The Modification of the Conserved GXXXG Motif of the Membrane-spanning Segment of Subunit g Destabilizes the Supramolecular Species of Yeast ATP Synthase
2005; Elsevier BV; Volume: 280; Issue: 32 Linguagem: Inglês
10.1074/jbc.m502140200
ISSN1083-351X
AutoresDiego M. Bustos, Jean Velours,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoThe supernumerary subunit g is found in all mitochondrial ATP synthases. Most of the conserved amino acid residues are present in the membrane C-terminal part of the protein that contains a dimerization motif GXXXG. In yeast, alteration of this motif leads to the loss of subunit g and of supramolecular structures of the ATP synthase with concomitant appearance of anomalous mitochondrial morphologies. Disulfide bond formation involving an engineered cysteine in position 109 of subunit g and the endogenous cysteine 28 of subunit e promoted g + g, e + g, and e + e adducts, thus revealing the proximity in the mitochondrial membrane of several subunits e and g. Disulfide bond formation between two subunits g in mitochondria increased the stability of an oligomeric structure of the ATP synthase in digitonin extracts. These data suggest the participation of the dimerization motif of subunit g in the formation of supramolecular structures and is in favor of the existence of ATP synthase associations, in the inner mitochondrial membrane, whose masses are higher than those of ATP synthase dimers. The supernumerary subunit g is found in all mitochondrial ATP synthases. Most of the conserved amino acid residues are present in the membrane C-terminal part of the protein that contains a dimerization motif GXXXG. In yeast, alteration of this motif leads to the loss of subunit g and of supramolecular structures of the ATP synthase with concomitant appearance of anomalous mitochondrial morphologies. Disulfide bond formation involving an engineered cysteine in position 109 of subunit g and the endogenous cysteine 28 of subunit e promoted g + g, e + g, and e + e adducts, thus revealing the proximity in the mitochondrial membrane of several subunits e and g. Disulfide bond formation between two subunits g in mitochondria increased the stability of an oligomeric structure of the ATP synthase in digitonin extracts. These data suggest the participation of the dimerization motif of subunit g in the formation of supramolecular structures and is in favor of the existence of ATP synthase associations, in the inner mitochondrial membrane, whose masses are higher than those of ATP synthase dimers. F0F1-ATP synthase is a molecular rotary motor that is responsible for aerobic synthesis of ATP. It exhibits a headpiece (catalytic sector), a base piece (membrane sector), and two connecting stalks. Sector F1 containing the headpiece is a water-soluble unit that retains the ability to hydrolyze ATP when in soluble form. F0 is embedded in the membrane and is mainly composed of hydrophobic subunits forming a specific proton-conducting pathway. When the F1 and F0 sectors are coupled, the enzyme functions as a reversible H+-transporting ATPase or ATP synthase (1Senior A. Nadanaciva S. Weber J. Biochim. Biophys. Acta. 2002; 1553: 188-211Crossref PubMed Scopus (331) Google Scholar, 2Fillingame R.H. Science. 1999; 286: 1687-1688Crossref PubMed Scopus (56) Google Scholar, 3Pedersen P.L. Ko Y.H. Hong S. J. Bioenerg. Biomembr. 2000; 32: 423-432Crossref PubMed Scopus (22) Google Scholar, 4Stock D. Gibbons C. Arechaga I. Leslie A.G. Walker J.E. Curr. Opin. Struct. Biol. 2000; 10: 672-679Crossref PubMed Scopus (259) Google Scholar). The two connecting stalks are made of components from F1 and F0. The central stalk is a part of the rotor of the enzyme and the second stalk, which is part of the stator, connects F1 and hydrophobic membranous components of the enzyme probably via a flexible region (1Senior A. Nadanaciva S. Weber J. Biochim. Biophys. Acta. 2002; 1553: 188-211Crossref PubMed Scopus (331) Google Scholar). High resolution x-ray crystallographic data have led to solving the structure of F1 from different sources (5Hausrath A.C. Gruber G. Matthews B.W. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13697-13702Crossref PubMed Scopus (82) Google Scholar, 6Bianchet M.A. Hullihen J. Pedersen P.L. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11065-11070Crossref PubMed Scopus (224) Google Scholar, 7Gibbons C. Montgomery M.G. Leslie A.G. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (434) Google Scholar, 8Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2736) Google Scholar, 9Stock D. Leslie A.G. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1079) Google Scholar) and the F1-c10-ring from Saccharomyces cerevisiae (9Stock D. Leslie A.G. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1079) Google Scholar).The mitochondrial F0 of mammals is composed of 10 different subunits (10Collinson I.R. Runswick M.J. Buchanan S.K. Fearnley I.M. Skehel J.M. van Raaij M.J. Griffiths D.E. Walker J.E. Biochemistry. 1994; 33: 7971-7978Crossref PubMed Scopus (162) Google Scholar), all identified in the S. cerevisiae enzyme (11Velours J. Arselin G. J. Bioenerg. Biomembr. 2000; 32: 383-390Crossref PubMed Scopus (91) Google Scholar, 12Arnold I. Bauer M.F. Brunner M. Neupert W. Stuart R.A. FEBS Lett. 1997; 411: 195-200Crossref PubMed Scopus (69) Google Scholar, 13Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schagger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (361) Google Scholar). Some of these subunits are not required for ATP synthesis function, but are involved in the dimerization/oligomerization of the mitochondrial ATP synthase (13Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schagger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (361) Google Scholar, 14Soubannier 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, 15Paumard P. Vaillier J. Coulary B. Schaeffer J. Soubannier V. Mueller D.M. Brethes D. di Rago J.P. Velours J. EMBO J. 2002; 21: 221-230Crossref PubMed Scopus (579) Google Scholar). For example, null mutants of subunits g and e abolish the ability of ATP synthase to make supramolecular structures. These subunits are small hydrophobic proteins that have only one spanning segment with a N terminus inside the matrix, and the C terminus in the intermembrane space (12Arnold I. Bauer M.F. Brunner M. Neupert W. Stuart R.A. FEBS Lett. 1997; 411: 195-200Crossref PubMed Scopus (69) Google Scholar, 16Belogrudov G.I. Tomich J.M. Hatefi Y. J. Biol. Chem. 1996; 271: 20340-20345Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) with a membrane-spanning segment probably located at the interface between two ATP synthase monomers (16Belogrudov G.I. Tomich J.M. Hatefi Y. J. Biol. Chem. 1996; 271: 20340-20345Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar).The subunits g and e have a conserved putative dimerization GXXXG motif located in the membrane-spanning segment. In subunit e, its alteration led to the loss of subunit g and the loss of dimeric and oligomeric forms of the yeast ATP synthase. The presence of a cysteine residue (Cys28) placed after the membrane domain of subunit e made it possible to establish, by cross-linking experiments, that two subunits e are close to each other in the membrane. This disulfide bond was shown to significantly stabilize ATP synthase dimerization/oligomerization in intact mitochondria (17Arselin 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). The subunit e also contains a putative coiled-coil region in its C-terminal part, which is involved in the stabilization of the dimeric forms of the detergent-solubilized ATP synthase complexes (18Everard-Gigot V. Dunn C. Dolan B. Brunner S. Jensen R. Stuart R. Eukaryotic Cells. 2005; 4: 346-355Crossref PubMed Scopus (54) Google Scholar). The study of subunits g and e is also important because the dimerization/oligomerization process of ATP synthase complex is linked to cristae biogenesis and mitochondrial morphology (13Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schagger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (361) Google Scholar, 15Paumard P. Vaillier J. Coulary B. Schaeffer J. Soubannier V. Mueller D.M. Brethes D. di Rago J.P. Velours J. EMBO J. 2002; 21: 221-230Crossref PubMed Scopus (579) Google Scholar).The most highly studied, and apparently widespread, mode of helix-helix association is mediated by the so-called GXXXG motif, which is known to act as a universal scaffold for the assembly of two transmembrane helices (19Russ W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (775) Google Scholar). The GXXXG is a motif where two glycine residues are separated by any three amino acids on a helical framework. This arrangement of glycine residues allows the close approach of interacting helices, whereupon extensive packing interactions take place between pairs of surrounding residues. Despite the high occurrence of the GXXXG motif in transmembrane helices, the transmembrane peptide of glycophorin A is the only dimer mediated by a GXXXG motif for which the structure has been determined to high resolution (20Senes A. Ubarretxena-Belandia I. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9056-9061Crossref PubMed Scopus (435) Google Scholar). Thus, it is not clear whether alternate structures for transmembrane dimers exist. Moreover, it is not known how residues surrounding GXXXG motifs “tailor” the affinity of their helix-helix interactions for required structural and functional purposes (21Melnyk R.A. Kim S. Curran A.R. Engelman D.M. Bowie J.U. Deber C.M. J. Biol. Chem. 2004; 279: 16591-16597Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar).Here, site-specific mutagenesis was used to modify the membranous domain, and especially the GXXXG domain of subunit g of the ATP synthase, to determine its interaction with different subunits of the complex and its participation in the edification of supramolecular ATP synthase species.EXPERIMENTAL PROCEDURESMaterials—Digitonin was from Sigma. Oligonucleotides were purchased from MWG-BIOTECH. All other reagents were of reagent grade quality.Yeast Strains and Nucleic Acid Techniques—The S. cerevisiae strain D273-10B/A/H/U (MATα, met6, ura3, his3) was the wild-type strain. The yeast mutants with a point mutation were named as (name of the subunit) (one-letter code of wild-type residue) (residue number) (mutant residue) (i.e. eC28S). The null mutant in the ATP20 gene (Δg) was constructed by PCR-based mutagenesis, and the kanr gene was removed. The gene ATP20 encoding subunit g was obtained by PCR amplification of genomic DNA and the resulting 1178-bp EcoRI-XhoI DNA fragment was cloned in the shuttle vector pRS313. A 1586-bp PvuII-EcoRV DNA fragment containing kanr was isolated from the pUG6 vector and inserted into the EcoRV site in the 3′ region of the ATP20 gene. The mutations gQ93A, gY98A, gG101L, g102A, gG105L, and gC75S/L109C were introduced by a PCR mutagenesis procedure into the resulting vector. The strains containing modified versions of subunit g were obtained by integration at the chromosomic locus of a 2777-bp EcoRI-ApaI DNA fragment, bearing the mutated versions of the ATP20 gene, in the Δg strain, and were selected for their resistance to geneticin. The strain containing the subunit gC75S/L109C(His)6 was constructed according to the following strategy. Two complementary oligonucleotides, 5′-TATAAACATCACCACCACCACCACCACCACTAAGCTTTT-3′ and 5′-GAATTAAAAAGCTTAGTGGTGGTGGTGGTGGTGGTGATG-3′, were used to introduce the (His)6 sequence into the C terminus of subunit gC75S/L109C by the PCR mutagenesis procedure. The point mutant eC28S was constructed by integration of the mutated version of the TIM11 gene at the chromosomic locus in the deleted-disrupted yeast strain (17Arselin 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). All strains containing the subunit eC28S and different mutations in subunit g were constructed by integration of the mutated versions of the ATP20 gene at the chromosomic locus in the Δg/eC28S yeast strain.Biochemical Procedures—Cells were grown aerobically at 28 °C in a complete liquid medium containing 2% lactate as carbon source and harvested in logarithmic growth phase. The rho- cell production in cultures was measured on glycerol plates supplemented with 0.1% glucose. Mitochondria were prepared from protoplasts as previously described. Protein amounts were determined according to Lowry et al. (22Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) in the presence of 5% SDS using bovine serum albumin as standard. The ATPase activity was measured at pH 8.4 in the presence of 0.375% Triton X-100 to remove the endogenous inhibitor of F1 (23Velours J. Vaillier J. Paumard P. Soubannier V. Lai-Zhang J. Mueller D.M. J. Biol. Chem. 2001; 276: 8602-8607Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar).Cross-linking Experiments—Mitochondria isolated from wild-type and mutant cells were washed by centrifugation in 0.6 m mannitol, 50 mm Hepes, pH 7.4, containing 0.25 mm phenylmethylsulfonyl fluoride. The pellet was suspended at a protein concentration of 5 mg ml-1 in 0.1 m mannitol, 50 mm Hepes, pH 7.4, containing either 5 mm EDTA and 5 mm NEM 1The abbreviations used are: NEM, N-ethylmaleimide; BN-PAGE, blue native-polyacrylamide gel electrophoresis. for the control experiment or 2 mm CuCl2 for cross-linking experiments. Incubations were performed at 4 °C for 30 min, and the reaction was stopped by addition of 5 mm EDTA and 5 mm NEM. Mitochondrial membranes were then dissociated in the presence of 20 mm NEM for SDS-gel electrophoresis and Western blot analysis. For BN-PAGE analyses of cross-linked products, mitochondrial membranes were centrifuged at 10,000 × g for 10 min at 4 °C after incubation with either 5 mm NEM or 2 mm CuCl2. Then proteins were extracted with a digitonin solution containing 5 mm NEM.Electrophoretic and Western Blot Analyses—Molecular mass markers (Benchmark Prestained Protein Ladder) were from Invitrogen. BN-PAGE and SDS-gel electrophoreses were performed as described in Ref. 24Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10442) Google Scholar, Western blot analyses were described previously (25Arselin G. Vaillier J. Graves P.V. Velours J. J. Biol. Chem. 1996; 271: 20284-20290Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Nitrocellulose membranes (Membrane Protean BA83, 0.2 μm from Schleicher & Schuell) were used. Polyclonal antibodies against subunits g, e, and i were raised against amino acid residues 31-45, 69-82, and 39-51, respectively. Antibodies against subunits g, e, and i were used with dilutions of 1:5,000, 1:10,000, and 1:50,000, respectively. Membranes were incubated with peroxidase-labeled antibodies and visualized with 10 ml of 100 mm Tris-HCl, pH 8.5, 20 μm p-coumaric acid, 1.25 mm luminol, and 16.5 mm H2O2. When the visualization for subunit g and subunit e was necessary, membranes were stripped with 2% SDS, 100 mm 2-mercaptoethanol, and 67.5 mm Tris-HCl, pH 6.7, for 20 min at 60 °C before incubation with the second antibody. BN-PAGE experiments were performed as described previously (26Schägger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1886) Google Scholar). Mitochondria (1 mg of protein) were incubated for 30 min at 4 °C with 0.1 ml of digitonin solution with the indicated digitonin/protein ratio. The extracts were centrifuged at 4 °C for 15 min at 40,000 × g, and aliquots (30 μl) were loaded on the top of a 3-13% polyacrylamide slab gel. After electrophoresis, the gel was incubated in a solution of 5 mm ATP, 5 mm MgCl2, 0.05% lead acetate, 50 mm glycine/NaOH, pH 8.4, to reveal the ATPase activity (27Grandier-Vazeille X. Guerin M. Anal. Biochem. 1996; 242: 248-254Crossref PubMed Scopus (62) Google Scholar).RESULTSThe Dimerization Motif GXXXG of the Membrane-spanning Segment of Subunit g—The subunit g is 115 amino acids long, exposing the main part of the sequence in the matricial space of mitochondria and a short C-terminal domain (the last 10 residues) in the intermembrane space. Although this supernumerary subunit is not involved in ATP synthesis, its association with the ATP synthase complex and its participation in the dimerization of the mitochondrial ATP synthase has been well documented (15Paumard P. Vaillier J. Coulary B. Schaeffer J. Soubannier V. Mueller D.M. Brethes D. di Rago J.P. Velours J. EMBO J. 2002; 21: 221-230Crossref PubMed Scopus (579) Google Scholar). Subunit g has been identified in numerous organisms and a multiple alignment shows a limited number of fully conserved amino acid residues all located in the C-terminal part (Fig. 1A). They are the Gly101 and Gly105 residues (Fig. 1B) that lay in a predicted membrane-spanning segment beginning with Leu85 and ending with Gly105. The Tyr112, which is in the intermembrane space, is also conserved. In addition, positions 93, 98, 102, and 106, which display E/Q, F/Y, E/Q, and K/R residues, respectively are semiconservative. The transmembrane segment of subunit g displays a dimerization motif, GXXXG, found in glycophorin A (19Russ W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (775) Google Scholar) at position GEIIG (residues 101 to 105). The full conservation of the glycine residues suggested their involvement in a transmembrane helix-helix interaction. To address whether the conserved amino acid residues have a role in a dimerization process of the ATP synthase, several mutants were constructed. Mitochondria were prepared from the truncated mutants gV100stop, gR106stop, and gY112stop, from the gG101L and gG105L strains where the small residue was replaced by a large residue, and from mutant g102A having an alanine residue inserted after Gly101 to disrupt a helix-helix packing interface. The phenotypes and ATPase activities are reported in Table I.Table IPhenotypic analyses of the yeast strains used The wild-type strain was D273-10B/A/H/U. Yeast cells were grown on complete medium containing 2% lactate as carbon source. The doubling time was calculated for a 10-h period during the logarithmic growing phase. The rho- cell production in cultures was measured on glycerol plates supplemented with 0.1% glucose. ATPase activities and sensitivity to oligomycin (6 μg/ml) were measured in the presence of Triton X-100 to remove the F1 inhibitor.StrainsDoubling time% of rho- cells in culturesATPase activitiesControl+ OligomycinInhibitionmin%μmol of Pi/min/mg of protein%Wild type18216.68 ± 0.290.51 ± 0.1092gC75S/L109C16816.78 ± 0.030.53 ± 0.0592eC28S/gC75S/L109C17026.66 ± 0.190.60 ± 0.0691gQ93A244256.49 ± 0.302.02 ± 0.0369gQ93E18616.60 ± 0.090.51 ± 0.0992gY98A364376.98 ± 0.062.36 ± 0.0666gV100stop238286.98 ± 0.052.83 ± 0.0259gG101L283296.67 ± 0.052.58 ± 0.0861g102A306306.72 ± 0.372.78 ± 0.0859gG105L283276.87 ± 0.052.78 ± 0.0259gR106stop250176.03 ± 0.422.47 ± 0.0959gY112stop17816.56 ± 0.020.45 ± 0.0493 Open table in a new tab With the exception of the mutant gY112stop all other mutants cited above displayed an increase in the doubling time with lactate as the carbon source. In addition, a spontaneous conversion into rho- cells was observed. As reported previously (15Paumard P. Vaillier J. Coulary B. Schaeffer J. Soubannier V. Mueller D.M. Brethes D. di Rago J.P. Velours J. EMBO J. 2002; 21: 221-230Crossref PubMed Scopus (579) Google Scholar, 17Arselin 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), there is a correlation between the increase in the spontaneous rho- cell conversion (rho- cells that are devoid of oxidative phosphorylation are unable to grow with lactate as carbon source) and the increase in the generation time of mutant strains. It is also possible to correlate the increase in the spontaneous rho- cell conversion with the increase in the insensitivity of the mitochondrial ATPase activity of the five mutants toward oligomycin (an inhibitor of membranous domain of the mitochondrial ATP synthase) under the experimental conditions used (pH 8.4 and Triton X-100) (Table I).If the GXXXG motif is juxtaposed in a dimer interface, facilitating close helix-helix approach, a right-handed or a left-handed dimer could be obtained. In a right-handed dimer the semiconservative Gln93 residue could be near the Gln93 in the other subunit g of the dimer, making an interhelical hydrogen bond, an interaction capable of mediating the association of membrane-embedded helices (28Dawson J.P. Melnyk R.A. Deber C.M. Engelman D.M. J. Mol. Biol. 2003; 331: 255-262Crossref PubMed Scopus (72) Google Scholar). In a left-handed dimer, the Tyr98, which is also a semiconservative residue, could be involved (Fig. 1B). Two additional mutations in these semiconservative positions were constructed. The Gln93 was replaced either for an alanine residue or a glutamate residue, an amino acid residue found in subunits g of other species. Whereas substitution of Gln93 by a glutamate did not alter the doubling time and did not increase the rho- cell conversion, gQ93A replacement affected generation time, conversion into rho- cells, and sensitivity to the F0 inhibitor. When Tyr98 was replaced by alanine the effects observed under the doubling time and the conversion into rho- cells were significantly more pronounced than in all other mutations (Table I).Conserved Amino Acid Residues of Subunit g Are Essential for the Presence of Subunit g in the Mitochondrial Membranes—Subunit g is an unstable protein that disappears upon alteration of either subunits e or subunit 4 (b) (14Soubannier 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). As a consequence, the presence of subunit g in mutant mitochondria was examined by Western blot analysis. The blots were probed with polyclonal antibodies raised against subunits g or e. Subunit i, which is a component of the yeast F0, was used as control. The gQ93A, gY98A, gG101L, g102A, and gG105L mutant mitochondria were fully devoid of subunit g (Fig. 2), and subunit e was found principally as a dimer, resulting from the oxidation of Cys28, an observation that has been already reported in mutants devoid of subunit g (29Giraud M.F. Paumard P. Soubannier V. Vaillier J. Arselin G. Salin B. Schaeffer J. Brethes D. di Rago J.P. Velours J. Biochim. Biophys. Acta. 2002; 1555: 174-180Crossref PubMed Scopus (97) Google Scholar). In addition, an unidentified product involving subunit e was observed in the molecular mass range of 17 kDa. A small amount of subunit g was found in gV100stop and gR106stop mitochondria, thus showing that a subunit g at least 106 amino acids long is required for its presence in the mitochondrial membrane. However, the truncated mutant gY112stop has no alterations in physiological parameters (Table I). It shows a normal amount of the modified subunit g as revealed by Western blot (not shown), indicating that conserved residue Tyr112 is not involved in a stabilizing function. Whereas an alanine residue could not replace Gln93, a glutamate residue (gQ93E) altered neither the generation time (Table I) nor the presence of subunit g in the mitochondrial membrane. These data are in agreement with the presence of a glutamate residue at this position in other subunits g (Fig. 1A).Fig. 2Mitochondria isolated from yeast cells having mutations in the membrane-spanning segment of subunit g are deficient in subunit g. Mitochondria isolated from wild-type (lane 1) gV100stop (lane 2), gR106stop (lane 3), null-mutant Δg (lane 4), null-mutant Δg containing the nonintegrative vector pRS313 bearing the ATP20 gene (lane 5), gQ93A (lane 6), gQ93E (lane 7), gY98A (lane 8), gG101L (lane 9), g102A (lane 10), and gG105L (lane 11) were treated with NEM as described under “Experimental Procedures” to prevent disulfide bond formation during the dissociation with SDS. Aliquots (50 μg of protein) were analyzed by Western blot. The blots were incubated with antibodies raised against subunits g and i (A), washed as described under “Experimental Procedures,” and probed again with antibodies raised against subunits e and i (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT)It has been previously reported that the absence of subunit g in the null mutant in the ATP20 gene leads to the loss of supramolecular structures of the ATP synthase (13Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schagger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (361) Google Scholar). Therefore, the presence of supramolecular species of the ATP synthase in the mitochondrial digitonin extracts of gV100stop and gR106stop mutants was examined by BN-PAGE. The digitonin extracts were loaded on a 3-13% acrylamide slab gel, and the mitochondrial complexes were separated under native conditions. The gel was incubated with ATP-Mg2+ and Pb2+ to reveal the ATPase activity (Fig. 3). As reported previously, the wild-type digitonin extracts contained the dimeric and oligomeric forms of the enzyme that were destabilized upon increasing the digitonin-to-protein ratio (13Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schagger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (361) Google Scholar, 29Giraud M.F. Paumard P. Soubannier V. Vaillier J. Arselin G. Salin B. Schaeffer J. Brethes D. di Rago J.P. Velours J. Biochim. Biophys. Acta. 2002; 1555: 174-180Crossref PubMed Scopus (97) Google Scholar). The mitochondrial digitonin extracts of mutant strains did not display any oligomeric form of the ATP synthase. Whatever the digitonin-to-protein ratio used, the monomeric form of the enzyme was predominant, although a small amount of dimeric form was found, as already observed for null mutants devoid of either subunit e or subunit g (13Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schagger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (361) Google Scholar, 30Brunner S. Everard-Gigot V. Stuart R.A. J. Biol. Chem. 2002; 277: 48484-48489Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar).Fig. 3Yeast ATP synthase neither dimerize nor oligomerize upon alterations of the membrane-spanning segment of subunit g. Mitochondria were isolated from wild-type (wt), gR106stop, and gV100stop strains. Digitonin extracts were prepared with the indicated digitonin-to-protein ratios and analyzed by BN-PAGE. The gel was incubated with ATP-Mg2+ and Pb2+ to reveal ATPase activity. %T, acrylamide concentration.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We have already reported that the loss of either subunits e or g led to the loss of mitochondrial cristae, thus indicating a relationship between the presence of supramolecular structures of the yeast ATP synthase and normal mitochondrial cristae morphology (15Paumard P. Vaillier J. Coulary B. Schaeffer J. Soubannier V. Mueller D.M. Brethes D. di Rago J.P. Velours J. EMBO J. 2002; 21: 221-230Crossref PubMed Scopus (579) Google Scholar). Transmission electron microscopy experiments indicated the presence of so-called onion-like structures in gV100stop and gR106stop cells, already described in ATP20 and TIM11 null mutant cells (Fig. 4).Fig. 4Mitochondria isolated from strains with alterations in the membrane-spanning segment of subunit g are defective in mitochondrial morphology. Transmission electron microscopy of yeast cell sections of gV100stop (A) and gR106stop (B) strains. m, mitochondria. The small and large bars indicate 0.1 and 0.5 μm, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Dimerization of Subunit g in the Inner Mitochondrial Membrane—The involvement of the GXXXG motif of subunit g in the formation of a homodimer was investigated. Because the two glycine residues are essential for the structure of this motif, a target was chosen outside the motif but in the alignment of the glycine residues if this part of the protein were a α-helix-like glycophorin A (19Russ W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (775) Google Scholar). As a consequence, a cysteine residue was placed at position 109. In addition, the unique endogenous Cys75 of subunit g was replaced by a serine residue. To prevent disulfide bond formation between subunits e and g, the unique endogenous Cys28 of subunit e was also replaced by a serine residue. Western blot analysis of CuCl2-treated eC28S/gC75S/L109C mitochondria showed the presence of a 26-kDa band that was absent in mutant gC75S, thus indicating the involvement of L109C in the formation of a g - g adduct (Fig. 5, lanes 1 and 2). To demonstrate that the 26-kDa band corresponded to a homodimer of subunit g resulting from the formation of a disulfide bond between the two subunit g, the two following strains were constructed: strain eC28S/gC75S/L109C(His)6 contained a (His)6 tag in the C-terminal part of subunit g. The second mutant was constructed from the eC28S/gC75S/L109C strain by complementation with a pRS316 shuttle vector bearing the gene encoding
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