Kristina. In Science advances, 7(22). United States, 2021.
  • [ 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.
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  Determinism and Contingencies Shaped the Evolution of Mitochondrial Protein Import. Rout, Samuel; Oeljeklaus, Silke; Makki, Abhijith; Tachezy, Jan; Warscheid, Bettina; Schneider, André. In Proceedings of the National Academy of Sciences of the United States of America, 118(6). United States, 2021.
  • [ 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.
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  DIMA: Data-Driven Selection of an Imputation Algorithm. Egert, Janine; Brombacher, Eva; Warscheid, Bettina; Kreutz, Clemens. In Journal of proteome research, 20(7), pp. 3489–3496. United States, 2021.
  • [ 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.
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  Quantitative High-Confidence Human Mitochondrial Proteome and Its Dynamics in Cellular Context. Morgenstern, Marcel; Peikert, Christian D.; Lübbert, Philipp; Suppanz, Ida; Klemm, Cinzia; Alka, Oliver; Steiert, Conny; Naumenko, Nataliia; Schendzielorz, Alexander; Melchionda, Laura; Mühlhäuser, Wignand W. D.; Knapp, Bettina; Busch, Jakob D.; Stiller, Sebastian B.; Dannenmaier, Stefan; Lindau, Caroline; Licheva, Mariya; Eickhorst, Christopher; Galbusera, Riccardo; Zerbes, Ralf M.; Ryan, Michael T.; Kraft, Claudine; Kozjak-Pavlovic, Vera; Drepper, Friedel; Dennerlein, Sven; Oeljeklaus, Silke; Pfanner, Nikolaus; Wiedemann, Nils; Warscheid, Bettina. In Cell metabolism, 33(12), pp. 2464–2483.e18. United States, 2021.
  • [ 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.
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  Maintaining Proteostasis under Mechanical Stress. Höhfeld, Jörg; Benzing, Thomas; Bloch, Wilhelm; Fürst, Dieter O.; Gehlert, Sebastian; Hesse, Michael; Hoffmann, Bernd; Hoppe, Thorsten; Huesgen, Pitter F.; Köhn, Maja; Kolanus, Waldemar; Merkel, Rudolf; Niessen, Carien M.; Pokrzywa, Wojciech; Rinschen, Markus M.; Wachten, Dagmar; Warscheid, Bettina. In EMBO reports, 22(8), p. e52507. England, 2021.
  • [ 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.
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  Quantitative Proteomics Identifies PTP1B as Modulator of B Cell Antigen Receptor Signaling. Schwarz, Jennifer J.; Grundmann, Lorenz; Kokot, Thomas; Kläsener, Kathrin; Fotteler, Sandra; Medgyesi, David; Köhn, Maja; Reth, Michael; Warscheid, Bettina. In Life science alliance, 4(11). United States, 2021.
  • [ 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.
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  Defining the Interactome of the Human Mitochondrial Ribosome Identifies SMIM4 and TMEM223 as Respiratory Chain Assembly Factors. Dennerlein, Sven; Poerschke, Sabine; Oeljeklaus, Silke; Wang, Cong; Richter-Dennerlein, Ricarda; Sattmann, Johannes; Bauermeister, Diana; Hanitsch, Elisa; Stoldt, Stefan; Langer, Thomas; Jakobs, Stefan; Warscheid, Bettina; Rehling, Peter. In eLife, 10. England, 2021.
  • [ 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.
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  Quantitative Proteomics Identifies the Universally Conserved ATPase Ola1p as a Positive Regulator of Heat Shock Response in Saccharomyces Cerevisiae. Dannenmaier, Stefan; Desroches Altamirano, Christine; Schüler, Lisa; Zhang, Ying; Hummel, Johannes; Milanov, Martin; Oeljeklaus, Silke; Koch, Hans-Georg; Rospert, Sabine; Alberti, Simon; Warscheid, Bettina. In The Journal of biological chemistry, 297(5), p. 101050. United States, 2021.
  • [ 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.
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  DNA Repair Protein APE1 Degrades Dysfunctional Abasic mRNA in Mitochondria Affecting Oxidative Phosphorylation. Barchiesi, Arianna; Bazzani, Veronica; Jabczynska, Agata; Borowski, Lukasz S.; Oeljeklaus, Silke; Warscheid, Bettina; Chacinska, Agnieszka; Szczesny, Roman J.; Vascotto, Carlo. In Journal of molecular biology, 433(18), p. 167125. Netherlands, 2021.
  • [ 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.

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  Towards the Molecular Architecture of the Peroxisomal Receptor Docking Complex. Lill, Pascal; Hansen, Tobias; Wendscheck, Daniel; Klink, Bjoern Udo; Jeziorek, Tomasz; Vismpas, Dimitrios; Miehling, Jonas; Bender, Julian; Schummer, Andreas; Drepper, Friedel; Girzalsky, Wolfgang; Warscheid, Bettina; Erdmann, Ralf; Gatsogiannis, Christos. In Proceedings of the National Academy of Sciences of the United States of America, 117(52), pp. 33216–33224. United States, 2020.
  • [ 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.
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  A Combinatorial Native MS and LC-MS/MS Approach Reveals High Intrinsic Phosphorylation of Human Tau but Minimal Levels of Other Key Modifications. Drepper, Friedel; Biernat, Jacek; Kaniyappan, Senthilvelrajan; Meyer, Helmut E.; Mandelkow, Eva Maria; Warscheid, Bettina; Mandelkow, Eckhard. In The Journal of biological chemistry, 295(52), pp. 18213–18225. United States, 2020.
  • [ 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.
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  Phosphoproteomics Identifies Dual-Site Phosphorylation in an Extended Basophilic Motif Regulating FILIP1-mediated Degradation of Filamin-C. Reimann, Lena; Schwäble, Anja N.; Fricke, Anna L.; Mühlhäuser, Wignand W. D.; Leber, Yvonne; Lohanadan, Keerthika; Puchinger, Martin G.; Schäuble, Sascha; Faessler, Erik; Wiese, Heike; Reichenbach, Christa; Knapp, Bettina; Peikert, Christian D.; Drepper, Friedel; Hahn, Udo; Kreutz, Clemens; van der Ven, Peter F. M.; Radziwill, Gerald; Djinovi’c}}-Carugo, Kristina; Fürst, Dieter O.; Warscheid, Bettina. In Communications biology, 3(1), p. 253. England, 2020.
  • [ 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.
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  COA6 Facilitates Cytochrome c Oxidase Biogenesis as Thiol-reductase for Copper Metallochaperones in Mitochondria. Pacheu-Grau, David; Wasilewski, Michal; Oeljeklaus, Silke; Gibhardt, Christine Silvia; Aich, Abhishek; Chudenkova, Margarita; Dennerlein, Sven; Deckers, Markus; Bogeski, Ivan; Warscheid, Bettina; Chacinska, Agnieszka; Rehling, Peter. In Journal of molecular biology, 432(7), pp. 2067–2079. Netherlands, 2020.
  • [ 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.
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  Defining the Substrate Spectrum of the TIM22 Complex Identifies Pyruvate Carrier Subunits as Unconventional Cargos. Gomkale, Ridhima; Cruz-Zaragoza, Luis Daniel; Suppanz, Ida; Guiard, Bernard; Montoya, Julio; Callegari, Sylvie; Pacheu-Grau, David; Warscheid, Bettina; Rehling, Peter. In Current biology : CB, 30(6), pp. 1119–1127.e5. England, 2020.
  • [ 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.
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  Homologue Replacement in the Import Motor of the Mitochondrial Inner Membrane of Trypanosomes. von Känel, Corinne; Muñoz-Gómez, Sergio A.; Oeljeklaus, Silke; Wenger, Christoph; Warscheid, Bettina; Wideman, Jeremy G.; Harsman, Anke; Schneider, Andre. In eLife, 9. England, 2020.
  • [ 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.
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  Pex14p Phosphorylation Modulates Import of Citrate Synthase 2 Into Peroxisomes in Saccharomyces Cerevisiae. Schummer, Andreas; Maier, Renate; Gabay-Maskit, Shiran; Hansen, Tobias; Mühlhäuser, Wignand W. D.; Suppanz, Ida; Fadel, Amir; Schuldiner, Maya; Girzalsky, Wolfgang; Oeljeklaus, Silke; Zalckvar, Einat; Erdmann, Ralf; Warscheid, Bettina. In Frontiers in cell and developmental biology, 8, p. 549451. Switzerland, 2020.
  • [ 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.
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  Cullin 3 Is Crucial for Pro-B Cell Proliferation, Interacts with CD22, and Controls CD22 Internalization on B Cells. Meyer, Sarah J.; Böser, Alexander; Korn, Marina A.; Koller, Claudia; Bertocci, Barbara; Reimann, Lena; Warscheid, Bettina; Nitschke, Lars. In Journal of immunology (Baltimore, Md. : 1950), 204(12), pp. 3360–3374. United States, 2020.
  • [ 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.

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  Mitochondrial Protein Translocation-Associated Degradation. Maa}rtensson, Christoph U.; Priesnitz, Chantal; Song, Jiyao; Ellenrieder, Lars; Doan, Kim Nguyen; Boos, Felix; Floerchinger, Alessia; Zufall, Nicole; Oeljeklaus, Silke; Warscheid, Bettina; Becker, Thomas. In Nature, 569(7758), pp. 679–683. England, 2019.
  • [ 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.
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  Light-Controlled Affinity Purification of Protein Complexes Exemplified by the Resting ZAP70 Interactome. Hörner, Maximilian; Eble, Julian; Yousefi, O. Sascha; Schwarz, Jennifer; Warscheid, Bettina; Weber, Wilfried; Schamel, Wolfgang W. A. In Frontiers in immunology, 10, p. 226. Switzerland, 2019.
  • [ 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.
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  The Deubiquitination of the PTS1-import Receptor Pex5p Is Required for Peroxisomal Matrix Protein Import. El Magraoui, Fouzi; Brinkmeier, Rebecca; Mastalski, Thomas; Hupperich, Alexander; Strehl, Christofer; Schwerter, Daniel; Girzalsky, Wolfgang; Meyer, Helmut E.; Warscheid, Bettina; Erdmann, Ralf; Platta, Harald W. In Biochimica et biophysica acta. Molecular cell research, 1866(2), pp. 199–213. Netherlands, 2019.
  • [ 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.
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  The Highly Diverged Trypanosomal MICOS Complex Is Organized in a Nonessential Integral Membrane and an Essential Peripheral Module. Eichenberger, Claudia; Oeljeklaus, Silke; Bruggisser, Julia; Mani, Jan; Haenni, Beat; Kaurov, Iosif; Niemann, Moritz; Zuber, Benoît; Lukev s, Julius; Hashimi, Hassan; Warscheid, Bettina; Schimanski, Bernd; Schneider, André. In Molecular microbiology, 112(6), pp. 1731–1743. England, 2019.
  • [ 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.
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  Mitochondrial Proteins: From Biogenesis to Functional Networks. Pfanner, Nikolaus; Warscheid, Bettina; Wiedemann, Nils. In Nature reviews. Molecular cell biology, 20(5), pp. 267–284. England, 2019.
  • [ 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.
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  Noncompetitive Binding of PpiD and YidC to the SecYEG Translocon Expands the Global View on the SecYEG Interactome in Escherichia Coli. Jauss, Benjamin; Petriman, Narcis-Adrian; Drepper, Friedel; Franz, Lisa; Sachelaru, Ilie; Welte, Thomas; Steinberg, Ruth; Warscheid, Bettina; Koch, Hans-Georg. In The Journal of biological chemistry, 294(50), pp. 19167–19183. United States, 2019.
  • [ 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.
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  Surface-Exposed Domains of TatB Involved in the Structural and Functional Assembly of the Tat Translocase in Escherichia Coli. Fröbel, Julia; Blümmel, Anne-Sophie; Drepper, Friedel; Warscheid, Bettina; Müller, Matthias. In The Journal of biological chemistry, 294(38), pp. 13902–13914. United States, 2019.
  • [ 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.
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  Evolutionary Divergent PEX3 Is Essential for Glycosome Biogenesis and Survival of Trypanosomatid Parasites. Kalel, Vishal C.; Li, Mengqiao; Gaussmann, Stefan; Delhommel, Florent; Schäfer, Ann-Britt; Tippler, Bettina; Jung, Martin; Maier, Renate; Oeljeklaus, Silke; Schliebs, Wolfgang; Warscheid, Bettina; Sattler, Michael; Erdmann, Ralf. In Biochimica et biophysica acta. Molecular cell research, 1866(12), p. 118520. Netherlands, 2019.
  • [ 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.

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  Quantitative Proteomics Identifies Redox Switches for Global Translation Modulation by Mitochondrially Produced Reactive Oxygen Species. Topf, Ulrike; Suppanz, Ida; Samluk, Lukasz; Wrobel, Lidia; Böser, Alexander; Sakowska, Paulina; Knapp, Bettina; Pietrzyk, Martyna K.; Chacinska, Agnieszka; Warscheid, Bettina. In Nature communications, 9(1), p. 324. England, 2018.
  • [ 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.
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  Recruitment of Cytosolic J-Proteins by TOM Receptors Promotes Mitochondrial Protein Biogenesis. Opaliński, Lukasz; Song, Jiyao; Priesnitz, Chantal; Wenz, Lena-Sophie; Oeljeklaus, Silke; Warscheid, Bettina; Pfanner, Nikolaus; Becker, Thomas. In Cell reports, 25(8), pp. 2036–2043.e5. United States, 2018.
  • [ 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.
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  The Interaction Network of the YidC Insertase with the SecYEG Translocon, SRP and the SRP Receptor FtsY. Petriman, Narcis-Adrian; Jauß, Benjamin; Hufnagel, Antonia; Franz, Lisa; Sachelaru, Ilie; Drepper, Friedel; Warscheid, Bettina; Koch, Hans-Georg. In Scientific reports, 8(1), p. 578. England, 2018.
  • [ 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.
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  Using Pull Down Strategies to Analyze the Interactome of Peroxisomal Membrane Proteins in Human Cells. Klümper, Jessica; Oeljeklaus, Silke; Warscheid, Bettina; Erdmann, Ralf; Schliebs, Wolfgang. In Sub-cellular biochemistry, 89, pp. 261–285. United States, 2018.
  • [ 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.
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  The Mitochondrial TMEM177 Associates with COX20 during COX2 Biogenesis. Lorenzi, Isotta; Oeljeklaus, Silke; Aich, Abhishek; Ronsör, Christin; Callegari, Sylvie; Dudek, Jan; Warscheid, Bettina; Dennerlein, Sven; Rehling, Peter. In Biochimica et biophysica acta. Molecular cell research, 1865(2), pp. 323–333. Netherlands, 2018.
  • [ 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.
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  Vps39 Interacts with Tom40 to Establish One of Two Functionally Distinct Vacuole-Mitochondria Contact Sites. González Montoro, Ayelén; Auffarth, Kathrin; Hönscher, Carina; Bohnert, Maria; Becker, Thomas; Warscheid, Bettina; Reggiori, Fulvio; van der Laan, Martin; Fröhlich, Florian; Ungermann, Christian. In Developmental cell, 45(5), pp. 621–636.e7. United States, 2018.
  • [ 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.
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  Complete Native Stable Isotope Labeling by Amino Acids of Saccharomyces Cerevisiae for Global Proteomic Analysis. Dannenmaier, Stefan; Stiller, Sebastian B.; Morgenstern, Marcel; Lübbert, Philipp; Oeljeklaus, Silke; Wiedemann, Nils; Warscheid, Bettina. In Analytical chemistry, 90(17), pp. 10501–10509. United States, 2018.
  • [ 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).
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  The Diverged Trypanosome MICOS Complex as a Hub for Mitochondrial Cristae Shaping and Protein Import. Kaurov, Iosif; Vancová, Marie; Schimanski, Bernd; Cadena, Lawrence Rudy; Heller, Jiv rí; Bíl’y}}, Tomáv s; Potv ev sil, David; Eichenberger, Claudia; Bruce, Hannah; Oeljeklaus, Silke; Warscheid, Bettina; Zdráhal, Zbynv ek; Schneider, André; Lukev s, Julius; Hashimi, Hassan. In Current biology : CB, 28(21), pp. 3393–3407.e5. England, 2018.
  • [ 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.
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  Defining the Mammalian Peroxisomal Proteome. Yifrach, Eden; Fischer, Sven; Oeljeklaus, Silke; Schuldiner, Maya; Zalckvar, Einat; Warscheid, Bettina. In Sub-cellular biochemistry, 89, pp. 47–66. United States, 2018.
  • [ 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.

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  Definition of a High-Confidence Mitochondrial Proteome at Quantitative Scale. Morgenstern, Marcel; Stiller, Sebastian B.; Lübbert, Philipp; Peikert, Christian D.; Dannenmaier, Stefan; Drepper, Friedel; Weill, Uri; Höß, Philipp; Feuerstein, Reinhild; Gebert, Michael; Bohnert, Maria; van der Laan, Martin; Schuldiner, Maya; Schütze, Conny; Oeljeklaus, Silke; Pfanner, Nikolaus; Wiedemann, Nils; Warscheid, Bettina. In Cell reports, 19(13), pp. 2836–2852. United States, 2017.
  • [ 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.
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  Study of Peroxisomal Protein Phosphorylation by Functional Proteomics. Schummer, Andreas; Fischer, Sven; Oeljeklaus, Silke; Warscheid, Bettina. In Methods in molecular biology (Clifton, N.J.), 1595, pp. 267–289. United States, 2017.
  • [ 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.
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  Phospho-Ubiquitin-PARK2 Complex as a Marker for Mitophagy Defects. Callegari, Sylvie; Oeljeklaus, Silke; Warscheid, Bettina; Dennerlein, Sven; Thumm, Michael; Rehling, Peter; Dudek, Jan. In Autophagy, 13(1), pp. 201–211. United States, 2017.
  • [ 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.
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  AKT-dependent Phosphorylation of the SAM Domain Induces Oligomerization and Activation of the Scaffold Protein CNK1. Fischer, Adrian; Weber, Wilfried; Warscheid, Bettina; Radziwill, Gerald. In Biochimica et biophysica acta. Molecular cell research, 1864(1), pp. 89–100. Netherlands, 2017.
  • [ 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.
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  Expanding the Archaellum Regulatory Network - the Eukaryotic Protein Kinases ArnC and ArnD Influence Motility of Sulfolobus Acidocaldarius. Hoffmann, Lena; Schummer, Andreas; Reimann, Julia; Haurat, Maria F.; Wilson, Amanda J.; Beeby, Morgan; Warscheid, Bettina; Albers, Sonja-V. In MicrobiologyOpen, 6(1). England, 2017.
  • [ 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.
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  PLK1 (Polo like Kinase 1) Inhibits MTOR Complex 1 and Promotes Autophagy. Ruf, Stefanie; Heberle, Alexander Martin; Langelaar-Makkinje, Miriam; Gelino, Sara; Wilkinson, Deepti; Gerbeth, Carolin; Schwarz, Jennifer Jasmin; Holzwarth, Birgit; Warscheid, Bettina; Meisinger, Chris; van Vugt, Marcel A. T. M.; Baumeister, Ralf; Hansen, Malene; Thedieck, Kathrin. In Autophagy, 13(3), pp. 486–505. United States, 2017.
  • [ 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.
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  Myofibrillar Z-discs Are a Protein Phosphorylation Hot Spot with Protein Kinase C ({{PKC\($\alpha$\)}}) Modulating Protein Dynamics. Reimann, Lena; Wiese, Heike; Leber, Yvonne; Schwäble, Anja N.; Fricke, Anna L.; Rohland, Anne; Knapp, Bettina; Peikert, Christian D.; Drepper, Friedel; van der Ven, Peter F. M.; Radziwill, Gerald; Fürst, Dieter O.; Warscheid, Bettina. In Molecular & cellular proteomics : MCP, 16(3), pp. 346–367. United States, 2017.
  • [ 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.
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  Mic10, a Core Subunit of the Mitochondrial Contact Site and Cristae Organizing System, Interacts with the Dimeric F(1)F(o)-ATP Synthase. Rampelt, Heike; Bohnert, Maria; Zerbes, Ralf M.; Horvath, Susanne E.; Warscheid, Bettina; Pfanner, Nikolaus; van der Laan, Martin. In Journal of molecular biology, 429(8), pp. 1162–1170. Netherlands, 2017.
  • [ 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.
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  Identification of Novel STAT6-Regulated Proteins in Mouse B Cells by Comparative Transcriptome and Proteome Analysis. Mokada-Gopal, Lavanya; Boeser, Alexander; Lehmann, Christian H. K.; Drepper, Friedel; Dudziak, Diana; Warscheid, Bettina; Voehringer, David. In Journal of immunology (Baltimore, Md. : 1950), 198(9), pp. 3737–3745. United States, 2017.
  • [ 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.
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  Charting Organellar Importomes by Quantitative Mass Spectrometry. Peikert, Christian D.; Mani, Jan; Morgenstern, Marcel; Käser, Sandro; Knapp, Bettina; Wenger, Christoph; Harsman, Anke; Oeljeklaus, Silke; Schneider, André; Warscheid, Bettina. In Nature communications, 8, p. 15272. England, 2017.
  • [ 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.
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  Corrigendum: The Non-Canonical Mitochondrial Inner Membrane Presequence Translocase of Trypanosomatids Contains Two Essential Rhomboid-like Proteins. Harsman, Anke; Oeljeklaus, Silke; Wenger, Christoph; Huot, Jonathan L.; Warscheid, Bettina; Schneider, André. In Nature communications, 8, p. 16123. England, 2017.
  • [ Abstract ]
  • [ DOI ]
  This corrects the article DOI: 10.1038/ncomms13707.
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  A Trypanosomal Orthologue of an Intermembrane Space Chaperone Has a Non-Canonical Function in Biogenesis of the Single Mitochondrial Inner Membrane Protein Translocase. Wenger, Christoph; Oeljeklaus, Silke; Warscheid, Bettina; Schneider, André; Harsman, Anke. In PLoS pathogens, 13(8), p. e1006550. United States, 2017.
  • [ 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.
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  The BTG2-PRMT1 Module Limits Pre-B Cell Expansion by Regulating the CDK4-Cyclin-D3 Complex. Dolezal, Elmar; Infantino, Simona; Drepper, Friedel; Börsig, Theresa; Singh, Aparajita; Wossning, Thomas; Fiala, Gina J.; Minguet, Susana; Warscheid, Bettina; Tarlinton, David M.; Jumaa, Hassan; Medgyesi, David; Reth, Michael. In Nature immunology, 18(8), pp. 911–920. United States, 2017.
  • [ 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.
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  tRNAs and Proteins Use the Same Import Channel for Translocation across the Mitochondrial Outer Membrane of Trypanosomes. Niemann, Moritz; Harsman, Anke; Mani, Jan; Peikert, Christian D.; Oeljeklaus, Silke; Warscheid, Bettina; Wagner, Richard; Schneider, André. In Proceedings of the National Academy of Sciences of the United States of America, 114(37), pp. E7679-E7687. United States, 2017.
  • [ 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.
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  Saccharomyces Cerevisiae Cells Lacking Pex3 Contain Membrane Vesicles That Harbor a Subset of Peroxisomal Membrane Proteins. Wróblewska, Justyna P.; Cruz-Zaragoza, Luis Daniel; Yuan, Wei; Schummer, Andreas; Chuartzman, Silvia G.; de Boer, Rinse; Oeljeklaus, Silke; Schuldiner, Maya; Zalckvar, Einat; Warscheid, Bettina; Erdmann, Ralf; van der Klei, Ida J. In Biochimica et biophysica acta. Molecular cell research, 1864(10), pp. 1656–1667. Netherlands, 2017.
  • [ 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.
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  INA Complex Liaises the F(1)F(o)-ATP Synthase Membrane Motor Modules. Naumenko, Nataliia; Morgenstern, Marcel; Rucktäschel, Robert; Warscheid, Bettina; Rehling, Peter. In Nature communications, 8(1), p. 1237. England, 2017.
  • [ 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.
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  Identification of New Channels by Systematic Analysis of the Mitochondrial Outer Membrane. Krüger, Vivien; Becker, Thomas; Becker, Lars; Montilla-Martinez, Malayko; Ellenrieder, Lars; Vögtle, F.-Nora; Meyer, Helmut E.; Ryan, Michael T.; Wiedemann, Nils; Warscheid, Bettina; Pfanner, Nikolaus; Wagner, Richard; Meisinger, Chris. In The Journal of cell biology, 216(11), pp. 3485–3495. United States, 2017.
  • [ 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.
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  Biogenesis of the Mitochondrial DNA Inheritance Machinery in the Mitochondrial Outer Membrane of Trypanosoma Brucei. Käser, Sandro; Willemin, Mathilde; Schnarwiler, Felix; Schimanski, Bernd; Poveda-Huertes, Daniel; Oeljeklaus, Silke; Haenni, Beat; Zuber, Benoît; Warscheid, Bettina; Meisinger, Chris; Schneider, André. In PLoS pathogens, 13(12), p. e1006808. United States, 2017.
  • [ 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.
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  Structural Features of the TatC Membrane Protein That Determine Docking and Insertion of a Twin-Arginine Signal Peptide. Blümmel, Anne-Sophie; Drepper, Friedel; Knapp, Bettina; Eimer, Ekaterina; Warscheid, Bettina; Müller, Matthias; Fröbel, Julia. In The Journal of biological chemistry, 292(52), pp. 21320–21329. United States, 2017.
  • [ 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.
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  Membrane Localization of Acetylated CNK1 Mediates a Positive Feedback on RAF/ERK Signaling. Fischer, Adrian; Mühlhäuser, Wignand W. D.; Warscheid, Bettina; Radziwill, Gerald. In Science advances, 3(8), p. e1700475. United States, 2017.
  • [ 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.

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  Outer Membrane Protein Functions as Integrator of Protein Import and DNA Inheritance in Mitochondria. Käser, Sandro; Oeljeklaus, Silke; T\’y}}{v c, Jiv rí; Vaughan, Sue; Warscheid, Bettina; Schneider, André. In Proceedings of the National Academy of Sciences of the United States of America, 113(31), pp. E4467–4475. United States, 2016.
  • [ 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.
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  Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry. Renz, Christian; Oeljeklaus, Silke; Grinhagens, Sören; Warscheid, Bettina; Johnsson, Nils; Gronemeyer, Thomas. In PloS one, 11(2), p. e0148340. United States, 2016.
  • [ 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.
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  Redox Activation of the Universally Conserved ATPase YchF by Thioredoxin 1. Hannemann, Liya; Suppanz, Ida; Ba, Qiaorui; MacInnes, Katherine; Drepper, Friedel; Warscheid, Bettina; Koch, Hans-Georg. In Antioxidants & redox signaling, 24(3), pp. 141–156. United States, 2016.
  • [ 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.
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  Regulation of Peroxisome Dynamics by Phosphorylation. Oeljeklaus, Silke; Schummer, Andreas; Mastalski, Thomas; Platta, Harald W.; Warscheid, Bettina. In Biochimica et biophysica acta, 1863(5), pp. 1027–1037. Netherlands, 2016.
  • [ 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.
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  Mitochondrial OXA Translocase Plays a Major Role in Biogenesis of Inner-Membrane Proteins. Stiller, Sebastian B.; Höpker, Jan; Oeljeklaus, Silke; Schütze, Conny; Schrempp, Sandra G.; Vent-Schmidt, Jens; Horvath, Susanne E.; Frazier, Ann E.; Gebert, Natalia; van der Laan, Martin; Bohnert, Maria; Warscheid, Bettina; Pfanner, Nikolaus; Wiedemann, Nils. In Cell metabolism, 23(5), pp. 901–908. United States, 2016.
  • [ 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.
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  The Hsp70 Homolog Ssb and the 14-3-3 Protein Bmh1 Jointly Regulate Transcription of Glucose Repressed Genes in Saccharomyces Cerevisiae. Hübscher, Volker; Mudholkar, Kaivalya; Chiabudini, Marco; Fitzke, Edith; Wölfle, Tina; Pfeifer, Dietmar; Drepper, Friedel; Warscheid, Bettina; Rospert, Sabine. In Nucleic acids research, 44(12), pp. 5629–5645. England, 2016.
  • [ 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.
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  Optogenetic Clustering of CNK1 Reveals Mechanistic Insights in RAF and AKT Signalling Controlling Cell Fate Decisions. Fischer, Adrian; Warscheid, Bettina; Weber, Wilfried; Radziwill, Gerald. In Scientific reports, 6, p. 38155. England, 2016.
  • [ 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.
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  Xilmass: A New Approach toward the Identification of Cross-Linked Peptides. Yilmaz, c Sule; Drepper, Friedel; Hulstaert, Niels; v Cerniv c, Mav sa; Gevaert, Kris; Economou, Anastassios; Warscheid, Bettina; Martens, Lennart; Vandermarliere, Elien. In Analytical chemistry, 88(20), pp. 9949–9957. United States, 2016.
  • [ 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.
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  Mitochondrial Protein Synthesis Adapts to Influx of Nuclear-Encoded Protein. Richter-Dennerlein, Ricarda; Oeljeklaus, Silke; Lorenzi, Isotta; Ronsör, Christin; Bareth, Bettina; Schendzielorz, Alexander Benjamin; Wang, Cong; Warscheid, Bettina; Rehling, Peter; Dennerlein, Sven. In Cell, 167(2), pp. 471–483.e10. United States, 2016.
  • [ 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.
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  A Systems Study Reveals Concurrent Activation of AMPK and mTOR by Amino Acids. Dalle Pezze, Piero; Ruf, Stefanie; Sonntag, Annika G.; Langelaar-Makkinje, Miriam; Hall, Philip; Heberle, Alexander M.; Razquin Navas, Patricia; van Eunen, Karen; Tölle, Regine C.; Schwarz, Jennifer J.; Wiese, Heike; Warscheid, Bettina; Deitersen, Jana; Stork, Björn; Fäßler, Erik; Schäuble, Sascha; Hahn, Udo; Horvatovich, Peter; Shanley, Daryl P.; Thedieck, Kathrin. In Nature communications, 7, p. 13254. England, 2016.
  • [ 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.
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  Ribosome-Associated Mba1 Escorts Cox2 from Insertion Machinery to Maturing Assembly Intermediates. Lorenzi, Isotta; Oeljeklaus, Silke; Ronsör, Christin; Bareth, Bettina; Warscheid, Bettina; Rehling, Peter; Dennerlein, Sven. In Molecular and cellular biology, 36(22), pp. 2782–2793. United States, 2016.
  • [ 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.
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  The Non-Canonical Mitochondrial Inner Membrane Presequence Translocase of Trypanosomatids Contains Two Essential Rhomboid-like Proteins. Harsman, Anke; Oeljeklaus, Silke; Wenger, Christoph; Huot, Jonathan L.; Warscheid, Bettina; Schneider, André. In Nature communications, 7, p. 13707. England, 2016.
  • [ 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.
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  Pex17p-Dependent Assembly of Pex14p/Dyn2p-subcomplexes of the Peroxisomal Protein Import Machinery. Chan, Anna; Schummer, Andreas; Fischer, Sven; Schröter, Thomas; Cruz-Zaragoza, Luis Daniel; Bender, Julian; Drepper, Friedel; Oeljeklaus, Silke; Kunau, Wolf-H.; Girzalsky, Wolfgang; Warscheid, Bettina; Erdmann, Ralf. In European journal of cell biology, 95(12), pp. 585–597. Germany, 2016.
  • [ 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.

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  Differential Tyrosine Phosphorylation Controls the Function of CNK1 as a Molecular Switch in Signal Transduction. Fischer, Adrian; Brummer, Tilman; Warscheid, Bettina; Radziwill, Gerald. In Biochimica et biophysica acta, 1853(11 Pt A), pp. 2847–2855. Netherlands, 2015.
  • [ 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.
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  Ribosome Binding Induces Repositioning of the Signal Recognition Particle Receptor on the Translocon. Kuhn, Patrick; Draycheva, Albena; Vogt, Andreas; Petriman, Narcis-Adrian; Sturm, Lukas; Drepper, Friedel; Warscheid, Bettina; Wintermeyer, Wolfgang; Koch, Hans-Georg. In The Journal of cell biology, 211(1), pp. 91–104. United States, 2015.
  • [ 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.