The Modification of the Conserved GXXXG Motif of the Membrane-spanning Segment of Subunit g Destabilizes the Supramolecular Species of Yeast ATP Synthase*

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.

F 0 F 1 -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 F 1 containing the headpiece is a water-soluble unit that retains the ability to hydrolyze ATP when in soluble form. F 0 is embedded in the membrane and is mainly composed of hydrophobic subunits forming a specific protonconducting pathway. When the F 1 and F 0 sectors are coupled, the enzyme functions as a reversible H ϩ -transporting ATPase or ATP synthase (1)(2)(3)(4). The two connecting stalks are made of components from F 1 and F 0 . The central stalk is a part of the rotor of the enzyme and the second stalk, which is part of the stator, connects F 1 and hydrophobic membranous components of the enzyme probably via a flexible region (1). High resolution x-ray crystallographic data have led to solving the structure of F 1 from different sources (5)(6)(7)(8)(9) and the F 1 -c 10 -ring from Saccharomyces cerevisiae (9).
The mitochondrial F 0 of mammals is composed of 10 different subunits (10), all identified in the S. cerevisiae enzyme (11)(12)(13). Some of these subunits are not required for ATP synthesis function, but are involved in the dimerization/oligomerization of the mitochondrial ATP synthase (13)(14)(15). 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 (12,16) with a membrane-spanning segment probably located at the interface between two ATP synthase monomers (16).
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 (Cys 28 ) 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 (17). 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 detergentsolubilized ATP synthase complexes (18). 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 (13,15).
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 (19). 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 (20). 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 (21).
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 differ-ent subunits of the complex and its participation in the edification of supramolecular ATP synthase species.

EXPERIMENTAL PROCEDURES
Materials-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 (MAT␣, 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 (⌬g) was constructed by PCR-based mutagenesis, and the kan r 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 kan r was isolated from the pUG6 vector and inserted into the EcoRV site in the 3Ј 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 ⌬g 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, 5Ј-TATAAACATCACCACCACCACCACCACCACTA-AGCTTTT-3Ј and 5Ј-GAATTAAAAAGCTTAGTGGTGGTGGTGGTGG-TGGTGATG-3Ј, 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 deleteddisrupted yeast strain (17). 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 ⌬g/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 Ϫ 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. (22) 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 F 1 (23).
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 Ϫ1 in 0.1 M mannitol, 50 mM Hepes, pH 7.4, containing either 5 mM EDTA and 5 mM NEM 1 for the control experiment or 2 mM CuCl 2 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 ϫ g for 10 min at 4°C after incubation with either 5 mM NEM or 2 mM CuCl 2 . 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. 24, Western blot analyses were described previously (25). Nitrocellulose membranes (Membrane Protean BA83, 0.2 m 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 M p-coumaric acid, 1.25 mM luminol, and 16.5 mM H 2 O 2 . 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 experi-ments were performed as described previously (26). 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 ϫ g, and aliquots (30 l) 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 MgCl 2 , 0.05% lead acetate, 50 mM glycine/NaOH, pH 8.4, to reveal the ATPase activity (27).

RESULTS
The 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 (15). 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 Gly 101 and Gly 105 residues ( Fig.  1B) that lay in a predicted membrane-spanning segment beginning with Leu 85 and ending with Gly 105 . The Tyr 112 , 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 (19) 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 Gly 101 to disrupt a helix-helix packing interface. The phenotypes and ATPase activities are reported in Table I.
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 Ϫ cells was observed. As reported previously (15,17), there is a correlation between the increase in the spontaneous rho Ϫ cell conversion (rho Ϫ 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 Ϫ 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 lefthanded dimer could be obtained. In a right-handed dimer the semiconservative Gln 93 residue could be near the Gln 93 in the other subunit g of the dimer, making an interhelical hydrogen bond, an interaction capable of mediating the association of membrane-embedded helices (28). In a left-handed dimer, the Tyr 98 , which is also a semiconservative residue, could be involved (Fig. 1B). Two additional mutations in these semiconservative positions were constructed. The Gln 93 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 Gln 93 by a glutamate did not alter the doubling time and did not increase the rho Ϫ cell conversion, gQ93A replacement affected generation time, conversion into rho Ϫ cells, and sensitivity to the F 0 inhibitor. When Tyr 98 was replaced by alanine the effects observed under the doubling time and the conversion into rho Ϫ 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) (14). 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 F 0 , 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 Cys 28 , an observation that has been already reported in mutants devoid of subunit g (29). 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 Tyr 112 is not involved in a stabilizing function. Whereas an alanine residue could not replace Gln 93 , 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).
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 (13). 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-Mg 2ϩ and Pb 2ϩ 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 (13,29). The mitochondrial digitonin extracts of mutant strains did not display any oligomeric form of the ATP synthase. Whatever the digitonin-toprotein 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 (13,30).
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 (15). 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).
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 ␣-helix-like glycophorin A (19). As a consequence, a cysteine residue was placed at position 109. In addition, the unique endogenous Cys 75 of subunit g was replaced by a serine residue. To prevent disulfide bond formation between subunits e and g, the unique endogenous Cys 28 of subunit e was also replaced by a serine residue. Western blot analysis of CuCl 2treated 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 Ϫ 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 gC75S/ L109C(His 6 ). For the last mutant, Western blot analysis of CuCl 2 -treated mitochondria displayed three bands in the 26-kDa region, which were gC75S/L109C plus gC75S/L109C; gC75S/ L109C plus gC75S/L109C(His) 6 ; and gC75S/L109C(His) 6 plus gC75S/L109C(His) 6 adducts because of their respective apparent molecular masses (Fig. 5, lane 4).
Subunits e and g Are in Close Proximity-Subunit e is another component of the yeast ATP synthase that is involved in the formation of the supramolecular species of the mitochondrial ATP synthase (17). The endogenous Cys 28 of subunit e, which is located at the frontier between the inner mitochondrial membrane and the intermembrane space, is able to homodimerize by oxidation (17). Because it is predicted that gL109 is likely to be located in the intermembrane space, a heterodimerization involving subunits e and gL109C could occur upon oxidation. Because the relative molecular masses of subunits e and g are very similar, the gC75S/L109C(His) 6 strain was used instead of the gC75S/L109C strain to increase the difference in the masses of adducts. Mitochondria of the gC75S/L109C(His) 6 strain and their digitonin extracts (0.75 g of digitonin/g of protein) were incubated in the presence or absence of CuCl 2 and analyzed by Western blot. The blots were probed first with polyclonal antibodies against subunits g and i (Fig. 6A), stripped, and probed again with antibodies against subunits e and i (Fig. 6B). Oxidation promoted a gC75S/ L109C(His) 6 homodimer, a e plus gC75S/L109C(His) 6 heterodimer, and a dimmer of subunit e in mitochondrial membranes, thus indicating that e and g subunits are neighbors in the yeast inner mitochondrial membrane as in bovine mitochondria (16). It appears also that the heterodimer of subunits e and g is more intense than the dimer of subunits e, probably indicating a smaller distance between the Cys 109 of subunit g and the Cys 28 of subunit e than between two Cys 28 . Another interpretation is that there are different accessibility to the antibodies against subunit e on heterodimers of subunits e and g and homodimers of subunits e. On the other hand, the g plus g and e plus e cross-linked products displayed similar intensities. When digitonin extracts were incubated with CuCl 2 , the eand gC75S/L109C(His) 6 subunits were cross-linked, whereas the formation of a disulfide bond between two subunits e and between two subunits g were decreased, thus showing different behavior of subunits g and e in mitochondrial membranes and in detergent extracts, but also the existence of digitonin-stable homodimers of subunits g and subunits e.
The Disulfide Bond Formation between Two Subunits g Stabilizes an Oligomeric Form of the Yeast ATP Synthase-In a previous paper (17), it has been reported that the formation of a disulfide bridge between two subunits e via the Cys 28 by oxidation of mitochondrial membranes leads to the stabilization of an oligomeric form of the ATP synthase in digitonin extracts. Similar experiments involving the mutation gL109C were performed. Mitochondria and digitonin extracts of the eC28S/gC75S/L109C strain were oxidized in the presence of CuCl 2 . Mitochondria were solubilized with digitonin, and detergent extracts were submitted to a BN-PAGE analysis. The digitonin-to-protein ratios of 0.75 and 2 g/g were chosen to extract the membranous proteins. With such ratios, F 1 F 0 oligomers, F 1 F 0 dimers, F 1 F 0 monomer, and F 1 were clearly observed. BN-PAGE analysis revealed the presence of an oligomeric form of the ATP synthase in digitonin extracts of CuCl 2 -treated mitochondria migrating at an acrylamide concentration of 4.8% (Fig. 7A), despite a digitonin-to-protein ratio of 2 g/g, i.e. conditions that fully destabilize the oligomeric forms of the yeast ATP synthase. On the other hand, oxidation of eC28S/gC75S/L109C digitonin extract (digitonin-to-protein ratio of 2 g/g) did not promote the oligomer formation (Fig. 7B).

The Homodimers of Subunits g and e Are Found Only with the Oligomeric Forms of the Yeast ATP Synthase, whereas the Heterodimers of Subunits g and e Are
Associated with the Dimeric Forms of ATP Synthase-To investigate the positions of different homo-and heterodimers of subunits e and g in the supramolecular species of ATP synthase, BN-PAGE analyses were performed with mitochondrial digitonin extracts obtained with a digitonin-to-protein ratio of 0.75 g/g. Under these conditions, only dimeric and oligomeric forms of ATP synthase were found (17). The gC75S/L109C(His) 6 mutant was used to increase the mass of the different adducts. After copper treatment of mitochondria and BN-PAGE analysis of digitonin extracts, gel slices were cut and submitted to SDS-gel electrophoresis in the second dimension. The proteins were transferred onto a nitrocellulose membrane that was probed with polyclonal antibodies against subunit g. Polyclonal antibodies directed against subunit i were also used as control to detect the position of the different forms of ATP synthases, because subunit i is strongly associated with the enzyme at digitonin-to-protein ratio of 0.75 g/g (17,31). Membranes were stripped and probed again with antibodies against subunits e and i. When copper-treated gC75S/L109C(His) 6 mitochondria were solubilized with digitonin, subunits g, e, and i were mainly associated with ATP synthases, and co-migrated with the dimeric and oligomeric forms during native electrophoresis (Fig. 8, A and B). However, the subunit g and subunit e homodimers were found in the oligomeric form of the ATP synthase not in the dimeric form. The heterodimers of subunit g and e were observed mainly in the dimeric form but also in the oligomeric form of the ATP synthase (Fig. 8, A and B). However, it should be noted that a large amount of the heterodimer of subunits e and g was dissociated from the enzyme because it migrated in the front of the BN-PAGE, thus indicating that severe constraints were produced by the cross-link of e and g subunits that led to the dissociation of the e ϩ g adduct from the supramolecular species.

DISCUSSION
The purpose of the present paper was to investigate the role of subunit g in the dimerization/oligomerization process of ATP synthase and to look for the molecular determinants leading to the association of ATP synthases in the inner mitochondrial membrane. Subunits g and e are two supernumerary subunits that are loosely bound to the ATP synthase because they are not present in the monomeric form of the yeast enzyme (15). They are not involved in ATP synthesis but most probably in the biogenesis and folding of cristae, because in their absence FIG. 6. Oxidation promotes the homodimerization of subunits  g and e and the heterodimerization of subunits g and e. Mitochondria (mit.) isolated from gC75S/L109C(His) 6 cells and the mitochondrial digitonin extracts obtained with a digitonin-to-protein ratio of 0.75 g/g (ext.) were incubated with different concentrations of CuCl 2 , as described under "Experimental Procedures." In the absence of CuCl 2 , incubation was performed in the presence of NEM and EDTA. After dissociation of samples, aliquots (50 g of protein) were analyzed by Western blot. The blots were incubated with polyclonal antibodies raised against subunits g and i (A), then membranes were washed and probed again with antibodies against subunits e and i (B). g* ϭ gC75S/L109C(His) 6 .
FIG. 7. The disulfide bond formation between two subunits g stabilizes an oligomeric form of the yeast ATP synthase. Mitochondria isolated from eC28S/gC75S/L109C cells were incubated in the absence or presence of CuCl 2 , before (A) or after (B) digitonin extraction. Digitonin extracts obtained with the indicated digitonin-to-protein ratios were submitted to BN-PAGE. The gel was incubated with ATP-Mg 2ϩ and Pb 2ϩ to reveal ATPase activity. NEM and EDTA were added in the control experiments instead of CuCl 2 . %T, acrylamide concentration. mitochondria display onion-like structures (15,32) at the same time as the supramolecular structures of the ATP synthase are lost. Like subunit e, subunit g has a membranous GXXXG dimerization motif that could be the basis of the dimerization/ oligomerization of mitochondrial ATP synthases.
The Involvement of the Membranous GXXXG Motif in the Dimerization/Oligomerization of Mitochondrial ATP Synthases-The involvement of the GXXXG motif of subunit e in the formation of supramolecular species of ATP synthase has recently been shown (17). Although, subunit g is closely related to subunit e, neither its dimerization nor its involvement in the architecture of supramolecular species of the ATP synthase has been reported so far. The GXXXG motif provides a basic scaffold responsible for mediating transmembrane helix-helix interactions. Indeed, early studies on glycophorin A have shown that the central GXXXG portion was the most crucial part of the interaction motif, as judged by its hypersensitivity to mutation, and its ability to singularly mediate the assembly of otherwise monomeric sequences. Although biased to this righthanded crossing motif, it was found that 80% of the highaffinity isolates contained the GXXXG motif (21).
Replacement of the first and last amino acid residues of the GXXXG motif of subunit g by leucine residues, the insertion of an alanine residue in the putative dimerization motif, and the truncation of subunit g after residues 99 and 105 led to the loss of subunit g, with the following consequences: a spontaneous homodimerization of subunit e by its unique cysteine residue, which was found loosely or not associated to the yeast ATP synthase in mutants devoid of subunit g (17), and a concomitant loss of supramolecular species of ATP synthase, highlighting the importance of the GXXXG motif and surrounding residues in the stability of subunit g in the mitochondrial membranes. We already know that this stability is dependant on the presence of subunit e and the first membrane-spanning segment of subunit 4 (b). To explore the environment of subunit g, the gC75S/L109C mutant was constructed, thus generating a target located in the intermembrane space. Incubation of mitochondrial membranes with copper led to a homodimer of subunit g and a heterodimer of subunits e and g resulting from disulfide bond formation between two gL109C and between gL109C and eC28, respectively. In addition, subunit e homodimer was found as described previously (17). Thus, these data reveal the proximity in the mitochondrial membrane of several subunits e and g that likely interact. An interaction domain between subunits e and g mediated by their respective GXXXG motifs was unlikely because the GXXXG motif of subunit e is located in the middle of the spanning segment (17) and that of subunit g in the C-terminal part of the spanning segment, the formation of the e ϩ g adduct by oxidation being the consequence of the proximity of the respective cysteine residues. The dimer formation of subunits g by the Cys 109 located on the same face as the GXXXG motif more likely indicates that this motif constitutes an interaction domain between two subunits g in the inner mitochondrial membrane. However, the amount of the g Ϫ g adduct was highly decreased in coppertreated digitonin extracts, whereas the amount of the e Ϫ g adduct was not. This indicates that the interface between subunits e and g was kept in the digitonin extracts, which contain supramolecular species of ATP synthase, whereas that between two subunits g and between two subunits e was not (Fig. 6). We hypothesize that the e Ϫ g adduct is the result of an intramolecular cross-linking and that the adduct between two subunits e and between two subunits g is the result of a cross-linking between ATP synthase dimers because the e Ϫ e and g Ϫ g adducts were found mainly associated with the oligomeric forms of ATP synthase (Fig. 7).
Oligomeric Forms of Mitochondrial ATP Synthase-Whether oligomeric forms of the ATP synthase exist in the inner mitochondrial membrane is still a matter of discussion. In yeast, oligomeric forms of ATP synthase have been found in mitochondrial digitonin extracts obtained with digitonin-to-protein ratios of 0.75 g/g, but they were absent at higher ratios. However, the formation of disulfide bonds either between subunits e via Cys 28 in wild type mitochondria (17) or between subunits g via the L109C residues promoted an oligomeric form of ATP synthase that was stabilized in digitonin extracts and which migrated at an acrylamide concentration of 4.8%, thus corresponding to a tetrameric form of the enzyme (15). The association by oxidation of ATP synthases in supramolecular structures higher than dimeric forms could result from the Brownian lateral diffusion of proteins in the inner mitochondrial membrane. However, (i) the experiments were performed at 4°C to decrease the diffusion, (ii) no other cross-links between the ATP synthase and other mitochondrial complexes have been identified so far, (iii) the oligomeric structures of ATP synthase exist without cross-linking in mitochondrial digitonin extracts, and (iv) cross-linked oligomeric arrangements of ATP synthase in yeast cells have been obtained during periods of respiratory growth (33). Taken together these data are in favor of the existence in the inner mitochondrial membrane of associations of ATP synthases whose masses are higher than those of ATP synthase dimers. Such oligomeric forms imply the existence of two different interfaces between ATP synthase monomers. On the basis of cross-linking data on mitochondrial membranes, it was proposed that two subunits 4 (subunit b) belonging to two neighboring ATP synthases participate at one interface (34). Recent data indicate that this interface more likely involves the peripheral stalk of the ATP synthase because homodimers of the yeast F 6 (subunit h) have been obtained by cross-linking (35). Moreover, a recent report showed that dimerization of the chloroplast ATP synthase of FIG. 8. Upon oxidation, the g ؉ g and e ؉ e dimers were associated only with the oligomeric forms of the ATP synthase, whereas the heterodimer of subunits e and g was mainly associated with dimeric forms of the enzyme. gC75S/L109C(His) 6 mitochondria were incubated with CuCl 2 as described under "Experimental Procedures." Mitochondrial digitonin extracts were obtained with a digitonin-to-protein ratio of 0.75 g/g and analyzed by BN-PAGE. A part of the gel was revealed by ATPase activity, corresponding slices were cut, incubated with 1% SDS, and submitted to SDS-gel electrophoresis (second dimension). The gel proteins were transferred onto a nitrocellulose membrane, which was probed with polyclonal antibodies raised against subunits g and i (A), then membranes were washed and probed again with antibodies against subunits e and i (B).
Clamydomonas reinhardtii (an enzyme devoid of subunits e and g) was mediated by the peripheral hydrophilic parts of subunits I (homologous to the mitochondrial b-subunit) associated with the ␣ 3 ␤ 3 hexamer (36). The second interface involves subunits e and g and it appears that the dimerization motifs in subunits g and e are essential for the stability of this interface. However, the two interfaces are not independent because the removal of the N-terminal part of subunit 4 leads to the loss of subunit g and the supramolecular species of ATP synthase, and the removal of subunits e or g leads also to the loss of supramolecular species of ATP synthase.
A Tentative Model of the Interaction between Subunits g and e-The mutation L109C was especially important, because in a normal ␣-helix it is located approximately on the same face of the GXXXG motif, and its cross-linking to another subunit g is proof that the GXXXG motif participates in the interface of subunit g dimers. Although, all structures and models known to date involve a right-handed crossing of two straight ␣-helices (20,21,(37)(38)(39), centered on a single GXXXG motif, a priori, we have no information enabling us to make a choice between the right-or left-handed dimer. In a right-handed dimer of subunit g, the semiconservative amino acid Gln 93 could be involved in stabilization of dimer by an intersubunit hydrogen bond, whereas, in a left-handed dimer the Tyr 98 could be implicated.
Recently, Dawson et al. (28) studied the function of polar residues in transmembrane sequences. They found that the interaction of two glutamines, mainly by hydrogen bonds, are primarily responsible for mediating the observed transmembrane associations, but affinities are modulated by the packing interactions provided by their surrounding residues. In this sense, mutant mitochondria gQ93E, where a polar residue has been changed by a negatively charged residue maintaining the possibility of hydrogen bonding interactions, has physiological and biochemical properties indistinguishable from the wildtype. This is not the case with mutant gQ93A, which cannot display hydrogen bonding interactions and loose subunit g. If two subunits g interact via a right-handed dimer involving gQ93, then Tyr 98 would participate to another interface between subunit g and another subunit. It has been demonstrated that subunits e and 4 (b) are in close relationship with subunit g (17). Indeed, it is possible to obtain a heterodimer between subunits g and e, by oxidation of endogenous eC28 and gC75S/L109C(His) 6 (Fig. 6). A simple model of subunits g and e shows that Tyr 98 is close to Phe 17 and Phe 18 in subunit e (Fig.  9). Tyr 98 is a semiconserved amino acid that is replaced by Phe in all other known subunits g (Fig. 1), a similar feature is observed for Phe 18 of subunit e, because it is replaced by Tyr in all other known subunits (see Ref. 17). This type of co-evolution has been described for many protein-protein interactions, where a mutated sequence could be retained if compensatory mutations that preserve the interaction occur in its interacting partners (40). Experiments are underway to define the interaction domains between subunits e and g.