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• W1977320434 abstract "The enzyme complexes involved in mitochondrial oxidative phosphorylation are organized into higher ordered assemblies termed supercomplexes. Subunits e and g (Su e and Su g, respectively) are catalytically nonessential subunits of the F1F0-ATP synthase whose presence is required to directly support the stable dimerization of the ATP synthase complex. We report here that Su g and Su e are also important for securing the correct organizational state of the cytochrome bc1-cytochrome oxidase (COX) supercomplex. Mitochondria isolated from the Δsu e and Δsu g null mutant strains exhibit decreased levels of COX enzyme activity but appear to have normal COX subunit protein levels. An altered stoichiometry of the cytochrome bc1-COX supercomplex was observed in mitochondria deficient in Su e and/or Su g, and a perturbation in the association of Cox4, a catalytically important subunit of the COX complex, was also detected. In addition, an increase in the level of the TIM23 translocase associated with the cytochrome bc1-COX supercomplex is observed in the absence of Su e and Su g. Together, our data highlight that a further level of complexity exists between the oxidative phosphorylation supercomplexes, whereby the organizational state of one complex, i.e. the ATP synthase, may influence that of another supercomplex, namely the cytochrome bc1-COX complex. The enzyme complexes involved in mitochondrial oxidative phosphorylation are organized into higher ordered assemblies termed supercomplexes. Subunits e and g (Su e and Su g, respectively) are catalytically nonessential subunits of the F1F0-ATP synthase whose presence is required to directly support the stable dimerization of the ATP synthase complex. We report here that Su g and Su e are also important for securing the correct organizational state of the cytochrome bc1-cytochrome oxidase (COX) supercomplex. Mitochondria isolated from the Δsu e and Δsu g null mutant strains exhibit decreased levels of COX enzyme activity but appear to have normal COX subunit protein levels. An altered stoichiometry of the cytochrome bc1-COX supercomplex was observed in mitochondria deficient in Su e and/or Su g, and a perturbation in the association of Cox4, a catalytically important subunit of the COX complex, was also detected. In addition, an increase in the level of the TIM23 translocase associated with the cytochrome bc1-COX supercomplex is observed in the absence of Su e and Su g. Together, our data highlight that a further level of complexity exists between the oxidative phosphorylation supercomplexes, whereby the organizational state of one complex, i.e. the ATP synthase, may influence that of another supercomplex, namely the cytochrome bc1-COX complex. ATP is produced within mitochondria through the oxygen-consuming process termed oxidative phosphorylation (OXPHOS). 2The abbreviations used are:OXPHOSoxidative phosphorylationCOXcytochrome oxidaseWTwild typeCN-PAGEclear native-PAGEBN-PAGEblue native-PAGE.2The abbreviations used are:OXPHOSoxidative phosphorylationCOXcytochrome oxidaseWTwild typeCN-PAGEclear native-PAGEBN-PAGEblue native-PAGE. The protein complexes involved in the mitochondrial OXPHOS pathway are large multisubunit enzymes, commonly referred to as complexes I–V. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome bc1-complex (or cytochrome c reductase)), and complex IV (cytochrome c oxidase, COX) compose the electron transport chain complexes and the F1F0-ATP synthase complex, which directly synthesizes ATP and is often referred to as complex V (1Saraste M. Science. 1999; 283: 1488-1493Crossref PubMed Scopus (1011) Google Scholar, 2Ackerman S.H. Tzagoloff A. Prog. Nucleic Acids Res. Mol. Biol. 2005; 80: 95-133Crossref PubMed Scopus (103) Google Scholar, 3Smeitink J.A. Zeviani M. Turnbull D.M. Jacobs H.T. Cell Metab. 2006; 3: 9-13Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Although complexes I–V can be detergent-solubilized and purified from the mitochondrial membranes as independent enzymatically active complexes, recent findings indicate that these complexes do not exist as physically separate entities within the mitochondrial inner membrane. Rather subpopulations of OXPHOS complexes physically associate with each other to form larger assemblies termed “OXPHOS supercomplexes” (4Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 5Boumans H. Berden J.A. Grivell L.A. Dam van K. Biochem. J. 1998; 331: 877-883Crossref PubMed Scopus (14) Google Scholar, 6Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schägger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (360) Google Scholar, 7Spannagel C. Vaillier J. Arselin G. Graves P.V. Grandier-Vazeille X. Velours J. Biochim. Biophys. Acta. 1998; 1414: 260-264Crossref PubMed Scopus (47) Google Scholar, 8Cruciat C. Brunner S. Baumann F. Neupert W. Stuart R.A. J. Biol. Chem. 2000; 275: 18093-18098Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 9Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schägger H. J. Biol. Chem. 2003; 278: 52873-52880Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar, 10Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1001) Google Scholar, 11Schägger H. IUBMB Life. 2001; 52: 119-128Crossref PubMed Scopus (162) Google Scholar, 12Schägger H. Biochim. Biophys. Acta. 2002; 1555: 154-159Crossref PubMed Scopus (301) Google Scholar, 13Dudkina N.V. Eubel H. Keegstra W. Boekema E.J. Braun H.P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3225-3229Crossref PubMed Scopus (261) Google Scholar, 14McKenzie M. Lazarou M. Thorburn D.R. Ryan M.T. J. Mol. Biol. 2006; 361: 462-469Crossref PubMed Scopus (317) Google Scholar, 15Dudkina N.V. Heinemeyer J. Sunderhaus S. Boekema E.J. Braun H.P. Trends Plant Sci. 2006; 11: 232-240Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 16Heinemeyer J. Braun H.P. Boekema E.J. Kouril R. J. Biol. Chem. 2007; 282: 12240-12248Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 17Boekema E.J. Braun H.P. J. Biol. Chem. 2007; 282: 1-4Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Evidence for the formation of complex III–IV supercomplex (termed here the cytochrome bc1-COX supercomplex) and complex I–III–IV supercomplex, as well as dimeric/oligomeric F1F0-ATP synthase complexes, has been reported (4Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 5Boumans H. Berden J.A. Grivell L.A. Dam van K. Biochem. J. 1998; 331: 877-883Crossref PubMed Scopus (14) Google Scholar, 6Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schägger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (360) Google Scholar, 7Spannagel C. Vaillier J. Arselin G. Graves P.V. Grandier-Vazeille X. Velours J. Biochim. Biophys. Acta. 1998; 1414: 260-264Crossref PubMed Scopus (47) Google Scholar, 8Cruciat C. Brunner S. Baumann F. Neupert W. Stuart R.A. J. Biol. Chem. 2000; 275: 18093-18098Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 9Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schägger H. J. Biol. Chem. 2003; 278: 52873-52880Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar, 10Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1001) Google Scholar, 11Schägger H. IUBMB Life. 2001; 52: 119-128Crossref PubMed Scopus (162) Google Scholar, 12Schägger H. Biochim. Biophys. Acta. 2002; 1555: 154-159Crossref PubMed Scopus (301) Google Scholar, 13Dudkina N.V. Eubel H. Keegstra W. Boekema E.J. Braun H.P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3225-3229Crossref PubMed Scopus (261) Google Scholar, 14McKenzie M. Lazarou M. Thorburn D.R. Ryan M.T. J. Mol. Biol. 2006; 361: 462-469Crossref PubMed Scopus (317) Google Scholar, 15Dudkina N.V. Heinemeyer J. Sunderhaus S. Boekema E.J. Braun H.P. Trends Plant Sci. 2006; 11: 232-240Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 16Heinemeyer J. Braun H.P. Boekema E.J. Kouril R. J. Biol. Chem. 2007; 282: 12240-12248Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 17Boekema E.J. Braun H.P. J. Biol. Chem. 2007; 282: 1-4Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). oxidative phosphorylation cytochrome oxidase wild type clear native-PAGE blue native-PAGE. oxidative phosphorylation cytochrome oxidase wild type clear native-PAGE blue native-PAGE. Formation of OXPHOS supercomplexes in mitochondria has been conserved throughout evolution, and determination of their functional relevance represents a new developing area of mitochondrial biogenesis research. The supercomplex organizational state of the OXPHOS complexes may confer an enzymatic advantage to these complexes as it has been proposed to enable direct substrate channeling between the complexes (4Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 5Boumans H. Berden J.A. Grivell L.A. Dam van K. Biochem. J. 1998; 331: 877-883Crossref PubMed Scopus (14) Google Scholar, 12Schägger H. Biochim. Biophys. Acta. 2002; 1555: 154-159Crossref PubMed Scopus (301) Google Scholar, 17Boekema E.J. Braun H.P. J. Biol. Chem. 2007; 282: 1-4Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). In addition the supercomplex organizational state of the ATP synthase plays a role in establishing the architecture of the mitochondrial inner membrane, the principal site of the OXPHOS activity (18Paumard P. Vaillier J. Coulary B. Schaeffer J. Soubannier V. Mueller D.M. Brethes D. Rago di J.P. Velours J. EMBO J. 2002; 21: 221-230Crossref PubMed Scopus (577) Google Scholar, 19Giraud M.F. Paumard P. Soubannier V. Vaillier J. Arselin G. Salin B. Schaeffer J. Brethes D. Rago di J.P. Velours J. Biochim. Biophys. Acta. 2002; 1555: 174-180Crossref PubMed Scopus (97) Google Scholar, 20Gavin P.D. Prescott M. Luff S.E. Devenish R.J. J. Cell Sci. 2004; 117: 2333-2343Crossref PubMed Scopus (63) Google Scholar, 21Arselin G. Vaillier J. Salin B. Schaeffer J. Giraud M.F. Dautant A. Brethes D. Velours J. J. Biol. Chem. 2004; 279: 40392-40399Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 22Allen R.D. Protoplasma. 1995; 189: 1-8Crossref Scopus (90) Google Scholar, 23Everard-Gigot V. Dunn C.D. Dolan B.M. Brunner S. Jensen R.E. Stuart R.A. Eukaryot. Cell. 2005; 4: 346-355Crossref PubMed Scopus (54) Google Scholar, 24Bornhovd C. Vogel F. Neupert W. Reichert A.S. J. Biol. Chem. 2006; 281: 13990-13998Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 25Gilkerson R.W. Selker J.M.L. Capaldi R.A. FEBS Lett. 2003; 546: 355-358Crossref PubMed Scopus (211) Google Scholar, 26Wurm C.A. Jakobs S. FEBS Lett. 2006; 580: 5628-5634Crossref PubMed Scopus (95) Google Scholar, 27Vogel F. Bornhovd C. Neupert W. Reichert A.S. J. Cell Biol. 2006; 175: 237-247Crossref PubMed Scopus (282) Google Scholar). Furthermore, a subset of the TIM23 translocase, involved in the import of nuclearly encoded proteins across the inner membrane, has been shown to exist in association with a subpopulation of the cytochrome bc1-COX supercomplex (28der Laan van M. Wiedemann N. Mick D.U. Guiard B. Rehling P. Pfanner N. Curr. Biol. 2006; 16: 2271-2276Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Finally, it has recently been demonstrated that a subpopulation of the cytochrome bc1-COX supercomplex can be found in association with the assembly factors Cox14 and Shy1, which are involved in the biogenesis of the COX complex (29Mick D.U. Wagner K. der Laan van M. Frazier A.E. Perschil I. Pawlas M. Meyer H.E. Warscheid B. Rehling P. EMBO J. 2007; 26: 4347-4358Crossref PubMed Scopus (104) Google Scholar). Thus it appears that a heterogeneity exists within populations of the cytochrome bc1-COX supercomplex and that different organizational states of these OXPHOS complexes may exist within one given mitochondrial type, thereby adding to the complexity of analyzing and understanding the role(s) of the OXPHOS supercomplex assembly states. The F1F0-ATP synthase complexes exist as higher ordered structures that, under mild detergent conditions, can be solubilized from mitochondrial membranes as dimeric ATP synthase complexes. To date, the dimeric assembly state of the ATP synthase has been best characterized from the yeast Saccharomyces cerevisiae, where it has been proposed that the ATP synthase dimers are solubilized from a larger network of ATP synthase oligomers within the mitochondrial inner membrane (6Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schägger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (360) Google Scholar, 7Spannagel C. Vaillier J. Arselin G. Graves P.V. Grandier-Vazeille X. Velours J. Biochim. Biophys. Acta. 1998; 1414: 260-264Crossref PubMed Scopus (47) Google Scholar, 10Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1001) Google Scholar, 18Paumard P. Vaillier J. Coulary B. Schaeffer J. Soubannier V. Mueller D.M. Brethes D. Rago di J.P. Velours J. EMBO J. 2002; 21: 221-230Crossref PubMed Scopus (577) Google Scholar, 19Giraud M.F. Paumard P. Soubannier V. Vaillier J. Arselin G. Salin B. Schaeffer J. Brethes D. Rago di J.P. Velours J. Biochim. Biophys. Acta. 2002; 1555: 174-180Crossref PubMed Scopus (97) Google Scholar, 20Gavin P.D. Prescott M. Luff S.E. Devenish R.J. J. Cell Sci. 2004; 117: 2333-2343Crossref PubMed Scopus (63) Google Scholar, 21Arselin G. Vaillier J. Salin B. Schaeffer J. Giraud M.F. Dautant A. Brethes D. Velours J. J. Biol. Chem. 2004; 279: 40392-40399Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 22Allen R.D. Protoplasma. 1995; 189: 1-8Crossref Scopus (90) Google Scholar, 23Everard-Gigot V. Dunn C.D. Dolan B.M. Brunner S. Jensen R.E. Stuart R.A. Eukaryot. Cell. 2005; 4: 346-355Crossref PubMed Scopus (54) Google Scholar, 24Bornhovd C. Vogel F. Neupert W. Reichert A.S. J. Biol. Chem. 2006; 281: 13990-13998Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 30Devenish R.J. Prescott M. Roucou X. Nagley P. Biochim. Biophys. Acta. 2000; 1458: 428-442Crossref PubMed Scopus (80) Google Scholar, 31Gavin P.D. Devenish R.J. Prescott M. Biochim. Biophys. Acta. 2003; 1607: 167-179Crossref PubMed Scopus (26) Google Scholar, 32Arnold I. Bauer M.F. Brunner M. Neupert W. Stuart R.A. FEBS Lett. 1997; 411: 195-200Crossref PubMed Scopus (69) Google Scholar, 33Brunner S. Everard-Gigot V. Stuart R.A. J. Biol. Chem. 2002; 277: 48484-48489Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 34Dienhart M. Pfeiffer K. Schägger H. Stuart R.A. J. Biol. Chem. 2002; 277: 39289-39295Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 35Arselin G. Giraud M.F. Dautant A. Vaillier J. Brethes D. Coulary-Salin B. Schaeffer J. Velours J. Eur. J. Biochem. 2003; 270: 1875-1884Crossref PubMed Scopus (111) Google Scholar, 36Soubannier V. Vaillier J. Paumard P. Coulary B. Schaeffer J. Velours J. J. Biol. Chem. 2002; 277: 10739-10745Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 37Saddar S. Stuart R.A. J. Biol. Chem. 2005; 280: 24435-24442Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 38Bustos D.M. Velours J. J. Biol. Chem. 2005; 280: 29004-29010Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The F1F0-ATP synthase complex can be divided into two regions, the membrane-embedded, H+-pumping F0 sector and the hydrophilic, catalytic F1 sector located in the mitochondrial matrix (2Ackerman S.H. Tzagoloff A. Prog. Nucleic Acids Res. Mol. Biol. 2005; 80: 95-133Crossref PubMed Scopus (103) Google Scholar). Dimerization of the ATP synthase complex involves F0-F0 interactions and in a manner involves an interface formed by the F0 sector subunits e, g, and 4 (Su e, Su g, and Atp4, respectively). Su e and Su g proteins (in contrast to Atp4) are not essential for the enzymatic activity of the ATP synthase complex, but like Atp4, Su e and Su g are required to support the stable dimerization of the ATP synthase complex. Current evidence indicates that in the absence of Su e and/or Su g, the ATP synthase dimers can still form in the mitochondrial membrane but that these dimers are not stable under detergent solubilization conditions; hence, monomeric complexes are predominantly observed in extracts of Δsu e and Δsu g mitochondria (30Devenish R.J. Prescott M. Roucou X. Nagley P. Biochim. Biophys. Acta. 2000; 1458: 428-442Crossref PubMed Scopus (80) Google Scholar, 31Gavin P.D. Devenish R.J. Prescott M. Biochim. Biophys. Acta. 2003; 1607: 167-179Crossref PubMed Scopus (26) Google Scholar, 32Arnold I. Bauer M.F. Brunner M. Neupert W. Stuart R.A. FEBS Lett. 1997; 411: 195-200Crossref PubMed Scopus (69) Google Scholar, 33Brunner S. Everard-Gigot V. Stuart R.A. J. Biol. Chem. 2002; 277: 48484-48489Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 34Dienhart M. Pfeiffer K. Schägger H. Stuart R.A. J. Biol. Chem. 2002; 277: 39289-39295Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 35Arselin G. Giraud M.F. Dautant A. Vaillier J. Brethes D. Coulary-Salin B. Schaeffer J. Velours J. Eur. J. Biochem. 2003; 270: 1875-1884Crossref PubMed Scopus (111) Google Scholar, 36Soubannier V. Vaillier J. Paumard P. Coulary B. Schaeffer J. Velours J. J. Biol. Chem. 2002; 277: 10739-10745Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 37Saddar S. Stuart R.A. J. Biol. Chem. 2005; 280: 24435-24442Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). As indicated earlier, the dimeric/oligomeric state of the ATP synthase plays a role in the development of the cristae membrane architecture (18Paumard P. Vaillier J. Coulary B. Schaeffer J. Soubannier V. Mueller D.M. Brethes D. Rago di J.P. Velours J. EMBO J. 2002; 21: 221-230Crossref PubMed Scopus (577) Google Scholar, 19Giraud M.F. Paumard P. Soubannier V. Vaillier J. Arselin G. Salin B. Schaeffer J. Brethes D. Rago di J.P. Velours J. Biochim. Biophys. Acta. 2002; 1555: 174-180Crossref PubMed Scopus (97) Google Scholar, 20Gavin P.D. Prescott M. Luff S.E. Devenish R.J. J. Cell Sci. 2004; 117: 2333-2343Crossref PubMed Scopus (63) Google Scholar, 21Arselin G. Vaillier J. Salin B. Schaeffer J. Giraud M.F. Dautant A. Brethes D. Velours J. J. Biol. Chem. 2004; 279: 40392-40399Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 22Allen R.D. Protoplasma. 1995; 189: 1-8Crossref Scopus (90) Google Scholar, 23Everard-Gigot V. Dunn C.D. Dolan B.M. Brunner S. Jensen R.E. Stuart R.A. Eukaryot. Cell. 2005; 4: 346-355Crossref PubMed Scopus (54) Google Scholar, 24Bornhovd C. Vogel F. Neupert W. Reichert A.S. J. Biol. Chem. 2006; 281: 13990-13998Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 25Gilkerson R.W. Selker J.M.L. Capaldi R.A. FEBS Lett. 2003; 546: 355-358Crossref PubMed Scopus (211) Google Scholar, 26Wurm C.A. Jakobs S. FEBS Lett. 2006; 580: 5628-5634Crossref PubMed Scopus (95) Google Scholar, 27Vogel F. Bornhovd C. Neupert W. Reichert A.S. J. Cell Biol. 2006; 175: 237-247Crossref PubMed Scopus (282) Google Scholar). Cristae membranes have been reported to be absent in the su e and su g null yeast mutants and instead the inner membrane has been shown to adopt a proliferated “onion-like” morphology (18Paumard P. Vaillier J. Coulary B. Schaeffer J. Soubannier V. Mueller D.M. Brethes D. Rago di J.P. Velours J. EMBO J. 2002; 21: 221-230Crossref PubMed Scopus (577) Google Scholar, 19Giraud M.F. Paumard P. Soubannier V. Vaillier J. Arselin G. Salin B. Schaeffer J. Brethes D. Rago di J.P. Velours J. Biochim. Biophys. Acta. 2002; 1555: 174-180Crossref PubMed Scopus (97) Google Scholar, 20Gavin P.D. Prescott M. Luff S.E. Devenish R.J. J. Cell Sci. 2004; 117: 2333-2343Crossref PubMed Scopus (63) Google Scholar). A number of reports in the literature have implicated that the assembly and/or stability of the cytochrome oxidase (COX) complex may be related to that of the ATP synthase complex (39Paul M.F. Velours J. de Chateaubodeau Arselin G. Aigle M. Guerin B. Eur. J. Biochem. 1989; 185: 163-171Crossref PubMed Scopus (80) Google Scholar, 40Rak M. Tetaud E. Duvezin-Caubet S. Ezkurdia N. Bietenhader M. Rytka J. Rago di J.P. J. Biol. Chem. 2007; 282: 34039-34047Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 41Rak M. Tetaud E. Godard F. Sagot I. Salin B. Duvezin-Caubet S. Slonimski P.P. Rytka J. Rago di J.P. J. Biol. Chem. 2007; 282: 10853-10864Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 42Boyle G.M. Roucou X. Nagley P. Devenish R.J. Prescott M. Eur. J. Biochem. 1999; 262: 315-323Crossref PubMed Scopus (47) Google Scholar). As ATP synthase assembly mutants often exhibit instability in their mtDNA (and hence coding capacity for key COX subunits), it could be argued that the reduced COX activity could merely be due to partial loss of the mtDNA in these strains. Reduced COX enzyme activity, however, was also recently reported in atp6 mutants, where retention of the mtDNA had been ensured by growing the cells under appropriate selective pressure, thereby suggesting that loss of the ATP synthase complex and/or enzyme activity can negatively impact the COX complex in a direct manner (40Rak M. Tetaud E. Duvezin-Caubet S. Ezkurdia N. Bietenhader M. Rytka J. Rago di J.P. J. Biol. Chem. 2007; 282: 34039-34047Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 41Rak M. Tetaud E. Godard F. Sagot I. Salin B. Duvezin-Caubet S. Slonimski P.P. Rytka J. Rago di J.P. J. Biol. Chem. 2007; 282: 10853-10864Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Furthermore, the F0 sector subunit Su g has also been reported to be required for maximal COX enzyme activity despite the fact that spectral analysis indicated that Δsu g mitochondria display a normal cytochrome content (42Boyle G.M. Roucou X. Nagley P. Devenish R.J. Prescott M. Eur. J. Biochem. 1999; 262: 315-323Crossref PubMed Scopus (47) Google Scholar). As Su g is not an essential subunit of the ATP synthase complex, this observation also suggests that the Su e/Su g proteins themselves, rather than the enzymatic activity of the ATP synthase complex, may directly impact the enzymatic activity of the COX complex. In this study, we have further pursued the relationship between the ATP synthase dimer-specific subunits, Su g and Su e, and the COX complex. We demonstrate here that the presence of both Su g and Su e are directly required for the establishment of maximal COX enzyme activity. Furthermore, in the absence of Su g and Su e, we observed an alteration in the assembly state of the cytochrome bc1 complex, whereby the stoichiometry of the cytochrome bc1-COX complex was altered, and free cytochrome bc1 complexes were observed. In addition, an increased level of cytochrome bc1-COX complexes associated with the TIM23 translocase of the inner membrane was also observed in the absence of Su g and/or Su e. Taken together our findings indicate that the absence of Su g and Su e not only affects the dimeric state of the ATP synthase but also causes an imbalance in the higher ordered organizational states of other OXPHOS supercomplexes and their association with the TIM23 machinery. Yeast Strains and Growth Conditions—S. cerevisiae strains used in this study are wild type (WT) (W303-1A, Mata, leu2, trp1, ura3, his3, ade2), Δsu g (W303-1A, ATP20::HIS3), Δsu e (W303-1A, TIM11::HIS3), Δcox4 (W303-1A, COX4::TRP1), Δcyt1 (W303-1A, CYT1::HIS3), Δimp1 (W303-1A, IMP1::HIS3), Δsdh2 (BY4739α, SDH2::KANr) and Δsdh4 (BY4739α, SDH4::KANr) (6Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schägger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (360) Google Scholar, 8Cruciat C. Brunner S. Baumann F. Neupert W. Stuart R.A. J. Biol. Chem. 2000; 275: 18093-18098Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 32Arnold I. Bauer M.F. Brunner M. Neupert W. Stuart R.A. FEBS Lett. 1997; 411: 195-200Crossref PubMed Scopus (69) Google Scholar). Yeast strains were maintained and cultured using standard protocols at 30 °C on YP-Gly (YP, 0.5% lactate media supplemented with 3% glycerol) (WT, Δsu g, and Δsu e) or YP-Gal (YP, 0.5% lactate media supplemented with 2% galactose) (Δcox 4, Δcyt1, Δimp1, Δsdh2 and Δsdh4) as indicated. Rho0/rho– Cell Conversion Assay—Rho0/rho– assay was performed as described earlier (23Everard-Gigot V. Dunn C.D. Dolan B.M. Brunner S. Jensen R.E. Stuart R.A. Eukaryot. Cell. 2005; 4: 346-355Crossref PubMed Scopus (54) Google Scholar). Briefly, yeast strains were grown on YP-lactate medium supplemented with 3% glycerol or 2% galactose overnight at 30 °C. The following day, equal numbers of cells were plated from each strain onto YPD (2% glucose) plates or YP-Gly plates containing 0.1% galactose. Following incubation at 30 °C, the colonies were counted, and the number of rho0/– cells (i.e. petite cells) was calculated and expressed as a percentage of total cells. Clear Native-Gel Electrophoresis—CN-PAGE analysis of the F1F0-ATP synthase and cytochrome bc1-COX supercomplexes was performed essentially as described previously (23Everard-Gigot V. Dunn C.D. Dolan B.M. Brunner S. Jensen R.E. Stuart R.A. Eukaryot. Cell. 2005; 4: 346-355Crossref PubMed Scopus (54) Google Scholar, 37Saddar S. Stuart R.A. J. Biol. Chem. 2005; 280: 24435-24442Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Mitochondria (200 μg of protein) were solubilized in lysis buffer (34 mm potassium acetate, 34 mm HEPES-KOH, pH 7.4, 11.4% glycerol, and 1 mm phenylmethylsulfonyl fluoride) with digitonin for 30 min on ice and then were subjected to a clarifying spin of 30,000 × g for 30 min at 4 °C. The supernatant from each sample was analyzed on a 3.5–10% gradient gel, Western-blotted, and immunedecorated with subunit-specific antibodies as indicated. Two-dimensional Blue Native-SDS-PAGE—Mitochondria were solubilized with 0.5% digitonin and analyzed by blue native (BN)-gel electrophoresis as described previously (6Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schägger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (360) Google Scholar). Following BN-PAGE, the individual gel strips were excised and equilibrated with denaturing solution (1% (w/v) SDS, 1% β-mercaptoethanol) for 30 min at room temperature. The excised gel strip was then inserted on top of an SDS-polyacrylamide gel, sealed with agarose solution (0.7% (w/v) agarose, 0.5% (w/v) SDS, and 15 mm β-mercaptoethanol), and electrophoresed in the second dimension. Followed by Western blotting, the nitrocellulose membrane was immunedecorated with subunit-specific antibodies as indicated. Gel Filtration Analysis—Isolated mitochondria (1 mg of protein) were solubilized in 1% Triton X-100, 150 mm NaCl, 20 mm HEPES-KOH, pH 7.2, 2 mm phenylmethylsulfonyl fluoride buffer. Following a clarifying spin, the detergent extract was applied to a Superose 6 FPLC gel filtration column (25-ml column volume). Fractions (0.3 ml) were collected, precipitated with trichloroacetic acid, and analyzed by SDS-PAGE, Western blotting, and immunedecoration with Cox2- and Cox4-specific antisera. Miscellaneous—Mitochondrial isolation, protein determinations, and SDS-PAGE were performed according to published methods (43Herrmann J.M. Fölsch H. Neupert W. Stuart R.A. Celis J.E. Cell Biology: A Laboratory Handbook. Academic Press, San Diego1994: 538-544Google Scholar, 44Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar, 45Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Cytochrome c oxidase and cytochrome bc1 enzyme activities were performed on freshly isolated mitochondria, as described previously (8Cruciat C. Brunner S. Baumann F. Neupert W. Stuart R.A. J. Biol. Chem. 2000; 275: 18093-18098Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The Dimeric ATP Synthase Is Required for Maximal Cytochrome Oxidase Activity—A reduction in COX enzyme activity has been reported in yeast mutants deficient in Su g of the ATP synthase (42Boyle G.M. Roucou X. Nagley P. Devenish R.J. Prescott M. Eur. J. Biochem. 1999; 262: 315-323Crossref PubMed Scopus (47) Google Scholar). As yeast cells deficient in Su e and Su g are known to undergo a high frequency of spontaneous rho–/rho0 conversion (i.e. deletion and/or loss of mtDNA) when grown on fermentable carbon sources, the decrease in COX enzyme activity could simply have reflected the instability of the mtDNA in the Δsu g cells (23Everard-Gigot V. Dunn C.D. Dolan B.M. Brunner S. Jensen R.E. Stuart R.A. Eukaryot. Cell. 2005; 4: 346-355Crossref PubMed Scopus (54) Google Scholar, 37Saddar S. Stuart R.A. J. Biol. Chem. 2005; 280: 24435-24442Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Loss of mtDNA would result in a reduced coding capacity for the COX subunits, 1–3, the presence of which are essential for the enzyme activity of the COX complex. Growth of the Δsu e and Δsu g strains under nonfermentable carbon source (glycerol) conditions exerts a selective pressure on these strains to maintain the integrity of their mitochondrial genome, as demonstrated by the fact that ∼97% of the resulting Δsu e and Δsu g cells were determined to be rho+, i.e. contained functional mtDNA (Fig. 1A). On the other hand, overnight growth of these null mutant strains in a fermentable carbon source (galactose), as published previously (23Everard-Gigot V. Dunn C.D. Dolan B.M. Brunner S. Jensen R.E. Stuart R.A. Eukaryot. Cell. 2005; 4: 346-355Crossref PubMed Scopus (54) Google Scholar, 37Saddar S. Stuart R.A. J. Biol. Chem. 2005; 280: 24435-2444" @default.
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• W1977320434 title "The F1F0-ATP Synthase Complex Influences the Assembly State of the Cytochrome bc1-Cytochrome Oxidase Supercomplex and Its Association with the TIM23 Machinery" @default.
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