Su e of the Yeast F1Fo-ATP Synthase Forms Homodimers*

The yeast F1Fo-ATP synthase forms a dimeric complex in the mitochondrial inner membrane. Dimerization of two F1Fo monomeric complexes involves the physical association of two membrane-embedded Fo sectors and in a manner, which is dependent on the Fo subunit, Su e. Sequence analysis of Su e protein family members indicated the presence of a conserved coiled-coil motif. As this motif is often the basis for protein homodimerization events, it was hypothesized that Su e forms homodimers in the inner membrane and that formation of Su e dimers between two neighboring Focomplexes would facilitate dimerization of the F1Fo-ATP synthase complex (Arnold, I., Pfeiffer, K., Neupert, W., Stuart, R. A., and Schägger, H. (1998) EMBO J. 17, 7170–7178). Using a histidine-tagged derivative of yeast Su e, Su e-His12, combined with cross-linking and affinity purification approaches, we have directly demonstrated the ability of the yeast Su e protein to form homodimers. Functionality of the Su e-His12 derivative was confirmed by its ability to assemble into the ATP synthase complex and to support its dimerization in the Δsu e null mutant yeast cells. The close association of two neighboring Su e proteins was also demonstrated using cross-linking with Cu2+, which binds and cross-links a unique Cys residue in neighboring Su e proteins. Finally, we propose a model for the molecular basis of the homodimerization of the Su e proteins.

The yeast F 1 F o -ATP synthase forms a dimeric complex in the mitochondrial inner membrane. Dimerization of two F 1 F o monomeric complexes involves the physical association of two membrane-embedded F o sectors and in a manner, which is dependent on the F o subunit, Su e. Sequence analysis of Su e protein family members indicated the presence of a conserved coiled-coil motif. As this motif is often the basis for protein homodimerization events, it was hypothesized that Su e forms homodimers in the inner membrane and that formation of Su e dimers between two neighboring F o complexes would facilitate dimerization of the F 1 F o -ATP synthase complex (Arnold, I., Pfeiffer, K., Neupert, W., Stuart, R. A., and Schä gger, H. (1998) EMBO J. 17, 7170 -7178). Using a histidine-tagged derivative of yeast Su e, Su e-His 12 , combined with cross-linking and affinity purification approaches, we have directly demonstrated the ability of the yeast Su e protein to form homodimers. Functionality of the Su e-His 12 derivative was confirmed by its ability to assemble into the ATP synthase complex and to support its dimerization in the ⌬su e null mutant yeast cells. The close association of two neighboring Su e proteins was also demonstrated using cross-linking with Cu 2؉ , which binds and cross-links a unique Cys residue in neighboring Su e proteins. Finally, we propose a model for the molecular basis of the homodimerization of the Su e proteins.
Mitochondria, eukaryotic organelles, produce energy in the form of ATP (adenosine 5Ј-triphosphate) by a process termed oxidative phosphorylation. The F 1 F o -ATP synthase complex catalyzes the formation of this ATP from ADP (adenosine 5Јdiphosphate) and in a manner that is coupled to the transport of protons across the inner membrane from the intermembrane space to the matrix (1)(2)(3). In general, F 1 F o -ATP synthase complexes may be resolved into two oligomeric parts, the catalytic F 1 sector, which performs the ATP synthesis and hydrolysis reactions, and the membrane-embedded F o sector, which mediates the proton transport steps. Both the F o and the F 1 sectors are multisubunit protein complexes. The F 1 sector is composed entirely of nuclear-encoded subunits, whereas the membrane-bound F o sector is assembled from both nuclear and mitochondrially encoded proteins (1)(2)(3). The mitochondrial F 1 F o -ATP synthase complexes do not exist as physically independent entities, but rather the complexes appear to be associated together as a larger oligomeric network of complexes in the inner mitochondrial membrane (4,5). Consistent with an earlier hypothesis proposed by Allen (6), formation of this oligomeric F 1 F o -ATP synthase network has been shown to play a critical role in the modulation of the cristae morphology (5).
Formation of the ATP synthase oligomeric network appears to be dependent on the initial dimerization of two F 1 F o -ATP synthase complexes (5). Evidence for the formation of the mitochondrial F 1 F o -ATP synthase dimers, was first demonstrated in the yeast Saccharomyces cerevisiae mitochondria (4,7,8). The dimeric ATP synthase was initially identified following mild detergent solubilization of mitochondrial membranes and subsequent analysis by either size-exclusion chromatography or blue native gel electrophoresis (BN-PAGE) 1 (4,7). A comparison of the subunit composition of the dimeric and monomeric forms of the ATP synthase following the BN-PAGE led to the identification of a number of novel, dimer-specific subunits, termed e, g, and k (Su e, Su g, and Su k, respectively) (4). Deletion of the genes encoding Su e, Su g, or Su k, showed them to be all non-essential subunits of the F 1 F o -ATP synthase complex, as the resulting null mutants (⌬su e, ⌬su g, and ⌬su k, respectively) were viable on non-fermentable carbon sources indicating that their mitochondria were respiratory-competent (4). The Su e and Su g proteins, however, were shown to play an important role in the formation of the dimeric ATP synthase complex. As demonstrated by BN-PAGE analysis, formation of the dimeric ATP synthase was adversely affected in mitochondria isolated from the ⌬su e or ⌬su g null mutants (4). Formation of the Su e-mediated F 1 F o dimers appears to be a prerequisite for the assembly of the F 1 F o complexes into the larger oligomeric network (5). Failure to form this complex network in the ⌬su e null mutant resulted in morphological changes of the mitochondrial inner membrane, with a notable absence of the characteristic inner membrane cristae tubular network (5).
The presence of F 1 F o -ATP synthase dimers has subsequently been characterized in a number of eukaryotic mitochondria, including bovine and human (9). Consistently, the Su e and Su g proteins are conserved throughout eukaryotic evolution, found both in fungal and mammalian mitochondria. Su e and Su g are integral inner membrane proteins, indicating them to be components of the membrane-embedded F o sector (4,7). Consequently, dimerization of the yeast F 1 F o -ATP synthase had been proposed to involve the physical association of two membrane-embedded F o sectors, and in a manner that involves the Su e and Su g proteins (4). Previous work has supported a central role of Su e in this F 1 F o -ATP synthase dimerization event (4). Su e spans the inner membrane once via a single transmembrane segment, located at the extreme N-terminal region (approximately residues 1-25). The C-terminal region of Su e, the bulk of the protein (approximately 70 residues), thus protrudes into the intermembrane space (7). Sequence analysis of known Su e-family members has indicated the presence of a conserved putative coiled-coil motif in the Su e protein, which is located directly C-terminal to the transmembrane segment (7). As coiled-coil motifs can often be the basis for homodimerization events, we previously proposed that Su e protein forms homodimers in the inner membrane (4). Furthermore, formation of Su e-Su e homodimers, between two neighboring F o complexes was hypothesized to play an important role in facilitating dimerization of the F 1 F o -ATP synthase complex (4). In support of this model, it was previously shown that bovine Su e could be chemically cross-linked to a protein of a similar size to Su e, thus providing preliminary support for Su e-Su e dimers (10). Direct demonstration of homodimers of Sue, however, has been lacking to date.
In this present study we directly analyzed the ability of the yeast Su e protein to form homodimers in the mitochondrial inner membrane. We have expressed a histidine-tagged derivative of yeast Su e, Su e-His 12 in either wild type or ⌬su e null mutant yeast cells. We demonstrate the functionality of the tagged Su e derivative by its ability to assemble into the ATP synthase complex and support dimerization of the complex. Using a chemical cross-linking and affinity purification approach, we directly demonstrate the formation of Su e-Su e dimers in the mitochondrial membrane. The close association of two neighboring Su e proteins is also demonstrated using cross-linking with Cu 2ϩ , which binds and cross-links a single Cys residue in neighboring Su e proteins. Finally, taking this information together, we propose a model for the molecular basis of the homodimerization of the F 1 F o -ATP synthase Su e proteins.
Expression of Su e-His 12 -The open reading frame encoding Su e (ATP21/TIM11 gene) was amplified by PCR as a BamHI-XbaI fragment, whereby the translational stop codon of the Su e open reading frame was omitted. The PCR fragment, following restriction digest, was cloned into a yeast multicopy 2 expression plasmid, Yep51-GAL10 (LEU2), which contained the galactose-inducible GAL10 promoter, BamHI and XbaI cloning sites, and a sequence encoding 12 histidine residues and a STOP codon. The resulting plasmid, Yep51-Su e-His 12 was transformed either into the haploid wild type (W303-1A) or into the su e null mutant, ⌬su e, using the protocol described by (13) and leucine positive transformants were selected. Expression of Su e-His 12 in the wild type (WTϩ Su e-His 12 ) and the su e null mutant background (⌬su eϩ Su e-His 12 ) were verified by Western blotting.
Chemical Cross-linking of Su e Protein with DTNB-Isolated mitochondria (100 g of protein) were resuspended in SH buffer (0.6 M Sorbitol, 20 mM Hepes, pH 7.2) at a protein concentration of 0.5 mg/ml. Cross-linking was performed on ice for 30 min in the presence of the sulfhydryl-specific homo-bifunctional reagent 5,5Ј-dithiobis-(2-nitrobenzoic acid) (DTNB, 0.2 mM or 0.4 mM, as indicated.) Following quenching with cysteine, mitochondria were reisolated by centrifugation, lysed in the presence of SDS-containing sample buffer (without ␤-mercaptoethanol), and cooked at 95°C for 5 min. Samples were analyzed by SDS-PAGE using a 16% acrylamide, 0.6% bisacrylamide gel followed by Western blotting.
Affinity Purification of Su e-His 12 on Ni-NTA-Agarose following Lysis under Native Conditions-A batch method was used for the affinity purification of the histidine-tagged Su e protein using Ni-NTA beads (Qiagen). Mitochondria (600 g of protein) were carefully solubilized in 600 l of digitonin-lysis buffer (1% (w/v) digitonin, 150 mM potassium acetate, 30 mM Hepes (pH 7.4), 10 mM imidazole, 1 mM PMSF) for 30 min on ice. Following lysis, 600 l of lysis buffer without digitonin was added, and the samples were subjected to a clarifying spin by centrifugation for 30 min, at 226,000 ϫ g (TLA45 rotor, Beckman TL-100 ultracentrifuge). The supernatant was then added to 90 l of Ni-NTAagarose bead suspension, which had been washed previously in the digitonin-containing lysis buffer. Binding was performed for 60 min at 4°C under constant gentle rotation. The Ni-NTA beads were recovered by centrifugation, the supernatant was removed, and the non-bound proteins were precipitated by the addition of trichloroacetic acid. The Ni-NTA beads were washed three times with the digitonin lysis buffer, and the bound proteins were eluted following the addition of SDScontaining sample buffer containing 400 mM imidazole. Samples were shaken for 10 min at 4°C and then cooked at 95°C for 3 min. Following centrifugation the supernatant was removed and analyzed by SDS-PAGE and Western blotting. Immunedecoration of the resulting blot with Su e and F 1 ␣-subunit-specific antisera was performed.
BN-PAGE Analysis of the Dimeric F 1 F o -ATP Synthase-Mitochondria (200 g of protein) were lysed in 40 l of digitonin buffer (1% (w/v) digitonin, 50 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 10% glycerol 1 mM PMSF) for 30 min on ice and subjected to a clarifying spin (30 min, 226,000 ϫ g, TLA45 rotor, Beckman TL-100 ultracentrifuge). The supernatants were supplemented with 4 l of sample buffer (5% (w/v) Serva Blue G in 500 mM aminocaproic acid) prior to electrophoresis. Samples were then analyzed by BN-PAGE using 5-10% polyacrylamide gradient gels, essentially as described previously (4,14). Following electrophoresis, Western blotting to nitrocellulose was performed, and protein complexes were detected by immunedecoration. The calibration standards used in the BN-PAGE are as follows: bovine thyroglobulin (670 kDa), horse spleen apoferritin (443 kDa), and bovine serum albumin monomer (66 kDa). The dimeric and monomeric forms of the ATP synthase were detected by immunedecoration with F 1 ␣-subunit-specific antisera.
Cross-linking of Su e Protein with Cu 2ϩ -Isolated mitochondria (100 g of protein) were resuspended in SH buffer (0.6 M sorbitol, 20 mM Hepes, pH 7.5) at a protein concentration of 0.5 mg/ml, and cupric sulfate (CuSO 4 ) cross-linking was performed essentially as previously described (15). CuSO 4 was added at concentrations ranging from 0 -10 M, as indicated, and samples were incubated on ice for 30 min. Free sulfhydryl residues were then blocked by the addition of the cysteinemodifying reagent N-ethylmaleimide (10 mM), and excess Cu 2ϩ was quenched following the addition of EDTA (10 mM). Samples were further incubated on ice for 15 min, and mitochondria then were reisolated by centrifugation. Mitochondria were lysed with SDS-containing sample buffer containing N-ethylmaleimide and EDTA (both 10 mM) in the absence of the reductant ␤-mercaptoethanol. Samples were cooked at 95°C for 3 min and then analyzed by SDS-PAGE using an SDS-urea acrylamide gel followed by Western blotting. Miscellaneous-Protein determinations and SDS-PAGE were performed according to published methods (16,17). The Western blot analysis and immunedecoration were performed using available Su e antisera raised against a peptide corresponding to the C-terminal region of the Su e protein (7).

Su e Plays an Essential Role in the Dimerization of the F 1 F o -ATP Synthase Complex-
The assembly state of the F 1 F o -ATP synthase was analyzed in mitochondria isolated from both the su e and su g null mutant yeast strains, together with those isolated from the isogenic wild type strain (Fig. 1A). Following solubilization with digitonin, mitochondrial protein complexes were resolved by BN-PAGE followed by Western blotting. Im-munedecoration with an antibody specific for the F 1 ␣-subunit demonstrated the presence of both dimeric and monomeric forms of the ATP synthase in wild type mitochondria as previously reported (4).
In the su e null mutant mitochondria, the F 1 F o -ATP synthase was resolved as a monomeric complex, with a notable absence of the dimeric form (Fig. 1A). In addition, free F 1 complex was observed in the ⌬su e mitochondria, reflecting the apparent instability of the monomeric F 1 F o -ATP synthase in the absence of the Su e as previously reported (4). Analysis of the F 1 F o -ATP synthase complex in the ⌬su g null mutant mitochondria indicated that as in the ⌬su e mutant, the majority of the complex was present in its monomeric and free F 1 form. In contrast to the ⌬su e null mutant, however, a small fraction of the F 1 F o -ATP synthase complex was present as its dimeric form in mitochondria isolated from the ⌬su g null mutant (Fig. 1A). We conclude therefore that the F 1 F o -ATP synthase can form dimers in the absence of Su g, albeit with limited capacity. Western blots analysis of the mitochondria isolated from the su g null mutant demonstrated that the Su e protein was detectable in the ⌬su g null mutant mitochondria, although strongly reduced relative to the wild type control mitochondria (Fig. 1B). Thus reduced ability of the ATP synthase to form dimers in the ⌬su g mitochondria is most likely directly related to the reduced levels of Su e in these mitochondria (Fig. 1B). In addition the levels of Su k were strongly reduced, yet still detectable, in both the ⌬su e and ⌬su g mitochondria (Fig. 1B). Thus, although not essential for Su k, the presence of Su g and Su e appear to enhance the steady state levels of Su k. Thus taken together, we conclude that the Su e protein plays the central role in forming the F 1 F o -ATP synthase dimer and that Su g protein plays an accessory role, which may involve a stabilization affect on the Su e protein.
Expression and Functionality of the Su e-His 12 Derivative-To facilitate the further characterization of Su e, we constructed a derivative of Su e, which contained an additional 12 histidine residues at the C terminus of the protein. Expression of the resulting histidine-tagged Su e, Su e-His 12 , was achieved by cloning the extended Su e open reading frame encoding Su e-His 12 into a 2 vector, Yep51, which contained a galactose-inducible GAL10 promoter. The Su e-His 12 protein was expressed both in the wild type (WT ϩ Su e-His 12 ) and su e null mutant, ⌬su e, (⌬su eϩSu e-His 12 ), genetic backgrounds. Western blot analysis of mitochondria isolated from the WT ϩ Su e-His 12 and ⌬su eϩSu e-His 12 strains confirmed the expression of the Su e-His 12 derivative ( Fig. 2A).
Following the successful expression of Su e-His 12 , our initial experiments were aimed at demonstrating that the addition of the histidine tag to Su e did not functionally compromise the Su e protein. To do so, mitochondria were isolated from the ⌬su e null mutant, the ⌬su eϩ Su e-His 12 strain, and the isogenic wild type background, and the steady state levels of the ATP synthase subunits Su g and Su k were analyzed. As previously described, the levels of Su g and Su k are significantly decreased in the absence of Su e, i.e. in the ⌬su e mitochondria (4). The functionality of the expressed Su e-His 12 was indicated from its ability to restore the levels of Su g and Su k in the ⌬su e mitochondria (Fig. 2B).
Functionality of the histidine-tagged Su e derivative was further tested by its ability to support the formation of the dimeric ATP synthase in the su e null mutant mitochondria (Fig. 2C). BN-PAGE analysis indicated the presence of the dimeric form of the F 1 F o -ATP synthase in the ⌬su eϩSu e-His 12 mitochondria. Furthermore, expression of the Su e-His 12 protein appeared to support the stabilization of the F 1 F o -ATP synthase by the noticeable lack of the free F 1 sector in the ⌬su eϩSu e-His 12 mitochondria, in contrast to the su e null mutant mitochondria and lacking the Su e-His 12 protein (Fig. 2C).
Assembly of Su e-His 12 into the F 1 F o -ATP Synthase Complex-The functionality of the Su e-His 12 derivative was further directly demonstrated by testing its ability to assemble together with other F 1 F o -ATP synthase complex subunits. Su e-His 12 was expressed in wild type yeast cells, and the mitochondria were isolated from these cells (WT ϩ Su e-His 12 ). Mitochondrial membrane proteins were solubilized with digitonin, a mild detergent known to maintain the assembled dimeric state of the F 1 F o -ATP synthase complex. Following solubilization in this manner, Su e-His 12 was affinity-purified via its histidine tag on a nickel-containing agarose matrix (Ni-NTA beads). Western blot analysis of the material bound to the Ni-NTA beads indicated that in addition to the Su e-His 12 protein both wild type Su e and the ␣ subunit of the F 1 sector (F 1 ␣) had been copurified. The co-purification of F 1 ␣ and Su e on the Ni-NTA beads was dependent on the presence of the Su e-His 12 protein as they were not observed bound to the Ni-NTA beads, following FIG. 1. The presence of Su e is essential for the formation of the F 1 F o -ATP synthase dimer. A, mitochondria isolated from wild type yeast (WT) or from yeast mutants deficient in either Su e (⌬su e) or Su g (⌬su g) were solubilized in digitonin as described under "Experimental Procedures." The dimeric state of the F 1 F o -ATP synthase was analyzed by BN-PAGE followed by Western blotting and decoration with an antiserum specific for the ␣-subunit of the F 1 sector. The positions of the dimeric (V dim. ) and the monomeric (V mon. ) ATP synthase and the free F 1 sector are indicated. B, mitochondria isolated from the ⌬su e, ⌬su g, and the ⌬su k null mutant strains and corresponding wild type (WT) strain were subjected to SDS-PAGE and analyzed by Western blotting for the presence of Su e and Su k proteins. their solubilization from wild type mitochondria, which did not harbor the Su e-His 12 protein (Fig. 3).
In conclusion, these data confirm the ability of the Su e-His 12 to assemble into the F 1 F o -ATP synthase complex. Most importantly, the co-purification of the wild type Su e protein with the histidine-tagged derivative indicates that the F 1 F o complex containing the Su e-His 12 protein must contain at least one additional Su e subunit. Indeed, preliminary stoichiometric analysis of the subunit composition of the dimeric F 1 F o -ATP synthase had suggested two Su e protein subunits/dimeric synthase (4).
Su e Forms a Homodimer-As previously mentioned the Su e proteins contain a conserved predicted coiled-coil motif, often the basis for homodimerization events. This, together with the observation that the Su e-His 12 -purified F 1 F o -ATP synthase complex contains more than one subunit of the Su e protein, led us to test the ability of Su e to form homodimers. We initially tested the ability of Su e (11 kDa) to be cross-linked to another protein of the same size. To do so, mitochondria were subjected to chemical cross-linking using DTNB, Ellman's reagent, a sulfhydryl-specific, reductant cleavable cross-linking reagent. Yeast Su e contains a single Cys residue (Cys-28), which according to hydropathy plots, is located proximal to the C-terminal end of the single transmembrane region of Su e, i.e. at the intermembrane space-side of the inner membrane. Following cross-linking, samples were analyzed by SDS-PAGE in the absence of a reducing agent and then by Western blotting. Using the Su e-specific antibody, Su e and resulting crosslinked adducts were identified (Fig. 4A). When cross-linking was performed in wild type mitochondria, a Su e cross-linked adduct of approximately 22 kDa, the predicted size for a Su e-Su e homodimer was observed. Cross-linking in ⌬su eϩSu e-His 12 mitochondria, resulted in the production of a 26-kDa adduct, a size in agreement for a Su e-His 12 -Su e-His 12 homodimer. Finally, cross-linking of Su e in the wild type mitochondria harboring also the Su e-His 12 protein (WTϩ Su e-His 12 ) resulted in a mixed population of Su e-specific adducts of 22, 24 and 26 kDa in molecular mass (Fig. 4A). From their predicted sizes, we postulate these three adducts to correspond to Su e-Su e, Su e-Su e-His 12 , and Su e-His 12 -Su e-His 12 homodimers, respectively.
To confirm directly that Su e does indeed form homodimers, wild type mitochondria harboring the Su e-His 12 protein were subjected to cross-linking with DTNB. Following cross-linking, the mitochondrial membranes were solubilized with the denaturing detergent SDS to disrupt any non-cross-linked complexes, and the Su e-His 12 protein and cross-linked partners were affinity-purified on Ni-NTA-agarose beads. Affinity-purified Su e and the cross-linked proteins were then incubated with the reductant ␤-mercaptoethanol to cleave the chemical cross-linker and were subsequently analyzed by SDS- PAGE   FIG. 2. Expression of the Su e-His 12 derivative. A, mitochondria (50 g of protein) isolated from the wild type (WT) and ⌬su e null mutant strains and the corresponding strains expressing the Su e-His 12 derivative, WT ϩ Su e-His 12 and ⌬su e ϩ Su e-His 12 , respectively, were subjected to SDS-PAGE and analyzed by Western blotting for the presence of Su e and the Su e-His 12 derivative using a Su e-specific antisera. B, mitochondria (50 g of protein) isolated from the wild type (WT), ⌬su e, and the ⌬su e ϩ Su e-His 12 yeast strains were analyzed by SDS-PAGE and Western blotting. The levels of Su g and Su k were analyzed following decoration with subunit-specific antisera as indicated. C, the dimeric and monomeric forms of the F 1 F o -ATP synthase in mitochondria isolated from the wild type (WT), ⌬su e, and the ⌬su e ϩ Su e-His 12 yeast strains were analyzed by BN-PAGE essentially as described in Fig. 1A. For abbreviations, see Fig. 1A.   FIG. 3. Affinity purification of the Su e-His 12 under native solubilization conditions. Mitochondria from the wild type strain expressing the Su e-His 12 derivative (WT ϩ Su e-His 12 ) and control wild type mitochondria were solubilized in digitonin buffer (see "Experimental Procedures"), and following a clarifying spin, the supernatants were incubated with Ni-NTA agarose beads for 60 min at 4°C. The Ni-NTA beads were then recovered by centrifugation, the supernatant was removed, and the non-bound proteins (Free) were precipitated by the addition of trichloroacetic acid. Following washing of the Ni-NTA beads, the bound proteins (Ni-bound) were eluted following the addition of SDS-containing sample buffer containing 400 mM imidazole. Samples were analyzed by SDS-PAGE, followed by Western blotting. Immunedecoration of the resulting blot with Su e and F 1 ␣-subunit-specific antisera was performed. Total, 10% of total solubilized proteins; Nibound, proteins bound and eluted from the Ni-NTA beads; Free, 10% of the supernatant containing the non-bound proteins. and Western blotting. Immunedecoration with Su e-specific antisera indicated that authentic Su e, in addition to the Su e-His 12 derivative, was bound to the Ni-NTA beads. Su e was not recovered on the Ni-NTA beads if the cross-linking with DTNB was performed in wild type mitochondria, which did not contain the Su e-His 12 derivative (Fig. 4B). Thus co-purification of the authentic Su e with the Ni-NTA beads under these denaturing conditions was dependent on the presence of the Su e-His 12 derivative. Furthermore, recovery of Su e on the Ni-NTA beads following SDS solubilization of the WTϩ Su e-His 12 mitochondria was not observed when the prior cross-linking step was omitted (results not shown). We conclude therefore that Su e can dimerize with the Su e-His 12 derivative, thus directly demonstrating the formation of Su e-homodimers in the mitochondrial membrane.
Cross-linking of Su e-Su e Homodimers with Cu 2ϩ -In addition we have used a second independent cross-linking approach, using Cu 2ϩ ions (15), to gain further evidence that the Cys residue from one Su e protein is in close physical proximity to the Cys residue of the neighboring Su e protein. In the presence of added Cu 2ϩ ions, cross-linking of Su e to an adduct of 22 kDa was observed (Fig. 5). A low level of this Su e-specific 22-kDa adduct was also observed in the absence of added Cu 2ϩ . Presumably the endogenous divalent cation level (no EDTA was present) may have supported the formation of this adduct. The mobility of the Su e-Cu 2ϩ cross-linked adduct was similar to that of the Su e-Su e homodimer, which had been crosslinked with DTNB. Taken together with the DTNB cross-linking data, we conclude therefore that the Su e forms homodimers, which are arranged in such a manner that the single Cys residue present in Su e is in close proximity to the Cys residue of the interacting Su e molecule. DISCUSSION The F 1 F o -ATP synthase forms dimeric complexes in the mitochondrial membrane (4,7). Evidence that these F 1 F o -ATP synthase dimers further assemble together to form a larger network of F 1 F o complexes in the mitochondrial inner membrane was recently presented (5). Our current data indicates that the initial ATP synthase dimerization event is supported through a physical interaction of neighboring F o sectors. We have recently demonstrated that dimerization of the F 1 F o -ATP synthase in intact mitochondria does not involve the natural inhibitor protein, Inh1, which was recently shown to bind to and promote dimerization of purified F 1 sectors in solution (18 -20). Rather, our data indicate that subunits e and g, Su e and Su g, are required for efficient ATP synthase dimer formation. In the absence of Su e, no dimer was observed and the F 1 F o -ATP synthase was present exclusively in its monomeric form. In contrast, in the ⌬su g mutant mitochondria, although strongly reduced in levels, a small percentage of F 1 F o complexes were present as dimers. The levels of Su e are strongly reduced (yet still detectable) in the ⌬su g mitochondria, thus indicating that Su g is involved in the stability of the Su e protein. We propose that Su e plays a central role in the formation of the dimeric F 1 F o -ATP synthase and that Su g plays a supporting role and one that involves stabilization of the Su e protein.
Su e family members contain a conserved coiled-coil motif in their hydrophilic region, just C-terminal to their transmembrane-spanning segments (7). As coiled-coil motifs are often the basis for protein-protein interactions, in particular homodimerization events, we proposed that the Su e mediated dimerization of the F 1 F o -ATP synthase complex involved the formation of Su e-Su e homodimers (4). Preliminary evidence for the presence of the Su e-Su e homodimers was reported earlier as the bovine Su e protein could be cross-linked to another protein of a molecular mass similar to that of Su e itself (10). Here, we have extended that initial observation and, using a chemical cross-linking approach combined with affinity purification, we FIG. 4. Chemical cross-linking of Su e-Su e homodimers. A, mitochondria (100 g of protein) isolated from the wild type (WT) and the ⌬su e null mutant strains and the corresponding strains expressing the Su e-His 12 derivative, WT ϩ Su e-His 12 and ⌬su e ϩ Su e-His 12 , respectively, were treated with the chemical cross-linker DTNB (0.4 mM) (ϩ) or were mock-treated and received Me 2 SO (Ϫ), as described under "Experimental Procedures." Following cross-linking, mitochondria were reisolated by centrifugation and subjected to non-reducing SDS-PAGE and Western blotting. Su e, Su e-His 12 , and their crosslinked adducts were identified following decoration of the resulting blot with Su e-specific antisera. Adducts with electrophoretic mobilities corresponding to Su e-Su e (e-e), Su e-Su e-His 12 (e-e His ) and Su e-His 12 -Su e-His 12 (e His -e His ) are indicated. A 17-kDa protein that crossreacts with the Su e antiserum (present also in the ⌬su e null mutant) is indicated (*). B, following cross-linking with DTNB (0.2 mM, see above), wild type (WT) mitochondria or wild type harboring Su e-His 12 (WT ϩ Su e-His 12 ) (1.5 mg of protein) were solubilized in SDS-lysis buffer, as described under "Experimental Procedures." Following dilution with a Triton X-100-containing buffer and a clarifying spin, the solubilized proteins were incubated with Ni-NTA-agarose beads. The binding and elution of the Su e-His 12 and cross-linked adducts was performed essentially as described above in Fig. 3. have directly demonstrated the formation of Su e-Su e homodimers in the yeast mitochondrial membrane. We have expressed a histidine-tagged Su e derivative, Su e-His 12 , in both wild type and ⌬su e mitochondria. The functionality of this Su e-His 12 derivative was shown 3-fold: (i) by its ability to restore steady-state levels of Su g and Su k when expressed in the ⌬su e yeast strain; (ii) by its ability to assemble and co-purify with the F 1 F o -ATP synthase complex; and finally, (iii) by its ability to support the dimerization of the ATP synthase in the ⌬su e mitochondria. When expressed in wild type cells, Su e-His 12 directly assembles and homodimerizes with the authentic Su e protein. Dimerization of Su e-His 12 with Su e was directly demonstrated by chemical cross-linking followed by their copurification on Ni-NTA agarose.
What is the molecular basis for the Su e-Su e homodimerization? The dimerization of the F 1 F o -ATP synthase is a conserved feature between the mitochondrial F 1 F o -ATP synthase complexes, and hence it is most likely to be supported by a conserved feature of the Su e protein family. For this reason, we consider it unlikely the Su e-Su e interaction is mediated by the extreme C-terminal region of Su e (the last approximately 30 amino acid residues). This C-terminal region (residues 70 -96) of the protein is unique to the yeast Su e homolog (7). Furthermore, the cross-linking of Su e proteins with Cu 2ϩ ions or with DTNB indicates that the unique Cys residue in Su e is in close proximity to the Cys of the associated Su e protein. As the Cys residue is located immediately after the transmembrane-spanning segment and at the interface with the coiledcoil region, it implies that these regions of the Su e polypeptide must be physically close in the Su e dimer. As mentioned previously, the predicted coiled-coil motif is a conserved feature between Su e protein family members. It is therefore highly possible that this region plays an important role in Su e function. Taking this together with the known involvement of coiled-coil regions in protein homodimerization events, it is possible that the Su e homodimerization involves coiled-coil interactions between neighboring Su e proteins. Alternatively, it is possible that the Su e homodimerization process involves helix-helix interactions between the transmembrane helices of interacting Su e subunits. Indeed a conserved GXXXG (G for glycine and X for any amino acid residue) motif has been described to be a frequently occurring sequence of residues that favor helix-helix interactions of transmembrane segments of dimerizing membrane proteins (21)(22)(23). Interestingly, a GXXXG motif is found in the transmembrane segment of the yeast Su e protein. The possible importance of this GXXXG motif in Su e-Su e homodimerization events is further suggested by the fact that the GXXXG motif is conserved between the transmembrane regions of the other Su e family members also. Thus taken together, it is plausible that the coiled-coil motif and/or the GXXXG motif in the transmembrane-spanning segment, rather than the extreme C-terminal region of Su e, play a direct role in the homodimerization of Su e. Experiments designed to test these possibilities are currently ongoing in our laboratory.