Topology and function of "stalk" proteins in the bovine mitochondrial H+-ATPase

Proton translocating ATPases comprise a hydrophilic sector F1, a membrane sector F0, and, in the case of bovine mitochondria, a connecting "stalk" which is believed to contain the oligomycin sensitivity-conferring protein (OSCP) and coupling factor 6 (F6). The present study was undertaken to verify the accessibility of F6 and OSCP to trypsin and to examine the functional consequences of such treatment. Our data show that F1 binds equally to trypsin-treated F0 and untreated F0, but the former complexes exhibit cold lability and only partial sensitivity to oligomycin. Furthermore, these complexes fail to exhibit ATP-driven proton translocation or ATP-32Pi exchange activity. Trypsinization of F0 does not, however, inhibit passive proton conductance through the membrane sector but actually enhances it. Immunological data indicate extensive degradation of OSCP under conditions where F6 proteolysis is insignificant. Intact H+-ATPase complexes are relatively resistant to both the structural and functional effects of trypsin. We conclude that OSCP is predominantly an extrinsic protein which is shielded by F1 in the native membrane. F6 may also be an extrinsic protein but is shielded from trypsinization by OSCP and/or other F0 polypeptides. The exposed, trypsin-sensitive segments of OSCP are not required for passive proton conductance through F0 but may be required for ATP-driven reactions. We propose that bovine mitochondrial OSCP is a functional analogue of subunit b in the Escherichia coli H+-ATPase.     

ATP synthase complex from bovine heart mitochondria: the oligomycin sensitivity conferring protein is essential for dicyclohexyl carbodiimide-sensitive ATPase.

The requirement of bovine heart mitochondrial oligomycin sensitivity conferring protein (OSCP) in conferring dicyclohexylcarbodiimide (DCCD)-sensitivity to membrane-bound F1 was investigated by using OSCP-depleted membrane fraction (UF0) of ATP synthase. The ATPase activity of UF0-F1 was completely insensitive to DCCD while that of UF0-F1-OSCP was inhibited 95% by 16 microM DCCD. Both UF0-F1 and UF0-F1-OSCP complexes bound 5 nmol [14C]DCCD/mg UF0, and all the radioactivity was found to be associated with the DCCD-binding proteolipid. The data suggest that OSCP may be necessary for transmitting not only energy-linked signals, but also signals induced by F0 inhibitory ligands in mitochondrial energy transduction.     

ATP synthase complex from bovine heart mitochondria. Subunit arrangement as revealed by nearest neighbor analysis and susceptibility to trypsin

The nearest neighbor relationships of bovine mitochondrial H(+)-ATPase subunits were investigated by the chemical cross-linking approach using the homobifunctional cleavable reagents dithiobis(succinimidyl propionate) and disuccinimidyl tartrate. Cross-linked proteins were resolved by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Individual subunits were detected by silver staining or by Western blotting and staining with subunit-specific antisera. Products larger than 80,000 daltons were not analyzed. Interactions between F1 subunits included cross-links between gamma and delta as well as gamma and epsilon subunits. Among F0 subunit interactions were observed cross-links of (i) coupling factor 6 (F6) with 8-, 20-, and 24-kDa proteins, (ii) oligomycin sensitivity-conferring protein (OSCP) with 24-kDa protein, and (iii) 20-kDa protein with 24-kDa protein. In addition, several cross-links among subunits involving F1 and F0 sectors were detected. These included cross-links between F6 and alpha, F6 and gamma, OSCP and alpha/beta, and 24-kDa protein and alpha/beta. Thus, OSCP, F6, and the 24-kDa protein were found to form cross-links with both F1 and F0 subunits. The surface accessibility of F0 subunits was investigated by subjecting aliquots of F0 to trypsin treatment. Our data demonstrated that the rate of degradation was in the order OSCP greater than 24-kDa protein greater than or equal to F6 greater than subunit 6. The degradation of subunits of F0 was prevented in intact or reconstituted F1-F0. Based on our present and previously published observations, a model of H(+)-ATPase has been proposed wherein OSCP, F6, and the 24-kDa protein are placed in the stalk region and the alpha and beta subunits of F1-ATPase have been extended down to the membrane surface to enclose the stalk segment.     

Efficient reconstitution of mitochondrial energy-transfer reactions from depleted membranes and F1-ATPase as a function of the amount of bound oligomycin sensitivity-conferring protein (OSCP)

Pig heart mitochondrial membranes depleted of F1 and OSCP by various treatments were analyzed for their content in alpha and beta subunits of F1 and in OSCP using monoclonal antibodies. Membrane treatments and conditions of rebinding of F1 and OSCP were optimized to reconstitute efficient NADH- and ATP-dependent proton fluxes, ATP synthesis and oligomycin-sensitive ATPase activity. F1 and OSCP can be rebound independently to depleted membranes but to avoid unspecific binding of F1 to depleted membranes (ASUA) which is not efficient for ATP synthesis, F1 must be rebound before the addition of OSCP. The rebinding of OSCP to depleted membranes reconstituted with F1 inhibits the ATPase activity of rebound F1, while it restores the ATP-driven proton flux measured by the quenching of ACMA fluorescence. The rebinding of OSCP also renders the ATPase activity of bound F1 sensitive to uncouplers. The rebinding of OSCP alone or F1 alone, does not modify the NADH-dependent proton flux, while the rebinding of both F1 and OSCP controls this flux, inducing an inhibition of the rate of NADH oxidation. Similarly, oligomycin, which seals the F0 channel even in the absence of F1 and OSCP, inhibits the rate of NADH oxidation. OSCP is required to adjust the fitting of F1 to F0 for a correct channelling of protons efficient for ATP synthesis. All reconstituted energy-transfer reactions reach their optimal value for the same amount of OSCP. This amount is consistent with a stoichiometry of two OSCP per F1 in the F0-F1 complex.     

Resolution and reconstitution of H+ -ATPase complex from beef heart mitochondria

Mitochondrial H+ -ATPase complex, purified by the lysolecithin extraction procedure, has been resolved into a "membrane" (NaBr-F0) and a "soluble" fraction by treatment with 3.5 M sodium bromide. The NaBr-F0 fraction is completely devoid of beta, delta, and epsilon subunits of the F, ATPase and largely devoid of alpha and gamma subunits of F1, where F0 is used to denote the membrane fraction and F1, coupling factor 1. This is confirmed by complete loss of ATPase and Pi-ATP exchange activities. The addition of F1 (400 micrograms X mg-1 F0) results in complete restoration of oligomycin sensitivity without any reduction in the F1-ATPase activity. Presumably, this is due to release of ATPase inhibitor protein from the F1-F0 complex consequent to sodium bromide extraction. Restoration of Pi-ATP exchange and H+ -pumping activities require coupling factor B in addition to F1-ATPase. The oligomycin-sensitive ATPase and 32Pi-ATP exchange activities in reconstituted F1-F0 have the same sensitivity to uncouplers and energy transfer inhibitors as in starting submitochondrial particles from the heavy layer of mitochondria and F1-F0 complex. The data suggest that the altered properties of NaBr-F0 observed in other laboratories are probably inherent to their F1-F0 preparations rather than to sodium bromide treatment itself. The H+ -ATPase (F1-F0) complex of all known prokaryotic (3, 8, 9, 10, 21, 32, 34) and eukaryotic (11, 26, 30, 33, 35-37) phosphorylating membranes contain two functionally and structurally distinct entities. The hydrophilic component F1, composed of five unlike subunits, shows ATPase activity that is cold labile as well as uncoupler- and oligomycin-insensitive. The membrane-bound hydrophobic component F0, having no energy-linked catalytic activity of its own, is indirectly assayed by its ability to regain oligomycin sensitive ATPase and Pi-ATP exchange activities on binding to F1-ATPase (33). The purest preparations of bovine heart mitochondrial F0 show seven or eight major components in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or SDS-PAGE (1, 2, 12, 14), ranging from 6 to 54 ku in molecular weight (12). The precise structure and polypeptide composition of mitochondrial F0 is not known. The F0 preparations from bovine heart reported so far have been derived from H+ -ATPase preparations isolated in the presence of cholate and deoxycholate (11, 33, 36, 37).(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions between the oligomycin sensitivity conferring protein (OSCP) and beef heart mitochondrial F1-ATPase. 1. Study of the binding parameters with a chemically radiolabeled OSCP

Upon treatment of beef heart mitochondrial oligomycin sensitivity conferring protein (OSCP) with [14C]-N-ethylmaleimide ( [14C]NEM) or dithiobis(nitro[14C] benzoate), 1 mol of either SH reagent was incorporated per mol of OSCP. Radiolabeling occurred at the level of the only cysteine residue, Cys-118, present in the OSCP sequence reported by Ovchinnikov et al. [Ovchinnikov, Y. A., Modyanov, N. N., Grinkevich, V. A., Aldanova, N. A., Trubetskaya, O. E., Nazimov, I. V., Hundal, T., & Ernster, L. (1984) FEBS Lett. 166, 19-22]; it did not alter the biological activity of OSCP tested in a reconstituted F0-F1 system that catalyzed oligomycin-sensitive ATPase activity or ATP-Pi exchange. The parameters of [14C]NEM-OSCP binding to isolated beef heart mitochondrial F1 were assessed by equilibrium dialysis. Addition of trace amounts of Tween 20 prevented unspecific adsorption of OSCP. The binding curves showed that each F1 possesses a high-affinity OSCP binding site (Kd = 0.08 microM) and two low-affinity OSCP binding sites (Kd = 6-8 microM). Binding of OSCP to the high-affinity site on F1 is probably responsible for the ability of OSCP to confer oligomycin sensitivity to F1 in the ATPase complex.     

Diethylstilbestrol. A novel F0-directed probe of the mitochondrial proton ATPase

At low concentrations, diethylstilbestrol (DES) is shown to be a potent F0-directed inhibitor of the F0F1-ATPase of rat liver mitochondria. In analogy to other F0-directed inhibitors, DES inhibits both the ATPase and ATP-dependent proton-translocation activities of the purified and membrane bound enzyme. When added at low concentrations with dicyclohexylcarbodiimide (DCCD), a covalent inhibitor, DES acts synergistically to inhibit ATPase activity of the complex. At higher concentrations, DES restores DCCD-inhibited ATPase activity. However, there is no restoration of ATP-dependent proton translocation. Under these conditions DCCD remains covalently bound to the F0F1-ATPase complex and F1 remains bound to Fo. Significantly, when the F0F1-ATPase is inhibited by the Fo-directed inhibitor venturicidin rather than DCCD, DES is also able to restore ATPase activity. In contrast, DES is unable to restore ATPase activity to F0F1 preparations inhibited by the Fo-directed inhibitors oligomycin or tricyclohexyltin. However, combinations of [DES + DCCD] or [DES + venturicidin] can restore ATPase activity to F0F1 preparations inhibited by either oligomycin or tricyclohexyltin. Results presented here indicate that the F0 moiety of the rat liver mitochondrial proton ATPase contains a distinct binding site for DES. In addition, they suggest that at saturating concentrations simultaneous occupancy of the DES binding site and sites for either DCCD or venturicidin promote "uncoupled" ATP hydrolysis.     

A fluorescent derivative of the oligomycin-sensitivity conferring protein (acrylodan-OSCP). Evidence for polarity changes in the environment of CYS118 of OSCP upon binding to mitochondrial F1

The fluorescent probe, 6-acryloyl-2-dimethylaminonaphtalene (acrylodan) was reacted with the oligomycin-sensitivity conferring protein (OSCP). Acrylodan bound covalently to the single cysteinyl residue of the protein. Acrylodan-OSCP was fully competent in conferring oligomycin sensitivity to the mitochondrial F0-F1 ATPase complex. The fluorescence emission peak of acrylodan-OSCP was blue-shifted compared to that of an acrylodan-mercaptoethanol adduct, which means that acrylodan experiences a hydrophobic environment in OSCP. Binding of acrylodan-OSCP to the isolated F1 was accompanied by a red shift of fluorescence. It was achieved in less than 1 s at 25 degrees C. The titration curve revealed one high affinity OSCP binding site per F1. Acrylodan-OSCP appears to be an interesting tool for studying the dynamics of structural changes within the mitochondrial ATPase complex.     

Photolabeling of mitochondrial F1-ATPase by an azido derivative of the oligomycin-sensitivity conferring protein

An azido derivative of the oligomycin sensitivity conferring protein (OSCP) was prepared by alkylation with the bifunctional reagent p-azido phenacyl bromide. Azido-OSCP was fully biologically active in the dark. Upon photoirradiation of a mixture of beef heart mitochondrial F1-ATPase and azido-OSCP, the resulting covalent photoproducts were separated by polyacrylamide gel electrophoresis in the presence of Na dodecyl sulfate and characterized by an immunochemical procedure. OSCP was found to react with the alpha and the beta subunits of F1 with strong preference for the alpha subunit.     

Heterologous expression, purification, and biochemistry of the oligomycin sensitivity conferring protein (OSCP) from yeast.

Yeast Saccharomyces cerevisiae oligomycin sensitivity conferring proteins (OSCP) have been expressed in Escherichia coli. Heterologous expression results in production of a protein that is identical to yeast mature OSCP, including the absence of the initiating methionine residue. Yeast OSCP expressed in E. coli has been purified to homogeneity and it is able to reconstitute oligomycin-sensitive ATPase using purified F1- and F1/OSCP-depleted membranes (electron transport particles (ETP). Binding of F1 to ETP is dependent on the addition of OSCP. Binding studies using 35S-OSCP indicated that OSCP binds to ETP with a Kd of 200 nM and a capacity of 420 pmol/mg particle protein, whereas OSCP does not interact with F1 in the absence of ETP. These data indicate that yeast OSCP must first form a specific complex with F0, which then binds F1 forming the functional complex. To identify functional domains in yeast OSCP, two deletion mutants have been made. Antibodies directed to these deletion products do not inhibit OSCP-dependent binding of F1 to ETP. However, antibodies directed against the last one-third of OSCP greatly reduce the oligomycin sensitivity of the reconstituted ATPase. These data suggest that OSCP is involved in a functional role in energy transduction or proton translocation and serves a structural role in the yeast mitochondrial ATP synthase.     

Interactions between the oligomycin sensitivity conferring protein (OSCP) and beef heart mitochondrial F1-ATPase. 2. Identification of the interacting F1 subunits by cross-linking

Interactions between oligomycin sensitivity conferring protein (OSCP) and subunits of beef heart mitochondrial F1-ATPase have been explored by cross-linking at an OSCP/F1 molar ratio close to 1 to ensure specific high-affinity binding of OSCP to F1 [see Dupuis et al. [Dupuis, A., Issartel, J.-P., Lunardi, J., Satre, M., & Vignais, P.V. (1985) Biochemistry (preceding paper in this issue)]]. Cross-links between F1 subunits and OSCP were established by means of two zero length cross-linkers, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and N-(ethoxycarbonyl)-2-ethoxydihydroquinoline. The cross-linked products were separated by sodium dodecyl suflate-polyacrylamide gel electrophoresis. Coomassie blue staining revealed two cross-linked products of Mr 75 000 and 80 000 which could result from the binding of OSCP to the alpha and beta subunits of F1. Definite identification of the cross-linked products was achieved by chemical labeling with specific radiolabeled reagents and by blotting on nitrocellulose filters followed by immunocharacterization with anti-alpha, anti-beta, and anti-OSCP antibodies. OSCP was found to cross-link with the alpha and beta subunits of F1.     

Effect of 2-hydroxy-5-nitrobenzyl bromide on proton translocation by the mitochondrial H+-ATPase

2-Hydroxy-5-nitrobenzyl bromide, a highly reactive reagent towards tryptophan residues in proteins, is shown to activate the passive proton flux through the inner mitochondrial membrane of bovine heart submitochondrial particles (ETPH). When added at low concentrations, the reagent increased both the ATPase activity of the particles and the passive proton transport rate through the membrane. The presence of oligomycin reduced the extent of the 2-Hydroxy-5-nitrobenzyl bromide action on the proton conductivity suggesting that it acted primarily on the H+-ATPase complex. Similar effects were observed on F1-depleted particles, whilst no effect was observed on the isolated F1-ATPase activity. The results suggest that polypeptides bearing tryptophan residues may be involved in the gating function of proton channels of the mitochondrial membrane and this is particularly evident for the F0F1-ATPase complex.     

Isolation and properties of OSCP and an F1-ATPase binding protein from rat liver mitochondria - evidence against OSCP as the linking "stalk" between F1 and the membrane.

Oligomycin Sensitivity Conferral Protein (OSCP) and an F₁-ATPase Binding Protein were isolated from F₁-depleted rat liver mitochondrial membrane. Their molecular weights on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea were 22,500 and 8,500 respectively. When incubated with liver TUA (trypsin, urea and ammonia-treated) submitochondrial particles, the binding protein was effective in the binding of F₁ to the particles with the resultant particle-bound ATPase activity not oligomycin sensitive. When OSCP was then incubated with the reconstituted membrane-bound ATPase, its activity became oligomycin sensitive. These results suggest that, first; the binding protein, but not OSCP, connects F₁-ATPase to the membrane of rat liver mitochondria and maybe to the “stalk”, if indeed there is a stalk in mitochondrial membrane ATPase complex; and second; the function of OSCP is solely to render the ATPase activity sensitive to oligomycin and other similar inhibitors.     

Recent developments on structural and functional aspects of the F1 sector of H+-linked ATPases

This review concerns the catalytic sector of F1 factor of the H+-dependent ATPases in mitochondria (MF1), bacteria (BF1) and chloroplasts (CF1). The three types of F1 have many similarities with respect to the structural parameters, subunit composition and catalytic mechanism. An alpha 3 beta 3 gamma delta epsilon stoichiometry is now accepted for MF1 and BF1; the alpha 2 beta 2 gamma 2 delta 2 epsilon 2 stoichiometry for CF1 remains as matter of debate. The major subunits alpha, beta and gamma are equivalent in MF1, BF1 and CF1; this is not the case for the minor subunits delta and epsilon. The delta subunit of MF1 corresponds to the epsilon subunit of BF1 and CF1, whereas the mitochondrial subunit equivalent to the delta subunit of BF1 and CF1 is probably the oligomycin sensitivity conferring protein (OSCP). The alpha beta gamma assembly is endowed with ATPase activity, beta being considered as the catalytic subunit and gamma as a proton gate. On the other hand, the delta and epsilon subunits of BF1 and CF1 most probably act as links between the F1 and F0 sectors of the ATPase complex. The natural mitochondrial ATPase inhibitor, which is a separate protein loosely attached to MF1, could have its counterpart in the epsilon subunit of BF1 and CF1. The generally accepted view that the catalytic subunit in the different F1 species is beta comes from a number of approaches, including chemical modification, specific photolabeling and, in the case of BF1, use of mutants. The alpha subunit also plays a central role in catalysis, since structural alteration of alpha by chemical modification or mutation results in loss of activity of the whole molecule of F1. The notion that the proton motive force generated by respiration is required for conformational changes of the F1 sector of the H+-ATPase complex has gained acceptance. During the course of ATP synthesis, conversion of bound ADP and Pi into bound ATP probably requires little energy input; only the release of the F1-bound ATP would consume energy. ADP and Pi most likely bind at one catalytic site of F1, while ATP is released at another site. This mechanism, which underlines the alternating cooperativity of subunits in F1, is supported by kinetic data and also by the demonstration of partial site reactivity in inactivation experiments performed with selective chemical modifiers. One obvious advantage of the alternating site mechanism is that the released ATP cannot bind to its original site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two-dimensional gel electrophoresis of membrane proteins using anionic and cationic detergents. Application to the study of mitochondrial F0-F1-ATPase

Polyacrylamide gel electrophoresis in the presence of a cationic detergent, tetradecyltrimethylammonium bromide (TDAB) has been compared to electrophoresis in the presence of an anionic detergent, sodium dodecyl sulfate (SDS). Although, in both systems, the peptides generally migrated as a function of their molecular weight, the TDAB electrophoresis permitted us to obtain a much better resolution of several peptides of the mitochondrial F0-F1-ATPase, especially for the alpha and beta subunits and for the oligomycin sensitivity conferring protein (OSCP). The differences between the two electrophoretic profiles have been used to devise a new technique of two-dimensional electrophoresis using successively anionic and cationic detergents. This method could be very useful in the case of membrane proteins, which are generally soluble only in the presence of powerful ionic detergents. It has been particularly successful in resolving the small peptides of the F0-F1-ATPase which were difficult to differentiate by other techniques in one- or two-dimensional polyacrylamide gel electrophoresis.     

Identification of subunits a, b, and c1 from Acetobacterium woodii Na+-F1F0-ATPase. Subunits c1, c2, AND c3 constitute a mixed c-oligomer

The Na(+)-F(1)F(0)-ATPase operon of Acetobacterium woodii was recently shown to contain, among eleven atp genes, those genes that encode subunit a and b, a gene encoding a 16-kDa proteolipid (subunit c(1)), and two genes encoding 8-kDa proteolipids (subunits c(2) and c(3)). Because subunits a, b, and c(1) were not found in previous enzyme preparations, we re-determined the subunit composition of the enzyme. The genes were overproduced, and specific antibodies were raised. Western blots revealed that subunits a, b, and c(1) are produced and localized in the cytoplasmic membrane. Membrane protein complexes were solubilized by dodecylmaltoside and separated by blue native-polyacrylamide gel electrophoresis, and the ATPase subunits were resolved by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analyses revealed the presence of subunits a, c(2), c(3), b, delta, alpha, gamma, beta, and epsilon. Biochemical and immunological analyses revealed that subunits c(1), c(2), and c(3) are all part of the c-oligomer, the first of a F(1)F(0)-ATPase that contains 8- and 16-kDa proteolipids.     

Interaction between the oligomycin sensitivity conferring protein and the F0 sector of the mitochondrial adenosinetriphosphatase complex: cooperative effect of the F1 sector

Beef heart mitchondrial oligomycin sensitivity conferring protein (OSCP) labeled with [14C]-N-ethylmaleimide ([14C]OSCP) at the only cysteine residue, Cys-118, present in the sequence [Ovchinnikov, Y. A., Modyanov, N. N., Grinkevich, V. A., Aldanova, N. A., Trubetskaya, O. E., Nazimov, I.V., Hundal, T., & Ernster, L. (1984) FEBS Lett. 166, 19-22] exhibits full biological activity in a reconstituted F0-F1 system [Dupuis, A., Issartel, J. P., Lunardi, J., Satre, M., & Vignais, P. V. (1985) Biochemistry 24, 728-733]. The binding parameters of [14C]OSCP with respect to the F0 sector of submitochondrial particles largely depleted of F1 and OSCP (AUA particles) have been explored. In the absence of added F1, a limited number of high-affinity OSCP binding sites were detected in the AUA particles (20-40 pmol/mg of particles); under these conditions, the low-affinity binding sites for OSCP were essentially not saturable. Addition of F1 to the particles promoted high-affinity binding for OSCP, with an apparent Kd of 5 nM, a value 16 times lower than the Kd relative to the binding of OSCP to F1 in the absence of particles. Saturation of the F1 and OSCP binding sites of AUA particles was attained with about 200 pmol of both F1 and OSCP added per milligram of particles. The oligomycin-dependent inhibition of F1-ATPase bound to AUA particles was assayed as a function of bound OSCP. At subsaturating concentrations of F1, the dose-effect curves were rectilinear until inhibition of ATPase activity by oligomycin was virtually complete, and maximal inhibition was obtained for an OSCP to F1 ratio of 1 (mol/mol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation at a Distance of Proton Conductance through the Saccharomyces cerevisiae Mitochondrial F1F0-ATP Synthase by Variants of the Oligomycin Sensitivity-Conferring Protein Containing Substitutions near the C-Terminus

We have sought to elucidate how the oligomycin sensitivity-conferring protein (OSCP) of the mitochondrial F₁F₀-ATP synthase (mtATPase) can influence proton channel function. Variants of OSCP, from the yeast Saccharomyces cerevisiae, having amino acid substitutions at a strictly conserved residue (Gly166) were expressed in place of normal OSCP. Cells expressing the OSCP variants were able to grow on nonfermentable substrates, albeit with some increase in generation time. Moreover, these strains exhibited increased sensitivity to oligomycin, suggestive of modification in functional interactions between the F₁ and F₀ sectors mediated by OSCP. Bioenergetic analysis of mitochondria from cells expressing OSCP variants indicated an increased respiratory rate under conditions of no net ATP synthesis. Using specific inhibitors of mtATPase, in conjunction with measurement of changes in mitochondrial transmembrane potential, it was revealed that this increased respiratory rate was a result of increased proton flux through the F₀ sector. This proton conductance, which is not coupled to phosphorylation, is exquisitely sensitive to inhibition by oligomycin. Nevertheless, the oxidative phosphorylation capacity of these mitochondria from cells expressing OSCP variants was no different to that of the control. These results suggest that the incorporation of OSCP variants into functional ATP synthase complexes can display effects in the control of proton flux through the F₀ sector, most likely mediated through altered protein—protein contacts within the enzyme complex. This conclusion is supported by data indicating impaired stability of solubilized mtATPase complexes that is not, however, reflected in the assembly of functional enzyme complexes in vivo. Given a location for OSCP atop the F₁-₃₃ hexamer that is distant from the proton channel, then the modulation of proton flux by OSCP must occur at a distance. We consider how subtle conformational changes in OSCP may be transmitted to F₀.     

ATP synthase complex from bovine heart mitochondria. Passive H+ conduction through mitochondrial coupling factor 6-depleted F0 complexes

Submitochondrial particles prepared by treatment of mitochondria with ammonia and silicotungstic acid were found to be deficient in coupling factor 6 according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting and had reduced ATP-Pi exchange activity. Requirement of coupling factor 6 for passive proton conductance through mitochondrial F0 was investigated by assaying the ability of depleted particles to sustain NADH-induced proton fluxes as measured by the quenching of 9-amino-6-chloro-2-methoxyacridine fluorescence. The depleted particles themselves showed negligible quenching, but the quenching increased markedly after treating the particles with oligomycin. The data show for the first time that coupling factor 6-depleted complexes have an active proton channel that can be blocked by oligomycin. Therefore, coupling factor 6 is not essential for inhibitor-sensitive proton conductance through mitochondrial F0.     

Complementation of a Schizosaccharomyces pombe mutant lacking the beta subunit of the mitochondrial ATPase by the ATP2 gene of Saccharomyces cerevisiae

A chimeric plasmid carrying the structural gene (ATP2) for the mitochondrial ATPase beta subunit of Saccharomyces cerevisiae has been used to complement a mutant of Schizosaccharomyces pombe lacking the beta subunit (Boutry, M., and Goffeau, A. (1982) Eur. J. Biochem. 125, 471-477). Transformation with ATP2 restored the growth rate of S. pombe mutant on glycerol as well as the mitochondrial ATPase and 32Pi-ATP exchange activities to approximately 20% of the parental strain. Mitochondria prepared from the transformant contained a normal amount of a hybrid F1-ATPase consisting of the S. cerevisiae beta subunit assembled with the remaining subunits of the S. pombe ATPase complex. The presence of the S. cerevisiae beta subunit in the S. pombe ATPase complex conferred a sensitivity to the energy transfer inhibitors citreoviridin and oligomycin which was like that of the intact S. cerevisiae enzyme. The S. cerevisiae beta subunit assembled into the hybrid ATPase complex was the same size as the mature subunit in S. cerevisiae. These data indicate that the mechanism of mitochondrial import and the assembly of the cytoplasmically synthesized subunits is similar or identical in these evolutionary divergent yeasts. In addition, this study provides a new approach for the construction of hybrid mitochondrial ATPase complexes which can be used to examine the function of selected subunits in energy transduction.