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• W1990625705 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; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In that study, titration with the complex III-specific inhibitor antimycin A was used to distinguish the organization of the respiratory chain into a single functional unit from independent localization of the redox components of the respiratory chain in the lipid bilayer connected by randomly diffusing small carriers, Coenzyme Q (CoQ) and cytochrome c, which behave in the membrane as homogeneous diffusible pools. It was found that the S. cerevisiae respiratory chain was titrated as a single functional unit (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), which is in good agreement with a supercomplex model in wild type mitochondria. Using the same kinetic analysis, we report here that the cytochrome c in the respiratory chain in intact mitochondria lacking cardiolipin displays pool behavior. This result is consistent with our previous observation based on the molecular organization of solubilized 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 demonstrates for the first time that cardiolipin plays an essential role in supercomplex formation in intact mitochondria, rather than merely providing added stability to the supercomplex. Yeast Strain and Growth Media—Wild type yeast strain DL1 and its CRD1 null derivatives (crd1Δ) lacking cardiolipin, YZD5 (15Zhang M. Su X. Mileykovskaya E. Amoscato A.A. Dowhan W. J. Biol. Chem. 2003; 278: 35204-35210Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), were grown at 30 °C in YPEG growth medium consisting of 1% Bacto yeast extract, 2% Bacto peptone, and 1% ethanol (v/v) and 3% glycerol as a carbon source. Mitochondrial Isolation—Mitochondria were isolated as previously described (16Glick B.S. Pon L.A. Methods Enzymol. 1995; 260: 213-223Crossref PubMed Scopus (287) Google Scholar) with the following modifications. Cells were grown in YPEG media to the mid-exponential phase of growth, harvested at 1500 × g (GSA rotor, Sorval) for 5 min at 4 °C, washed with cold water, and resuspended in 100 mm Tris-SO4 (pH 9.4) at a ratio of 2 ml/g wet weight cells. Thereafter, the cells were incubated with 10 mm dithiothreitol at 30 °C for 15 min with gentle shaking, centrifuged at 2000 × g (Sorval, SS-34 rotor here and later, if not specified) for 5 min, washed with 2 ml of 1 m cold sorbitol/1 g of cells, and digested with Zymolase-20T (25 mg/g of wet weight cells) in buffer containing 1 m sorbitol, 20 mm potassium Pi buffer (pH 7.2) at 37 °C for ∼30–40 min. Spheroplasts were washed with 1 m cold sorbitol and lysed at 4 °C by Dounce homogenization in 0.5 m sorbitol, 10 mm Tris-HCl (pH 7.5), 0.02% bovine serum albumin supplemented with 1/1000 volume of Protease Inhibitor Mixture Set IV (Calbiochem®). After homogenization, the samples were centrifuged at 1500 × g for 5 min, and then the supernatant was transferred to a new tube and centrifuged again at 1500 × g for 5 min. The supernatant was once again centrifuged at 12,000 × g for 10 min. Pellets were washed with 20 ml of SE buffer (250 mm sucrose, 10 mm MOPS, 1 mm EDTA (pH 7.2)) and centrifuged at 1500 × g for 5 min. Supernatant from the last centrifugation was centrifuged at 12,000 × g for 10 min at 4 °C. Mitochondria (pellets) were suspended in SE buffer and frozen in 0.1-ml aliquots at –80 °C. Protein concentration was measured using the Bradford method (Bio-Rad) with bovine serum albumin as the standard. Mitoplast Preparation—Isolated mitochondria in SE buffer were diluted 15-fold with hypotonic buffer (20 mm HEPES-KOH (pH 7.4)). After incubating on ice for 30 min, the mitoplasts were centrifuged at 14,000 × g for 10 min at 4 °C and redissolved in SE buffer (17Glick B.S. Brandt A. Cunningham K. Muller S. Hallberg R.L. Schatz G. Cell. 1992; 69: 809-822Abstract Full Text PDF PubMed Scopus (303) Google Scholar, 18Glick B.S. Methods Enzymol. 1995; 260: 224-231Crossref PubMed Scopus (56) Google Scholar). Changes in the ratio of mitochondrial outer membrane porin (voltage-dependent anion channel or VDAC) to subunit 2 of complex IV were estimated by Western blotting with antibody to these proteins (Molecular Probes) and were used as an indicator of mitoplast formation. CN-PAGE—Mitochondria (75 μg of protein) were solubilized in 15 μl of buffer containing 1% digitonin (w/v recrystallized digitonin), 50 mm potassium acetate, 30 mm HEPES-KOH (pH 7.4), 10% glycerol, 0.1 mg/ml α2-macroglobulin, 0.1% (v/v) protease inhibitor mixture set IV for 30 min on ice. After solubilization, the samples were centrifuged at 125,000 × g for 30 min (Beckman TL-100 ultracentrifuge, TLA100.3 rotor). The supernatant (15 μl) was transferred to a new tube, supplemented with 1.5 μl of sample buffer containing 500 mm aminocaproic acid, and subjected to electrophoresis in a 4–8% polyacrylamide gradient gel at 80 V for 1 h and then for 4 h at 200 V at 4 °C, cathode buffer (50 mm Tricine, 15 mm BisTris (pH 7.0)) and anode buffer (50 mm BisTris, pH 7.0). Low concentrations of the mild detergent digitonin (0.003 or 0.01%, w/v) were added into the gel to achieve separation of the protein complexes in the gel. After CN-PAGE, the gel was soaked in Western blotting buffer (20% methanol (v/v), 0.02% SDS, 20 mm Tris-HCl (pH 8.3), 0.15 m glycine) with gentle shaking for 60 min. Proteins were transferred to nitrocellulose sheets using a semidry apparatus (MilliBlot™-Graphite Electroblotter I, Millipore Corporation) at 40 V for 1.5 h at 4 °C. Cathode buffer was used as the buffer during transfer. Proteins were then specifically located using monoclonal antibody (Molecular Probes) specific for subunit 3 (Cox3p) of complex IV or polyclonal antibody (Gottfried Schatz, Biozentrum, Basel, Switzerland) specific for the cytochrome b component (Cobp) of complex III. Final detection was with secondary antibodies linked to horseradish peroxidase using SuperSignal® West Pico chemiluminescent substrate (Pierce) and a Bio-Rad Fluor-S™Max MultiImager for quantification. High molecular mass native markers from Amersham Biosciences were used as standards and included thyroglobulin (669 kDa) and ferritin (440 kDa). Cytochrome b Concentration in Mitochondria—The cytochrome b concentration was determined spectroscopically (Hitachi U-3000) from the absorption difference spectra. Either solid dithionite or 10 μm ferricyanide was added to either reduce or oxidize the cytochrome, respectively, and the visible difference spectrum (reduced versus oxidized) was determined. An absorbance coefficient of 28.0 for the maximum (at 563 nm) minus the minimum (at 577 nm) difference was used to determine cytochrome b concentration (19Berden J.A. Slater E.C. Biochim. Biophys. Acta. 1970; 216: 237-249Crossref PubMed Scopus (152) Google Scholar). Electron Transfer Activity of Complex III—Q2H2 (reduced CoQ2): cytochrome c oxidoreductase activity of isolated mitochondria (0.02–0.1 mg/ml) was determined at 30 °C by measuring the reduction of 18 μm yeast ferricytochrome c at 550 nm by 25 μm Q2H2 in 20 mm potassium Pi (pH 7.4), 2 mm EDTA, and 0.5 mm KCN in the presence of different concentrations of antimycin A (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) using a Hitachi U-3000 spectrophotometer. An extinction coefficient for reduced cytochrome c at 550 nm of 18.5 mm–1 cm–1 was used to calculate the reductase activity (20Kubota T. Yoshikawa S. Matsubara H. J. Biochem. (Tokyo). 1992; 111: 91-98Crossref PubMed Scopus (24) Google Scholar). Respiratory Activity of Isolated Mitochondria—Oxygen consumption rates were measured at 30 °C with a Clark oxygen electrode. Rates were determined from the slope of a plot of O2 concentration versus time. Mitochondria (0.03–0.06 mg/ml) were incubated in buffer containing 0.6 m sorbitol, 25 mm potassium Pi (pH 7.0), 1 mm EDTA, and 1 mm MgCl2 in a final volume of 3 ml. After 2 min of preincubation with antimycin A (dissolved in methanol), 0.5 mm NADH was added to the reaction chamber (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Control samples had an equal volume of methanol. Titrations with antimycin A were carried out at least three times. Definition and Kinetics of Pool Function of Cytochrome c—As established by Boumans et al. (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), the kinetic behavior of the respiratory chain of yeast is consistent with an ordered molecular assembly, which means that the number of subsequent acceptors a carrier, such as CoQ or cytochrome c, can reach is limited to 1. As organization of the system deviates from an ordered array, the number of subsequent acceptors increases from 1, dependent on the degree of pool function of a freely diffusing carrier. When cytochrome c is present as a pool, respiratory activity (Vo) can be described as a function of the maximum rate of reduction (Vred) of cytochrome c and the maximum rate of oxidation (Vox) of cytochrome c by equation 1 (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) previously derived in the study of another mobile carrier, CoQ (21Kroger A. Klingenberg M. Eur. J. Biochem. 1973; 39: 313-323Crossref PubMed Scopus (172) Google Scholar).Vo=Vred VoxVred+Vox(eq.1) Vx=Vred(1−x) VoxVred(1−x)+Vox(eq.2) VxVo=VredVo(1−x) VoxVoVredVo(1−x)+VoxVo(eq.3) A putative pool behavior of specifically cytochrome c can be studied by titration of the respiratory activity of yeast mitochondria with NADH as a substrate by the complex III-specific inhibitor antimycin A (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), which targets the quinone reduction site of the complex and binds in a 1:1 ratio (22Tsai A.L. Palmer G. Biochim. Biophys. Acta. 1982; 681: 484-495Crossref PubMed Scopus (58) Google Scholar). In this case, antimycin A titrates the reduction rate of cytochrome c (Vred). The respiratory chain of S. cerevisiae, unlike that of mammals, does not contain complex I (23de Vries S. Marres C.A. Biochim. Biophys. Acta. 1987; 895: 205-239Crossref PubMed Scopus (155) Google Scholar). Instead, inner membrane-bound NADH dehydrogenases Ndi1p (internal, faces the matrix) and Nde1p (external, faces the intermembrane space) are present, which transfer the electrons without translocation of protons across the membrane (24Marres C.A. de Vries S. Grivell L.A. Eur. J. Biochem. 1991; 195: 857-862Crossref PubMed Scopus (109) Google Scholar). When NADH is used as a substrate in isolated S. cerevisiae mitochondria, it reduces CoQ via external NADH dehydrogenase and provides the highest reduction rate for cytochrome c, which was previously established (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) to be a necessary prerequisite for measuring cytochrome c pool function. The titrated respiratory activity, Vx, can be described by equation 2, where Vx is the activity at a given antimycin A concentration and x is the fraction of complex III inhibited at a given antimycin A concentration (14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Equation 3 is derived from equation 2 by dividing both sides by Vo (initial respiratory activity without antimycin A) and was used to fit the data using the Kaleida Graph program. The y-axis on graphs (Fig. 3) represents the relative NADH oxidase activity (Vx/Vo). The x-axis represents the fractional saturation by antimycin A. Saturation with antimycin A was determined in the fitting procedure of the experimental data, with equation 3 as the concentration of antimycin A at which the curve crosses the x-scale (Vx/Vo = 0; complete inhibition) and was set to 1. Vred/Vo, the maximum rate of reduction (which is titrated with antimycin A) relative to the Vo, is referred to as Vtitr in Table I. The values of this coefficient for curves represented in Fig. 3 were obtained in the fitting procedure of the experimental data using equation 3 in the Kaleida Graph program. If the value for Vtitr is close to 1, the above hyperbolic relationship (equation 3) approximates a straight line relationship between relative NADH oxidase activity and relative saturation by antimycin A. This would be consistent with complex III, cytochrome c, and complex IV functioning as a single unit. The deviation of Vtitr from 1 reflects the degree of pool function of cytochrome c and represents its ability to provide interconnection between individual complexes III and IV randomly embedded in the membrane (for details, see Ref. 14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar).Table IReaction rates of NADH oxidation titrated with antimycin ABufferaBuffer was 0.6 m sorbitol, 1 mm EDTA, 1 mm MgCl2, pH adjusted to 7.0. plusVtitr (CRD1)Vtitr (crd1Δ)25 mM KPi1.07 ± 0.082.8 ± 0.325 mM KPi/50 mM TCAbpH adjusted to 7.0.3.0 ± 0.23.1 ± 0.4a Buffer was 0.6 m sorbitol, 1 mm EDTA, 1 mm MgCl2, pH adjusted to 7.0.b pH adjusted to 7.0. Open table in a new tab Cytochrome c Release from Mitochondria or Mitoplast—Mitochondria (5 mg/ml protein) or mitoplasts (5 mg/ml protein) isolated from both wild type and crd1Δ cells after exponential growth at 30 °C in YPEG were resuspended in mitochondrial respiratory buffer containing either 0.6 m sorbitol, 25 mm potassium Pi (pH 7.0), 1 mm EDTA, 1 mm MgCl2, or the above buffer plus 300 mm KCl (pH 7.0). The samples were supplemented with 0.5 mm NADH and the same concentration of methanol as described under “Experimental Procedures” to mimic the exact conditions of the oxygen consumption measurement. After incubation under the same condition as in the respiration experiment for the indicated time, the samples were centrifuged at 10,000 × g for 5 min. Supernatants were centrifuged once more to avoid mitochondrial contamination and concentrated by 10% trichloroacetic acid precipitation in the presence of 0.5% (w/v) bovine serum albumin. The resulting supernatants (released cytochrome c) and pellets (mitochondrial-bound cytochrome c) were analyzed by SDS-PAGE and Western blotting (15Zhang M. Su X. Mileykovskaya E. Amoscato A.A. Dowhan W. J. Biol. Chem. 2003; 278: 35204-35210Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) with a monoclonal anticytochrome c antibody (BD Biosciences) for the presence of cytochrome c. The same amount of total pellet protein in sufficient amounts for detection was loaded in each lane, whereas all the protein recovered from the supernatant was loaded in each lane. Lack of Supercomplex Formation between Complexes III and IV as Detected by CN-PAGE—The anionic dye Coomassie Blue used in BN-PAGE analysis may result in the dissociation of weakly 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). Therefore, a gentler technique of CN-PAGE in which the anionic dye is replaced by low levels of nonionic detergent was employed to verify 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 minimize nonspecific membrane protein aggregation in the absence of the anionic dye, low concentrations of mild detergent digitonin (0.01 or 0.003% w/v), which were much lower then those used for extraction (1%), were added during CN-PAGE. In CN-PAGE, the direction and mobility of protein complexes are determined by the intrinsic charge of the complexes and their apparent molecular size, whereas in BN-PAGE, the presence of the protein-bound anionic dye is assumed to normalize all protein components to the same charge to mass ratio, so that electrophoretic mobility within the gel is dependent only on apparent molecular size. Consistent 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, 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), digitonin extracts of mitochondria isolated from CRD1 cells and subjected to CN-PAGE (Fig. 1) showed similar supercomplex formation as detected by antibodies specific for a component of complex IV (Cox3p) and a component of complex III (Cobp) (Fig. 1, lanes 1 and 3). Digitonin extracts of mitochondria isolated from crd1Δ cells showed primarily bands in which mobilities were consistent with a homodimer of complex III (∼600 kDa) (Fig. 1, lane 4) and a homodimer of complex IV (∼400 kDa, Fig. 1, lane 2). No supercomplex was observed using CN-PAGE analysis of mitochondria lacking cardiolipin (Fig. 1, lanes 2 and 4), but trace amounts of intermediate molecular species containing both Cox3p and Cobp were present. In the experiment presented in Fig. 1, 0.003% (w/v) digitonin was introduced into the gel for CN-PAGE. A gel with 0.01% (w/v) digitonin displayed the same protein pattern (data not shown). Titration of Q2H2:Cytochrome c Oxidoreductase Activity in Mitochondria with Antimycin A—To establish the lack of supercomplex formation between complexes III and IV in cardiolipin-lacking (crd1Δ) intact mitochondria, as suggested by CN-PAGE, we used an independent kinetic approach that avoids membrane solubilization by digitonin and the introduction of charged dyes. We first compared the sensitivity of complex III to antimycin A in wild type and cardiolipin-deficient mitochondria. Antimycin A is a specific inhibitor of complex III, which binds to the complex in a 1:1 ratio (22Tsai A.L. Palmer G. Biochim. Biophys. Acta. 1982; 681: 484-495Crossref PubMed Scopus (58) Google Scholar). Because it was shown previously for purified complex III that delipidation can change the affinity of the complex for antimycin A (25Tsai A.L. Palmer G. Biochim. Biophys. Acta. 1986; 852: 100-105Crossref PubMed Scopus (16) Google Scholar), we tested whether antimycin A has the same inhibitory effect on complex III in mitochondria from wild type (CRD1) and crd1Δ strains. The concentration of cytochrome b was measured by absorption difference spectra as described under “Experimental Procedures.” Both CRD1 and crd1Δ mitochondria contained cytochrome b at a concentration of ∼0.2 nmol/mg protein. Mitochondria from both strains had a similar Q2H2:cytochrome c oxidoreductase activity (500–600 nmoles/min/mg). Under the conditions of our experiments, a linear relationship between antimycin A concentration and electron transfer activity was observed in both wild type, as reported by Ref. 14Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, and cardiolipin-deficient mitochondria. In both cases, the relative inhibition of complex III activity by antimycin A can be viewed directly as the relative saturation of the complex with the antimycin A, suggesting that lack of cardiolipin did not alter the sensitivity of complex III to antimycin A (Fig. 2). Pool Versus Non-Pool Behavior of Cytochrome c—The kinetic characterization of the electron carrier cytochrome c between complexes III and IV should provide evidence of whether mitochondria lacking cardiolipin contain a physiologically functional supercomplex or individual complexes. Mitochondria from both CRD1 and crd1Δ cells were analyzed for oxygen consumption using NADH as the substrate. If, as in wild type cells, complexes III and IV are associated into a supercomplex with channeling of cytochrome c, a linear relationship (Vtitr approaching 1) between the saturation of complex III with antimycin A and inhibition of the relative respiratory activity should be observed, because inhibition of complex III would reflect the inhibition of the whole supercomplex. If complexes III and IV exist as individual complexes, then cytochrome c would mediate electron transfer between them as a freely diffusible carrier producing interconnection between non-inhibited complexes III and comple" @default.
• W1990625705 created "2016-06-24" @default.
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• W1990625705 creator A5005928599 @default.
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• W1990625705 date "2005-08-01" @default.
• W1990625705 modified "2023-09-30" @default.
• W1990625705 title "Cardiolipin Is Essential for Organization of Complexes III and IV into a Supercomplex in Intact Yeast Mitochondria" @default.
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