Cardiolipin Is Essential for Organization of Complexes III and IV into a Supercomplex in Intact Yeast Mitochondria

Abstract

Digitonin extracts of mitochondria from cardiolipin-containing (wild type) and cardiolipin-lacking (crd1Δ mutant) Saccharomyces cerevisiae subjected to colorless native polyacrylamide gel electrophoresis in the presence of 0.003% digitonin displayed a supercomplex composed of homodimers of complexes III and IV in the former case but only the individual homodimers in the latter case. To avoid treatment with any detergent or dye, we compared organization of the respiratory chain in intact mitochondria from wild type and cardiolipin-lacking cells by using a functional analysis developed previously for the study of the organization of the respiratory chain of S. cerevisiae (Boumans, H., Grivell, L. A., and Berden, J. A. (1998) J. Biol. Chem. 273, 4872–4877). Dependence of the kinetics of NADH oxidation via complexes III, IV, and cytochrome c on the concentration of the complex III-specific inhibitor antimycin A was studied. A linear relationship between respiratory activity and saturation of complex III with antimycin A was obtained for wild type mitochondria consistent with single functional unit kinetics of the respiratory chain. Under the same conditions, cardiolipin-lacking mitochondria displayed a hyperbolic relationship indicating cytochrome c pool behavior. No release of cytochrome c from cardiolipin-lacking mitochondria or mitoplasts under our standard experimental conditions was detected. Identical cytochrome c pool behavior was observed for both wild type and cardiolipin-lacking mitochondria in the presence of a chaotropic agent, which disrupts the interaction between respiratory complexes. The results demonstrate that cardiolipin is essential for association of complexes III and IV into a supercomplex in intact yeast mitochondria. Digitonin extracts of mitochondria from cardiolipin-containing (wild type) and cardiolipin-lacking (crd1Δ mutant) Saccharomyces cerevisiae subjected to colorless native polyacrylamide gel electrophoresis in the presence of 0.003% digitonin displayed a supercomplex composed of homodimers of complexes III and IV in the former case but only the individual homodimers in the latter case. To avoid treatment with any detergent or dye, we compared organization of the respiratory chain in intact mitochondria from wild type and cardiolipin-lacking cells by using a functional analysis developed previously for the study of the organization of the respiratory chain of S. cerevisiae (Boumans, H., Grivell, L. A., and Berden, J. A. (1998) J. Biol. Chem. 273, 4872–4877). Dependence of the kinetics of NADH oxidation via complexes III, IV, and cytochrome c on the concentration of the complex III-specific inhibitor antimycin A was studied. A linear relationship between respiratory activity and saturation of complex III with antimycin A was obtained for wild type mitochondria consistent with single functional unit kinetics of the respiratory chain. Under the same conditions, cardiolipin-lacking mitochondria displayed a hyperbolic relationship indicating cytochrome c pool behavior. No release of cytochrome c from cardiolipin-lacking mitochondria or mitoplasts under our standard experimental conditions was detected. Identical cytochrome c pool behavior was observed for both wild type and cardiolipin-lacking mitochondria in the presence of a chaotropic agent, which disrupts the interaction between respiratory complexes. The results demonstrate that cardiolipin is essential for association of complexes III and IV into a supercomplex in intact yeast mitochondria. Cardiolipin, a phospholipid exclusively found in bacterial membranes and the inner membrane of mitochondria, supports structural integrity and modulates activity of many multimeric complexes of these energy-transducing membranes (1Schlame M. Rua D. Greenberg M.L. Prog. Lipid Res. 2000; 39: 257-288Crossref PubMed Scopus (665) Google Scholar, 2Palsdottir H. Hunte C. Biochim. Biophys. Acta. 2004; 1666: 2-18Crossref PubMed Scopus (333) Google Scholar, 3Dowhan W. Mileykovskaya E. Bogdanov M. Biochim. Biophys. Acta. 2004; 1666: 19-39Crossref PubMed Scopus (110) Google Scholar, 4Mileykovskaya E. Zhang M. Dowhan W. Biochemistry (Mosc.). 2005; 70: 154-158Crossref PubMed Scopus (77) Google Scholar). In particular, considerable evidence implicates the general requirement of cardiolipin for the activities and structure of cytochrome bc1 complex (complex III) 1The abbreviations used are: complex III, cytochrome bc1 complex; complex IV, cytochrome c oxidase; BN, blue native; CN, colorless native; CoQ, coenzyme Q; Q2H2, reduced CoQ2; YPEG, yeast extract, peptone, ethanol, glycerol; MOPS, 4-morpholinepropanesulfonic acid; Cox3p, subunit 3 of complex IV; Cobp, cytochrome b component of complex III. (5Gomez Jr., B. Robinson N.C. Biochemistry. 1999; 38: 9031-9038Crossref PubMed Scopus (130) Google Scholar, 6Lange C. Nett J.H. Trumpower B.L. Hunte C. EMBO J. 2001; 20: 6591-6600Crossref PubMed Scopus (366) Google Scholar) and cytochrome c oxidase (complex IV) (7Sedlak E. Robinson N.C. Biochemistry. 1999; 38: 14966-14972Crossref PubMed Scopus (132) Google Scholar). It was demonstrated by blue native (BN)-PAGE that complexes III and IV form a supercomplex in yeast and mammalian mitochondria composed of a heterodimer of individual homodimers of complex III and complex IV that remains intact after detergent solubilization (8Cruciat C.M. Brunner S. Baumann F. Neupert W. Stuart R.A. J. Biol. Chem. 2000; 275: 18093-18098Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 9Schagger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1049) Google Scholar). Using BN-PAGE analysis of digitonin extracts from Saccharomyces cerevisiae mitochondria, we previously found that, in contrast to wild type yeast, complexes III and IV do not associate to form a supercomplex in mitochondrial extracts from cardiolipin-lacking yeast mutants (crd1Δ). Expression in the crd1Δ mutant of a low copy plasmid carrying a “tet-off”-regulated CRD1 gene (that encodes cardiolipin synthase) allowed us to demonstrate that the level of the supercomplex in the digitonin extracts was dependent on the cardiolipin content of the mitochondria. We suggested a specific role for cardiolipin in supporting a critical interaction between complexes III and IV in vivo that cannot be substituted by the elevated levels of phosphatidylglycerol in the mutant (10Zhang M. Mileykovskaya E. Dowhan W. J. Biol. Chem. 2002; 277: 43553-43556Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). Although BN-PAGE analysis was developed to study native assembly of mitochondrial complexes (11Schagger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1918) Google Scholar), the possible dissociation of loosely bound subunits of the supercomplex caused by the anionic dye Coomassie Blue (Serva Blue G) in the absence of cardiolipin was suggested (12Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). Colorless native (CN)-PAGE might be a preferred method to identify membrane protein complexes in cardiolipin-lacking mutants, because low levels of nonionic detergent normally replace the anionic dye in the gel (13Krause F. Reifschneider N.H. Vocke D. Seelert H. Rexroth S. Dencher N.A. J. Biol. Chem. 2004; 279: 48369-48375Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In a comparative study, no significant amount of the supercomplex was detected in the mutant lacking cardiolipin when using BN-PAGE, whereas mainly supercomplex was found in the same mutant when CN-PAGE was employed in the absence of detergent (12Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). It was concluded that the anionic dye induced dissociation of the loosely associated complexes III and IV in the absence of cardiolipin and that the supercomplex still existed in the mutant mitochondria, although cardiolipin was essential for its stabilization. On the other hand, because no detergent or anionic dye was introduced during gel separation, the possibility of aggregation between detergent-solubilized membrane components resulting in loss of components was not excluded. In this study, we employed CN-PAGE at concentrations of digitonin in the gel that were much lower than that used to extract the supercomplex. Next, we investigated the cardiolipin-dependent association of complexes III and IV into a supercomplex in intact yeast mitochondria. To avoid treatment of mitochondria with any detergent or dye, we compared the organization of the respiratory chain in wild type and cardiolipin-lacking mitochondria using a functional, kinetic analysis developed previously for the study of the organization of the respiratory chain of S. cerevisiae (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In that study, with the complex III-specific inhibitor antimycin A was used to the organization of the respiratory chain into a single functional unit from of the components of the respiratory chain in the by and cytochrome which in the membrane It was found that the S. cerevisiae respiratory chain was a single functional unit (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), which is in with a supercomplex in wild type mitochondria. Using the same kinetic we that the cytochrome c in the respiratory chain in intact mitochondria lacking cardiolipin pool behavior. is consistent with our on the organization of components of the respiratory chain (10Zhang M. Mileykovskaya E. Dowhan W. J. Biol. Chem. 2002; 277: 43553-43556Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar) and for the that cardiolipin essential role in supercomplex in intact mitochondria, than to the supercomplex. and type yeast and its CRD1 lacking M. Mileykovskaya E. Dowhan W. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), were at in of yeast extract, peptone, and and a were previously L.A. PubMed Scopus Google Scholar) with the were in to the of at for at with and in at a of the cells were with at for with at and not for with of of and with of in at for were with and at by in with of IV the were at for and the was to a and at for The was at for were with of MOPS, and at for from the was at for at were in and in at concentration was using the method with the mitochondria in were with on for the mitoplasts were at for at and in Brandt A. K. S. G. Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). in the of mitochondrial membrane or to subunit of complex IV were by with to these and were used of of were in of digitonin inhibitor IV for on the were at for The was to a with of and subjected to electrophoresis in a polyacrylamide gel at for and for at at and concentrations of the detergent digitonin or were into the gel to of the protein complexes in the the gel was in with for were to using a at for at was used the during were using specific for subunit 3 of complex IV or specific for the cytochrome b component of complex III. was with to using and a for native from were used and and b in cytochrome b concentration was from the or was to or the and the was of for the the was used to cytochrome b concentration J.A. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). of cytochrome c activity of mitochondria was at by the of yeast c at by in and in the presence of concentrations of antimycin A (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) using a for reduced cytochrome c at of was used to the activity S. H. J. Biochem. PubMed Scopus Google Scholar). of were at with a were from the of a of concentration were in and in a of 3 of with antimycin A in NADH was to the (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of with antimycin A were at and of of by (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), the kinetic behavior of the respiratory chain of yeast is consistent with which that the of a or cytochrome is to organization of the from the of from dependent on the of pool of a cytochrome c is a respiratory activity be a of the of of cytochrome c and the of oxidation of cytochrome c by (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) previously in the study of A. M. J. Biochem. 39: PubMed Scopus Google A pool behavior of cytochrome c be by of the respiratory activity of yeast mitochondria with NADH a by the complex III-specific inhibitor antimycin A (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), which the of the complex and in a G. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). In this antimycin A the of cytochrome c The respiratory chain of S. that of not complex S. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). inner NADH the and the which the of the membrane S. Grivell L.A. J. Biochem. 1991; PubMed Scopus Google Scholar). NADH is used a in S. cerevisiae mitochondria, via NADH and the for cytochrome which was previously (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) to be a for cytochrome c pool The respiratory be by is the activity at a antimycin A concentration and is the of complex III at a antimycin A concentration (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). 3 is from by both by respiratory activity antimycin and was used to the using the The on the NADH oxidase activity The the saturation by antimycin A. with antimycin A was in the of the experimental with 3 the concentration of antimycin A at which the the and was to the of is with antimycin to the is to in The of this for in 3 were obtained in the of the experimental using 3 in the the for is to the hyperbolic relationship a relationship between NADH oxidase activity and saturation by antimycin A. be consistent with complex III, cytochrome and complex IV a single The of from the of pool of cytochrome c and its to between individual complexes III and IV in the membrane H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google of NADH oxidation with antimycin was to to was to to in a c from or or mitoplasts from both wild type and crd1Δ cells after at in were in mitochondrial respiratory or the The were with NADH and the same concentration of under to the conditions of the under the same in the for the the were at for were to avoid mitochondrial and by in the presence of The resulting cytochrome and cytochrome were by and M. Mileykovskaya E. Dowhan W. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) with a c for the presence of cytochrome The same amount of protein in for was in whereas the protein from the was in of between III and IV by anionic dye Coomassie Blue used in BN-PAGE analysis in the dissociation of bound complexes (12Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). a of CN-PAGE in which the anionic dye is by low levels of nonionic detergent was employed to the results obtained by BN-PAGE (10Zhang M. Mileykovskaya E. Dowhan W. J. Biol. Chem. 2002; 277: 43553-43556Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). To membrane protein aggregation in the absence of the anionic dye, low concentrations of detergent digitonin or 0.003% which were much lower used for were during In the and of protein complexes by the of the complexes and whereas in BN-PAGE, the presence of the anionic dye is to protein components to the same to that the gel is dependent only on with the results using BN-PAGE analysis (10Zhang M. Mileykovskaya E. Dowhan W. J. Biol. Chem. 2002; 277: 43553-43556Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar), digitonin extracts of mitochondria from CRD1 cells and subjected to CN-PAGE supercomplex detected by specific for a component of complex IV and a component of complex III and Digitonin extracts of mitochondria from crd1Δ cells in which were consistent with a of complex III and a of complex IV No supercomplex was observed using CN-PAGE analysis of mitochondria lacking cardiolipin and but of both and were In the in 0.003% digitonin was introduced into the gel for A gel with digitonin displayed the same protein not of c in with the of supercomplex between complexes III and IV in cardiolipin-lacking intact mitochondria, suggested by we used kinetic that membrane solubilization by digitonin and the of We compared the of complex III to antimycin A in wild type and mitochondria. A is a specific inhibitor of complex III, which to the complex in a G. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). was previously for complex III that the of the complex for antimycin A G. Biochim. Biophys. Acta. PubMed Scopus Google Scholar), we antimycin A the same on complex III in mitochondria from wild type and crd1Δ The concentration of cytochrome b was by under CRD1 and crd1Δ mitochondria cytochrome b at a concentration of from both a c activity Under the conditions of our a linear relationship between antimycin A concentration and activity was observed in both wild by H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, and mitochondria. In both the of complex III activity by antimycin A be the saturation of the complex with the antimycin that of cardiolipin not the of complex III to antimycin A of kinetic of the cytochrome c between complexes III and IV evidence of mitochondria lacking cardiolipin a functional supercomplex or individual complexes. from both CRD1 and crd1Δ cells were for using NADH the in wild type complexes III and IV associated into a supercomplex with of cytochrome a linear relationship between the saturation of complex III with antimycin A and of the respiratory activity be because of complex III the of the supercomplex. complexes III and IV individual cytochrome c between a between complexes III and complexes In this of any complex III assembly not of cytochrome c or complex IV and a hyperbolic be observed of the and H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). using standard with a linear relationship between respiratory activity and saturation with antimycin A was observed for wild type mitochondria results and the single functional unit of the chain of S. cerevisiae (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). is consistent with a cytochrome c from complex III to complex IV a associated supercomplex of Under the same conditions, mitochondria from crd1Δ cells displayed a hyperbolic indicating cytochrome c a with 3 standard was with a chaotropic to the respiratory supercomplex (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), hyperbolic were obtained in both CRD1 and and crd1Δ mitochondria and with from 3 and the in demonstrate the level of dissociation of the functional supercomplex composed of complexes III and IV and cytochrome c in crd1Δ mitochondria, which is with the level of dissociation in the presence of a chaotropic of of c from and to (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), cytochrome c in wild type mitochondria pool behavior under conditions, the treatment of mitochondria with a chaotropic or with of In the dissociation of the supercomplex composed of complexes III and IV was In the release of cytochrome c from the of the inner mitochondrial membrane into the was in our the hyperbolic between NADH oxidase activity and saturation of complex III with antimycin A in cardiolipin-lacking mitochondria be in dissociation of complexes III and IV in the case of the chaotropic or release of cytochrome c into the under experimental conditions in the case of No release of cytochrome c from wild type or cardiolipin-lacking mitochondria was observed after a treatment under the same used in the cytochrome c from crd1Δ mitochondria after of treatment and after for wild type mitochondria not results to previously M. V. M. S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) but the low conditions employed were lower of than used of under the conditions of our kinetic cytochrome c is still associated with mitochondria. To the possibility that the absence of cytochrome c release from mitochondria is a of the of the membrane in the absence of we the same using mitoplasts from wild type and cardiolipin-lacking mitochondria. In mitochondria with on for of the mitochondrial membrane protein in the from wild type and cardiolipin-lacking whereas of the mitochondrial inner membrane protein subunit of complex IV was still in the indicating that the membrane was to of the release of cytochrome c from the inner Under conditions, no release of cytochrome c from the mitochondrial inner membrane was detected with wild type or crd1Δ mitoplasts of cytochrome c was detected only under conditions not which that the pool behavior of cytochrome c detected in mitochondria, in was not because of the release of cytochrome c from the mitochondrial inner membrane but the dissociation of complexes III and IV in the absence of on BN-PAGE analysis of complexes III and IV from S. we previously concluded that these complexes do not form a supercomplex in detergent-solubilized extracts of mitochondria of cardiolipin (10Zhang M. Mileykovskaya E. Dowhan W. J. Biol. Chem. 2002; 277: 43553-43556Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar) do in extracts of mitochondria from wild type cells H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1049) Google Scholar). In a (12Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar), was concluded that cardiolipin supercomplex in wild type cells but that the supercomplex was still the form observed in cardiolipin-lacking mitochondria on analysis of detergent-solubilized complexes using The that the presence of anionic dye Coomassie Blue in the BN-PAGE detergent that a associated supercomplex lacking cardiolipin but not a complex we that the CN-PAGE employed digitonin or other in the cells on the of supercomplex displayed on the to complex that was by the of was lower for the cardiolipin-containing that complexes III and IV a supercomplex or were and were because of aggregation in the absence of detergent or anionic To these in the behavior of complexes III and IV in native gel we CN-PAGE analysis in the presence of low levels of digitonin in the gel to possible in contrast to the (12Pfeiffer K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). We that, the anionic dye in the BN-PAGE not the digitonin used for the of 0.003% digitonin in the gel aggregation of but not specific between complexes III and of low of digitonin in of complexes III and IV from wild type and cardiolipin-lacking mitochondria but supercomplex in the former case and homodimers of individual complexes III and IV in the latter we previously using BN-PAGE (10Zhang M. Mileykovskaya E. Dowhan W. J. Biol. Chem. 2002; 277: 43553-43556Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). The results from native electrophoresis M. Mileykovskaya E. Dowhan W. J. Biol. Chem. 2002; 277: 43553-43556Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, K. Gohil V. Stuart R.A. Hunte C. Brandt U. Greenberg M.L. Schagger H. J. Biol. Chem. 2003; 278: 52873-52880Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, H. Biochim. Biophys. Acta. 2002; PubMed Scopus Google Scholar, and this us to functional analysis of the organization of the respiratory chain of S. cerevisiae in intact mitochondria. analysis was used to between of organization of complexes of the respiratory organization of individual respiratory or assembly into M.L. C. G. J. Biochem. 2003; Google and analysis of cytochrome c behavior (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) in the study of the organization of the respiratory chain of S. cerevisiae is in with BN-PAGE results (8Cruciat C.M. Brunner S. Baumann F. Neupert W. Stuart R.A. J. Biol. Chem. 2000; 275: 18093-18098Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, M. Mileykovskaya E. Dowhan W. J. Biol. Chem. 2002; 277: 43553-43556Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar), which the organization of complexes III and IV in a supercomplex in wild type mitochondria. kinetic from this study that cytochrome c not a pool in wild type mitochondria. consistent with BN-PAGE and the respiratory chain in intact cardiolipin-lacking mitochondria with to cytochrome pool behavior. might to our observed pool behavior of cytochrome c in mitochondria. complexes III and IV not be associated with other in the absence of complexes III and IV still be associated with but the pool behavior be caused by the release of cytochrome c to the in the absence of was previously demonstrated that cytochrome c bound to the inner mitochondrial membrane of wild type and cardiolipin-lacking in mitochondria from S. cerevisiae after under low conditions M. V. M. S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). We that was no release of cytochrome c from the cardiolipin-lacking mitochondria or mitoplasts under our experimental conditions, which employed conditions than used previously M. V. M. S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). experimental evidence reduced of cytochrome c for the of the inner mitochondrial membrane the for pool behavior in the absence of The possibility that complexes III and IV still associate with other in the absence of cardiolipin but that cytochrome although bound to the its to from to the supercomplex is In any of cardiolipin results in the of the assembly of a functional supercomplex with of cytochrome c from complex III to IV the structural in supercomplex organization in the absence of with our structural from BN-PAGE and the results demonstrate significant dissociation of the supercomplex composed of complexes III and IV in intact mitochondria lacking The of the respiratory chain of yeast mitochondria into a functional and structural unit or B. PubMed Scopus Google Scholar) from organization of the respiratory chain of in which and cytochrome c pool behavior and BN-PAGE and CN-PAGE in of the individual complexes between and supercomplex organization H. Pfeiffer K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Google Scholar). A in the of of S. cerevisiae a supercomplex organization might a significant in the respiratory of the chain under conditions of when a in the level of in the membrane (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast to complexes III and IV into a supercomplex with the complex the yeast supercomplex is only between complexes III and IV H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1049) Google Scholar). might a specific role for cardiolipin in of in yeast mitochondria. We A. for in the with antimycin V. for critical on the of kinetic and for for of mitochondria