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• W3020222711 abstract "Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labeled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly because of the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8Δ mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase. Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labeled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly because of the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8Δ mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase. Mitochondria play key roles in energy production and metabolism of eukaryotic cells. Accordingly, mitochondrial dysfunction has been linked to a variety of human disorders and mitochondria fulfill central tasks in cell fate decisions such as the initiation of apoptosis (1Pfanner N. Warscheid B. Wiedemann N. Mitochondrial proteins: from biogenesis to functional networks.Nat. Rev. Mol. Cell Biol. 2019; 20: 267-284Crossref PubMed Scopus (138) Google Scholar, 2Palmieri F. Diseases caused by defects of mitochondrial carriers: a review.Biochim. Biophys. Acta. 2008; 1777: 564-578Crossref PubMed Scopus (171) Google Scholar, 3Alston C.L. Rocha M.C. Lax N.Z. Turnbull D.M. Taylor R.W. The genetics and pathology of mitochondrial disease.J. Pathol. 2017; 241: 236-250Crossref PubMed Scopus (136) Google Scholar, 4Martinou J.C. Youle R.J. Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics.Dev. Cell. 2011; 21: 92-101Abstract Full Text Full Text PDF PubMed Scopus (957) Google Scholar, 5Gomes L.C. Scorrano L. Mitochondrial morphology in mitophagy and macroautophagy.Biochim. Biophys. Acta. 2013; 1833: 205-212Crossref PubMed Scopus (156) Google Scholar, 6Gustafsson C.M. Falkenberg M. Larsson N.G. Maintenance and Expression of Mammalian Mitochondrial DNA.Annu. Rev. Biochem. 2016; 85: 133-160Crossref PubMed Scopus (211) Google Scholar). Hence, mitochondrial morphology, biogenesis, metabolism, and protein content have been studied extensively. Mass spectrometry (MS)-based proteomics have allowed the identification, localization, and quantitation of mitochondrial proteins in various species (7Sickmann A. Reinders J. Wagner Y. Joppich C. Zahedi R. Meyer H.E. Schonfisch B. Perschil I. Chacinska A. Guiard B. Rehling P. Pfanner N. Meisinger C. The proteome of Saccharomyces cerevisiae mitochondria.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13207-13212Crossref PubMed Scopus (671) Google Scholar, 8Reinders J. Zahedi R.P. Pfanner N. Meisinger C. Sickmann A. Toward the complete yeast mitochondrial proteome: multidimensional separation techniques for mitochondrial proteomics.J. Proteome Res. 2006; 5: 1543-1554Crossref PubMed Scopus (287) Google Scholar, 9Taylor S.W. Fahy E. Zhang B. Glenn G.M. Warnock D.E. Wiley S. Murphy A.N. Gaucher S.P. Capaldi R.A. 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The yeast mitochondrial proteome, a study of fermentative and respiratory growth.J. Biol. Chem. 2004; 279: 3956-3979Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and revealed their post-translational modifications (14Ohlmeier S. Hiltunen J.K. Bergmann U. Protein phosphorylation in mitochondria –a study on fermentative and respiratory growth of Saccharomyces cerevisiae.Electrophoresis. 2010; 31: 2869-2881Crossref PubMed Scopus (7) Google Scholar, 15Renvoise M. Bonhomme L. Davanture M. Valot B. Zivy M. Lemaire C. Quantitative variations of the mitochondrial proteome and phosphoproteome during fermentative and respiratory growth in Saccharomyces cerevisiae.J. Proteomics. 2014; 106: 140-150Crossref PubMed Scopus (0) Google Scholar). Based on these studies, mitochondria contain ∼1000 proteins in yeast (16Cherry J.M. Hong E.L. Amundsen C. Balakrishnan R. Binkley G. Chan E.T. Christie K.R. Costanzo M.C. Dwight S.S. Engel S.R. Fisk D.G. Hirschman J.E. Hitz B.C. Karra K. Krieger C.J. Miyasato S.R. Nash R.S. Park J. Skrzypek M.S. Simison M. Weng S. Wong E.D. Saccharomyces Genome Database: the genomics resource of budding yeast.Nucleic Acids Res. 2012; 40: D700-D705Crossref PubMed Scopus (931) Google Scholar) and up to 1500 in mammals (17Calvo S.E. Mootha V.K. The mitochondrial proteome and human disease.Annu. Rev. Genomics Hum. Genet. 2010; 11: 25-44Crossref PubMed Scopus (326) Google Scholar). Most mitochondrial proteins are organized into functional and metabolic networks, e.g. protein complexes. Prominent examples for this are the electron transport chain (ETC), the mitochondrial contact-site and cristae-organizing system (MICOS), the translocases of outer and inner membranes (TOM and TIM), the pyruvate dehydrogenase complex (PDH), or multienzyme complexes involved in the tricarboxylic acid cycle (TCA). The underlying protein-protein interactions have been investigated extensively by co-affinity purifications, blue native-polyacrylamide gel electrophoresis (BN-PAGE), or sucrose density gradient centrifugation, revealing large interconnected protein complexes (18Schagger H. Pfeiffer K. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria.EMBO J. 2000; 19: 1777-1783Crossref PubMed Google Scholar, 19Cruciat C.M. Brunner S. Baumann F. Neupert W. Stuart R.A. The cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supracomplex in yeast mitochondria.J. Biol. Chem. 2000; 275: 18093-18098Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 20Boekema E.J. Braun H.P. Supramolecular structure of the mitochondrial oxidative phosphorylation system.J. Biol. Chem. 2007; 282: 1-4Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 21Robinson Jr, J.B. Inman L. Sumegi B. Srere P.A. Further characterization of the Krebs tricarboxylic acid cycle metabolon.J. Biol. Chem. 1987; 262: 1786-1790Abstract Full Text PDF PubMed Google Scholar). These approaches have been complemented with more MS-focused approaches such as APEX (22Rhee H.W. Zou P. Udeshi N.D. Martell J.D. Mootha V.K. Carr S.A. Ting A.Y. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging.Science. 2013; 339: 1328-1331Crossref PubMed Scopus (509) Google Scholar), BioID (23Roux K.J. Kim D.I. Raida M. Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.J. Cell Biol. 2012; 196: 801-810Crossref PubMed Scopus (798) Google Scholar), or complexome profiling (24Wessels H.J. Vogel R.O. van den Heuvel L. Smeitink J.A. Rodenburg R.J. Nijtmans L.G. Farhoud M.H. LC-MS/MS as an alternative for SDS-PAGE in blue native analysis of protein complexes.Proteomics. 2009; 9: 4221-4228Crossref PubMed Scopus (59) Google Scholar) that expanded the number of potential protein-protein interactions, inter alia in mitochondria (25Liyanage S.U. Coyaud E. Laurent E.M. Hurren R. Maclean N. Wood S.R. Kazak L. Shamas-Din A. Holt I. Raught B. Schimmer A. Characterizing the mitochondrial DNA polymerase gamma interactome by BioID identifies Ruvbl2 localizes to the mitochondria.Mitochondrion. 2017; 32: 31-35Crossref PubMed Scopus (8) Google Scholar, 26Heide H. Bleier L. Steger M. Ackermann J. Drose S. Schwamb B. Zornig M. Reichert A.S. Koch I. Wittig I. Brandt U. Complexome profiling identifies TMEM126B as a component of the mitochondrial complex I assembly complex.Cell Metab. 2012; 16: 538-549Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 27Wessels H.J. Vogel R.O. Lightowlers R.N. Spelbrink J.N. Rodenburg R.J. van den Heuvel L.P. van Gool A.J. Gloerich J. Smeitink J.A. Nijtmans L.G. Analysis of 953 human proteins from a mitochondrial HEK293 fraction by complexome profiling.PLoS ONE. 2013; 8: e68340Crossref PubMed Scopus (35) Google Scholar). As an alternative approach, chemical cross-linking in combination with MS (XL-MS) has emerged as a powerful tool for the identification of protein-protein interactions (28Schmidt C. Urlaub H. Combining cryo-electron microscopy (cryo-EM) and cross-linking mass spectrometry (CX-MS) for structural elucidation of large protein assemblies.Curr. Opin. Struct. Biol. 2017; 46: 157-168Crossref PubMed Scopus (31) Google Scholar, 29Rappsilber J. The beginning of a beautiful friendship: cross-linking/mass spectrometry and modelling of proteins and multi-protein complexes.J. Struct. Biol. 2011; 173: 530-540Crossref PubMed Scopus (278) Google Scholar, 30Sinz A. Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions.Mass Spectrom. Rev. 2006; 25: 663-682Crossref PubMed Scopus (483) Google Scholar, 31Leitner A. Walzthoeni T. Kahraman A. Herzog F. Rinner O. Beck M. Aebersold R. Probing native protein structures by chemical cross-linking, mass spectrometry, and bioinformatics.Mol. Cell. Proteomics. 2010; 9: 1634-1649Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). Here, water-insoluble disuccinimidyl suberate (DSS) and its water-soluble derivative bis(sulfosuccinimidyl)suberate (BS3) are commonly used as protein-protein cross-linkers. Both contain N-hydroxysuccinimide (NHS) esters as bifunctional groups that react primarily with the ε-amino group of lysine residues in proteins. XL-MS allows not only the identification of cross-linked proteins that interact with each other but in addition provides the cross-linked peptides and thus topological information. Protein-protein cross-linking has mainly been applied to isolated protein complexes (32Maiolica A. Cittaro D. Borsotti D. Sennels L. Ciferri C. Tarricone C. Musacchio A. Rappsilber J. Structural analysis of multiprotein complexes by cross-linking, mass spectrometry, and database searching.Mol. Cell. Proteomics. 2007; 6: 2200-2211Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 33Herzog F. Kahraman A. Boehringer D. Mak R. Bracher A. Walzthoeni T. Leitner A. Beck M. Hartl F.U. Ban N. Malmstrom L. Aebersold R. Structural probing of a protein phosphatase 2A network by chemical cross-linking and mass spectrometry.Science. 2012; 337: 1348-1352Crossref PubMed Scopus (269) Google Scholar, 34Leitner A. Joachimiak L.A. Unverdorben P. Walzthoeni T. Frydman J. Forster F. Aebersold R. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 9455-9460Crossref PubMed Scopus (121) Google Scholar, 35Arlt C. Ihling C.H. Sinz A. Structure of full-length p53 tumor suppressor probed by chemical cross-linking and mass spectrometry.Proteomics. 2015; 15: 2746-2755Crossref PubMed Scopus (0) Google Scholar, 36Plaschka C. Larivière L. Wenzeck L. Seizl M. Hemann M. Tegunov D. Petrotchenko E.V. Borchers C.H. Baumeister W. Herzog F. Villa E. Cramer P. Architecture of the RNA polymerase II–Mediator core initiation complex.Nature. 2015; 518: 376-380Crossref PubMed Scopus (166) Google Scholar, 37Robinson P.J. Trnka M.J. Bushnell D.A. Davis R.E. Mattei P.-J. Burlingame A.L. Kornberg R.D. Structure of a complete mediator-RNA polymerase II pre-initiation complex.Cell. 2016; 166: 1411-1422.e1416Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 38Cretu C. Schmitzova J. Ponce-Salvatierra A. Dybkov O. De Laurentiis E.I. Sharma K. Will C.L. Urlaub H. Luhrmann R. Pena V. Molecular architecture of SF3b and structural consequences of its cancer-related mutations.Mol. Cell. 2016; 64: 307-319Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 39Vos S.M. Farnung L. Boehning M. Wigge C. Linden A. Urlaub H. Cramer P. Structure of activated transcription complex Pol II-DSIF-PAF-SPT6.Nature. 2018; 560: 607-612Crossref PubMed Scopus (86) Google Scholar, 40Sailer C. Offensperger F. Julier A. Kammer K.M. Walker-Gray R. Gold M.G. Scheffner M. Stengel F. Structural dynamics of the E6AP/UBE3A-E6-p53 enzyme-substrate complex.Nat. Commun. 2018; 9: 4441Crossref PubMed Scopus (15) Google Scholar), where the cross-linking results also allowed to map proteins into the respective 3D structures and to interrogate protein networks. In recent years in vivo cross-linking of bacteria, organelles or even entire eukaryotic cells has paved the way for global protein network analyses, by the application of MS-cleavable and enrichable cross-linkers (41Kaake R.M. Wang X. Burke A. Yu C. Kandur W. Yang Y. Novtisky E.J. Second T. Duan J. Kao A. Guan S. Vellucci D. Rychnovsky S.D. Huang L. A new in vivo cross-linking mass spectrometry platform to define protein-protein interactions in living cells.Mol. Cell. Proteomics. 2014; 13: 3533-3543Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 42Weisbrod C.R. Chavez J.D. Eng J.K. Yang L. Zheng C. Bruce J.E. In vivo protein interaction network identified with a novel real-time cross-linked peptide identification strategy.J. Proteome Res. 2013; 12: 1569-1579Crossref PubMed Scopus (96) Google Scholar, 43Götze M. Iacobucci C. Ihling C.H. Sinz A. A Simple Cross-Linking/Mass Spectrometry Workflow for Studying System-wide Protein Interactions.Analytical Chemistry. 2019; 91: 10236-10244Crossref PubMed Scopus (19) Google Scholar, 44Petrotchenko E.V. Serpa J.J. Borchers C.H. An isotopically coded CID-cleavable biotinylated cross-linker for structural proteomics.Mol. Cell. Proteomics. 2011; 10 (M110.001420)Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 45Kao A. Chiu C.L. Vellucci D. Yang Y. Patel V.R. Guan S. Randall A. Baldi P. Rychnovsky S.D. Huang L. Development of a novel cross-linking strategy for fast and accurate identification of cross-linked peptides of protein complexes.Mol. Cell. Proteomics. 2011; 10 (M110.002212)Abstract Full Text Full Text PDF Google Scholar, 46Liu F. Rijkers D.T. Post H. Heck A.J. Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry.Nat. Methods. 2015; 12: 1179-1184Crossref PubMed Scopus (223) Google Scholar). In previous studies mitochondria have been a target for XL-MS analyses (47Schweppe D.K. Chavez J.D. Lee C.F. Caudal A. Kruse S.E. Stuppard R. Marcinek D.J. Shadel G.S. Tian R. Bruce J.E. Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry.Proc. Natl. Acad. Sci. U.S.A. 2017; 114: 1732-1737Crossref PubMed Scopus (77) Google Scholar, 48Liu F. Lossl P. Rabbitts B.M. Balaban R.S. Heck A.J.R. The interactome of intact mitochondria by cross-linking mass spectrometry provides evidence for coexisting respiratory supercomplexes.Mol. Cell. Proteomics. 2018; 17: 216-232Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). These studies provided evidence for respirasome formation in the physiological context in mammalian mitochondria revealing supercomplexes of complex I (CI), complex III (CIII) and complex IV (CIV) of the ETC, which had been defined upon solubilization in BN-PAGE experiments (18Schagger H. Pfeiffer K. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria.EMBO J. 2000; 19: 1777-1783Crossref PubMed Google Scholar). Ryl et al. (49Ryl P.S.J. Bohlke-Schneider M. Lenz S. Fischer L. Budzinski L. Stuiver M. Mendes M.M.L. Sinn L. O'Reilly F.J. Rappsilber J. In situ structural restraints from crosslinking mass spectrometry in human mitochondria.J. Proteome Res. 2019; (https://doi.org/10.1021/acs.jproteome.9b00541)PubMed Google Scholar) emphasized the presence of a multitude of intra-cross-linked proteins in human mitochondria. In a very recent study of yeast mitochondria an enrichable, MS-cleavable and stable-isotope labeled cross-linker was used to investigate protein-protein interactions (50Makepeace K.A.T. Mohammed Y. Rudashevskaya E.L. Petrotchenko E.V. Voegtle F.N. Meisinger C. Sickmann A. Borchers C.H. Improving identification of in-organello protein-protein interactions using an affinity-enrichable, isotopically-coded, and mass spectrometry-cleavable chemical crosslinker.Mol. Cell. Proteomics. 2020; (https://doi.org/10.1074/mcp.RA119.001839)Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). Yeast is an established eukaryotic model organism for studying mitochondrial functions (51Altmann K. Durr M. Westermann B. Saccharomyces cerevisiae as a model organism to study mitochondrial biology: general considerations and basic procedures.Methods Mol. Biol. 2007; 372: 81-90Crossref PubMed Google Scholar). Its facultative anaerobic nature allows yeast cells to generate ATP through oxidative phosphorylation in the presence of oxygen, whereas in the presence of glucose ATP can be produced by fermentation (Crabtree effect (52Crabtree H.G. Observations on the carbohydrate metabolism of tumours.Biochem. J. 1929; 23: 536-545Crossref PubMed Google Scholar)). This change of metabolism affects the morphology of mitochondria in yeast (53Visser W. van Spronsen E.A. Nanninga N. Pronk J.T. Gijs Kuenen J. van Dijken J.P. Effects of growth conditions on mitochondrial morphology in Saccharomyces cerevisiae.Antonie Van Leeuwenhoek. 1995; 67: 243-253Crossref PubMed Scopus (0) Google Scholar, 54Egner A. Jakobs S. Hell S.W. Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 3370-3375Crossref PubMed Scopus (222) Google Scholar) and also the copy numbers of proteins (55Morgenstern M. Stiller S.B. Lubbert P. Peikert C.D. Dannenmaier S. Drepper F. Weill U. Hoss P. Feuerstein R. Gebert M. Bohnert M. van der Laan M. Schuldiner M. Schutze C. Oeljeklaus S. Pfanner N. Wiedemann N. Warscheid B. Definition of a high-confidence mitochondrial proteome at quantitative scale.Cell Rep. 2017; 19: 2836-2852Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) whose genes are repressed by glucose (56Entian K.D. Glucose repression: a complex regulatory system in yeast.Microbiol. Sci. 1986; 3: 366-371PubMed Google Scholar, 57Gancedo J.M. Carbon catabolite repression in yeast.Eur. J. Biochem. 1992; 206: 297-313Crossref PubMed Scopus (0) Google Scholar). Applying XL-MS to isolated yeast mitochondria makes it possible (1) to provide a protein interaction network of mitochondrial proteins including those of the outer (OM) and inner membrane (IM), intermembrane space (IMS) and matrix (M), (2) to establish a protein-protein interaction network for yeast growing on different carbon sources and (3) to identify hitherto unknown protein interactions. In this work, we cross-linked mitochondria derived from yeast grown on either a non-fermentable (glycerol) or a fermentable (glucose) carbon source with BS3. We identified 2100 unique residue-to-residue cross-links in the glycerol and 1787 cross-links in the glucose data set. Hence, we could demonstrate that a water-soluble but non-cleavable cross-linker such as BS3 is suitable for elucidating protein-protein interactions in complex samples. By applying a stable-isotope labeled cross-linking approach, we were able to quantify differences in protein-protein cross-links in mitochondria between the two growth conditions. However, the significance of this quantitative approach was limited because of high numbers of intraprotein links and different protein copy numbers induced by glucose-repression. The analysis of mitochondria derived from yeast grown on glycerol medium shows that Ndi1, the internal NADH:ubiquinone oxidoreductase, is directly associated to the CIII-CIV supercomplex of the yeast's ETC. Furthermore, we identified the so far uncharacterized protein Min8 as a new constituent of CIV. Yeast strains used in this study are derivatives of S. cerevisiae strain YPH499. Deletion of MIN8 (YPR010C-A) was achieved by homologous recombination of a HIS3MX6 cassette into the corresponding locus. Generation of HA-tagged strains was performed by chromosomal integration. Yeast cells were grown on rich medium (1% yeast extract, 2% peptone) supplemented with 2% glucose, 3% glycerol or 3% lactate. For growth tests, yeast cells from liquid cultures were adjusted to an OD600 of 1 and serial dilutions of the culture were spotted onto agar plates containing either glucose, glycerol or lactate as a carbon source and incubated at indicated temperatures. Mitochondria were isolated as previously described from yeast cells grown at 30 °C (58Meisinger C. Pfanner N. Truscott K.N. Isolation of yeast mitochondria.Methods Mol. Biol. 2006; 313: 33-39PubMed Google Scholar). Mitochondria used for mass spectrometric analyses of BS3 cross-linked proteins were further purified by tandem sucrose gradient centrifugation. Oxygen consumption was assessed using high-resolution respirometry (Oxygraph-2k, Oroboros Instruments, Innsbruck, Austria) in 2 ml of respiration buffer (225 mM Sucrose, 75 mm Mannitol, 10 mm Tris, 10 mm KH2PO4, 5 mm MgCl2, 10 mm KCl, pH 7.4) at 30 °C. Non-phosphorylating respiration (LEAK) was addressed using pyruvate (10 mm) and malate (2 mm). Adding ADP in a saturating concentration (1 mm, State3) followed by succinate (10 mm) determines the maximal capacity for oxidative phosphorylation (OXPHOS). Respiration was killed by addition of Antimycin A (5 μM) to prevent electron transfer from CIII to CIV and ascorbate (2 mM) followed by TMPD (N,N,N′,N′-Tetramethyl-p-phenylenediamine; 500 μM) were added to address OXPHOS capacity via CIV. To distinguish between respiration and auto-oxidation of TMPD/ascorbate, NaN3 (100 mM) was added to block the O2 binding site of CIV and the values were subtracted from the values after TMPD/ascorbate addition. Cox13 and Cox12 were amplified using appropriate primers including a 3′ overhang for SP6 recognition. PCR products were used for in vitro transcription. Respective proteins were synthesized in rabbit reticulocyte lysate (Promega, Madison, Wisconsin) in the presence of [35S] methionine. For assembly analyses, isolated mitochondria were incubated with radiolabeled proteins in import buffer (250 mm sucrose, 10 mm MOPS/KOH pH 7.2, 80 mm KCl, 2 mm KH2PO4, 5 mm MgCl2, 5 mm methionine, and 3% fatty acid-free BSA; import buffer for Cox12 without BSA), supplemented with 5 mm creatine phosphate and 0.1 mg/ml creatine kinase, in the presence of 2 mm ATP and 2 mm NADH for indicated times. Cox13 reactions were stopped on ice by dissipation of the membrane potential with 8 μm Antimycin A, 1 μm Valinomycin and 20 μm Oligomycin. For Cox12 assembly experiments the reactions were stopped by adding 50 mm iodoacetamide (IAA). All Samples were lysed in 0.6% n-Dodecyl β-d-maltoside (DDM) buffer for BN-PAGE. Isolation of cross-linked adducts were performed in WT and Min8HA expressing strains. For co-immunoprecipitation, Cox12-specific antisera as well as non-binding Pam18-antisera were bound to protein A-Sepharose (GE Healthcare, Chicago, Illinois). BS3 cross-linked and non-treated mitochondria were lysed in 20 mm Tris (pH 7.4), 80 mm NaCl, 0.5 mm EDTA, 0.5% Triton X-100, 0.1% SDS, 10% glycerol and 1 mm PMSF for 1 h at 4 °C. Lysates were cleared at 20,000 × g for 15 min at 4 °C and total samples were taken. Solubilized proteins were incubated for binding with Cox12, Pam18 and HA antibody-columns for 1 h at 4 °C. After intensive washing, the bound proteins were eluted by the application of 0.1 m glycine pH 2.8 and further analyzed by SDS-PAGE and Western blotting. Purified mitochondria of each condition were aliquoted in 1 mg portions and stored at −80 °C before use. Aliquots were thawed gently and spun down with 10,000 × g for 5 min at 4 °C. Bis(sulfosuccinimidyl)suberate (BS3, non-weight format, Thermo Fisher Scientific, Waltham, Massachusetts) was used as chemical cross-linker and resuspended in cross-linking buffer (10 mm HEPES, pH 7.5, 100 mm NaCl) to a concentration of 100 mm. After diluting the BS3 stock solution to 5 mm, pelleted mitochondria were resuspended in 200 μl cross-linking buffer including BS3 and incubated for 1 h at room temperature (RT) with gentle rotation. The reaction was quenched, and mitochondria were lysed by adding Tris, pH 8, to a final concentration of 50 mm and SDS to 2%. Proteins were boiled at 70 °C for 10 min and after chilling to room temperature precipitated by adding ice-cold acetone four times the sample volume, incubated at −80 °C for 2 h. This was repeated once with different isolations of mitochondria, resulting in two biological replicates per condition. For the comparison of BS3 with disuccinimidyl suberate (DSS), freshly prepared crude mitochondrial extract derived from yeast grown on glycerol medium was treated as described above with the following changes: isotopically labeled BS3-d4 (non-weight format, Thermo Fisher Scientific) and DSS (non-weight format, Thermo Fisher Scientific, resuspended in DMSO) were mixed in an equimolar ratio and used for the cross-linking reaction. The final concentration of each cross-linker was 1 mm. For the quantitation, purified mitochondria from yeast grown on glycerol and glucose, respectively, were cross-linked with BS3 and isotopically labeled BS3-d4 (non-weight format, Thermo Fisher Scientific) in a label-swap experiment. Therefore, 0.5 mg of each condition were cross-linked with either 5 mm BS3 or BS3-d4 as described above. After quenching the reaction, both samples were mixed in a 1:1 ratio according to protein amount (calculated by Pierce BCA Protein Assay Kit, Thermo Fisher Scientific) and the already mentioned lysis procedure was applied with the following changes: lysis was performed with 8 m urea without heating and supported by sonication (diagenode: Liège, Belgium Bioruptor, 3 × 30 s, 4 °C). Proteins were not precipitated. The label-swap experiment was repeated once, resulting in four replicates. Precipitated proteins were resuspended in 50 μl 8 m urea/50 mm ammonium bicarbonate, pH 8 (for the qualitative data s" @default.
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• W3020222711 date "2020-07-01" @default.
• W3020222711 modified "2023-10-16" @default.
• W3020222711 title "A Cross-linking Mass Spectrometry Approach Defines Protein Interactions in Yeast Mitochondria" @default.
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