Subunit interaction in the mitochondrial H+-translocating ATPase. The role of oligomycin sensitivity conferral protein and coupling factor 6 in ATPase binding and Pi-ATP exchange in mitochondrial membranes

Oligomycin sensitivity conferral protein, in the absence of coupling factor 6 (F6), is able to bind the ATPase to mitochondrial membranes with an apparent association constant of 10(6) M-1. The F6-dependent ATPase binding has an apparent association constant 1 to 2 orders of magnitude lower than that obtained with oligomycin sensitivity conferral protein. The oligomycin sensitivity conferral protein-dependent, membrane-bound ATPase activity is sensitive to rutamycin while the F6-dependent, membrane-bound ATPase activity is insensitive to rutamycin. F1-ATPase and Type II ATPase require F6 in addition to oligomycin sensitivity conferral protein and FB to reconstitute 32Pi-ATP exchange activity in silicotungstic acid particles. This F6 requirement for the 32Pi-ATP exchange is not related to the F6 effect on the ATPase binding. The Type I ATPase and therefore the 26,500-dalton subunit associated with it requires F6 and FB to reconstitute 32Pi-ATP exchange activity in silicotungstic acid particles. Oligomycin sensitivity conferral protein can be interchanged with the 26,500-dalton ATPase binding protein in the binding of the ATPase and the 32Pi-ATP exchange.     

Reconstitution of Oxidative Phosphorylation and of Oligomycin-Sensitive ATPase by Five- and Six-Subunit Forms of Pea Mitochondrial F(1)-ATPase

Five- and six-subunit forms of F₁-ATPase were purified from pea (Pisum sativum L. cv Homesteader) cotyledon submitochondrial particles. Apart from the usual complement of five subunits, the six-subunit enzyme contained an additional 26,500-dalton protein. Both forms of the F₁-ATPase were used to reconstitute oxidative phosphorylation in F₁-depleted (ASU) as well as in F₁ and oligomycin-sensitivity conferring protein (OSCP)-depleted (ASUA) bovine mitochondrial membranes. The six-subunit enzyme was considerably more efficient in reconstituting the ATP synthesis than the five-subunit enzyme. Both forms of the enzyme were also able to reconstitute the ATPase activity in ASU- as well as in ASUA-particles. There were substantial differences, however, in the oligomycin sensitivity of the ATPase bound to the ASUA-particles: 20 and 60% inhibition by oligomycin was obtained in the case of the five-subunit and six-subunit enzyme, respectively. We conclude, that the 26,500-dalton protein present in the six-subunit F₁-ATPase is responsible for the increase in oligomycin sensitivity of the bound enzyme and functions, therefore, as the plant OSCP.     

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.     

Reconstitution of mitochondrial oligomycin and dicyclohexylcarbodiimide-sensitive ATPase

1. Oligomycin and dicyclohexylcarbodiimide-sensitive ATPase was isolated from beef-heart mitochondria and treated with 3.5 M NaBr in order to remove F1. The residue, called F0, was found to consist of seven components. Five of these are stained by Coomassie blue after dodecylsulfate-polyacrylamide-gel electrophoresis. Two of them correspond to the oligomycin-sensitivity-conferring protein and coupling factor F6, with apparent molecular weights of 21,000 and 9,400, respectively. Three additional polypeptides of molecular weights 23,000, 10,500 and 8,600 were not identified with known proteins. Two components not stained with Coomassie blue were detected by autoradiography of the gels of F0 preincubated with [14C]dicyclohexylcarbodiimide. These two components probably represent monomeric and oligomeric forms of the dicyclohexylcarbodiimide-binding protein. 2. F0 induced an oligomycin and dicyclohexylcarbodiimide-sensitive enhancement of K+ + valinomycin-driven proton translocation across the membrane of artificial phospholipid vesicles. 3. The interaction of F0 with purified, soluble beef heart F1 was investigated. F0 was capable of binding F1 and conferring oligomycin and dicyclohexylcarbodiimide sensitivity and cold stability on its ATPase activity. Furthermore F0 was found to diminish the specific activity of F1-ATPase. A comparison of these effects at varying F0/F1 ratios shows that F0 binds F1 in both an oligomycin-sensitive and an oligomycin-insensitive manner, and that both types of binding involve a conferral of cold stability and a decrease in specific activity. High F0/F1 ratios favoured in oligomycin-sensitive type of binding, indicating that F1 binds preferentially to oligomycin-sensitivity-conferring sites. Treatment of ATPase complex with trypsin resulted in an F0 with a decreased proportion of oligomycin-sensitivity-conferring binding sites and a diminished ability to lower the specific activity an cold lability of F1. 4. Reconstitution of F0 treated with trypsin and F1, oligomycin-sensitivity-conferring protein and F6 showed that at a constant amount of F1 bound, both oligomycin-sensitivity-conferring protein and F6 increased the oligomycin sensitivity of ATPase activity. It was therefore concluded that both of these coupling factors are involved in the conferral of oligomycin sensitivity. 5. The effect of the order of addition of F1, oligomycin-sensitivity-conferring protein and F6 to F0 on the reconstitution of oligomycin-sensitive ATPase activity, and of F1 and oligomycin-sensitivity-conferring protein to submitochondrial particles on the reconstitution of respiratory control, was investigated. The highest values of oligomycin sensitivity and respiratory control were obtained when F1 was added as the first component, indicating that F1 plays a directing role in the organisation of the components.     

The biogenesis of rat liver mitochondrial ATPase. Subunit composition of the normal ATPase complex and of the deficient complex formed when mitochondrial protein synthesis is blocked.

1. An ATPase complex containing 12 subunits was isoalted from rat liver mitochondria. 2. In vivo inhibition of mitochondrial protein synthesis by the chloramphenicol analogue thiamphenicol leads to the formation of an oligomycin-insensitive membrane-bound ATPase complex in mitochondria of regenerating rat liver. 3. This oligomycin-insensitive, membrane-bound ATPase was isolated by the same procedure as the ATPase complex from regenerating livers of untreated animals. 4. SDS-polyacrylamide gel electrophoresis of in vivo labelled ATPase complexes from control and from thiamphenicol-treated rats reveals that three subunits out of the 12 are not synthesized or assembled when the mitochondrial translation activity is blocked. 5. From the subunits synthesized and assembled when mitochondrial pror (Fo) of the ATPase complex (subunit 5). 6. The oligomycin sensitivity-conferring protein seems absent in the ATPase complex formed in the presence of thiamphenicol.     

A novel method for the isolation of calsequestrin from porcine skeletal muscle sarcoplasmic reticulum

Bovine heart submitochondrial particles depleted of F1 by treatment with urea ("F1-depleted particles') were incubated with soluble F1-ATPase. The binding of F1 to the particles and the concomitant conferral of oligomycin sensitivity on the ATPase activity required the presence of cations in the incubation medium. NH4+, K+, Rb+, Na+ and Li+ promoted reconstitution maximally at 40-74 mM, guanidinium+ and Tris+ at 20-30 mM, and Ca2+ and Mg2+ at 3-5 mM. The particles exhibited a negative zeta-potential, as determined by microelectrophoresis, and this was neutralized by mono- and divalent cations in the same concentration range as that needed to promote F1 binding and reconstitution of oligomycin-sensitive ATPase. It is concluded that the cations act by neutralizing negative charges on the membrane surface, mainly negatively charged phospholipids. These results are discussed in relation to earlier findings reported in the literature with F1-depleted thylakoid membranes and with submitochondrial particles depleted of both F1 and the coupling proteins F6 and oligomycin sensitivity-conferring protein.     

On the mechanism of action of alkylguanidines on oxidative phosphorylation in mitochondria.

The effect of octylguanidine and oligomycin on the oxygen uptake of rat liver mitochondria and on the ATPase activity of "sonic" submitochondrial particles has been studied. 1. Octylguanidine inhibits state 3 respiration with glutamate-malate and succinate as substrates, but much lower concentrations are required to inhibit oxygen uptake with the former substrates. State 4 respiration is unaffected by octylguanidine. 2. The titration-curve for the octylguanidine inhibition of glutamate-malate oxidation is hyperbolic and apparently biphasic, half-maximal inhibition is obtained at 30 muM octylguanidine. The octylguanidine-curve for inhibition of succinate oxidation is sigmoid with half-maximal inhibition at about 250 muM. 3. Octylguanidine and oligomycin show additive inhibitory action on state 3 respiration with both glutamate plus malage and succinate as respiratory substrates. 4. Concentrations of oligomycin or octylguanidine, which added separately are ineffective on state 3 respiration, become inhibitory when the two inhibitors are added together. 5. Octylguanidine inhibits the ATPase activity of sonic submitochondrial particles with a hyperbolic titration-curve analogous to that obtained for oligomycin inhibition. The inhibitory actions of octylguanidine and oligomycin on the ATPase activity are additive. 6. It is concluded that octylguanidine acts directly on the ATPase complex and that its binding at the action site is mutually exclusive with the binding of oligomycin. A kinetic explanation is given for the reported higher sensitivity of site I phosphorylation to octylguanidine.     

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.     

Adenosine triphosphatase of rat liver mitochondria: detergent solubilization of an oligomycin- and dicyclohexylcarbodiimide-sensitive form of the enzyme.

The hydrolytic activity of the ATPase bound to purified inner membrane vesicles of rat liver mitochondria can be increased threefold by washing extensively with a high ionic strength phosphate buffer. The specific ATPase activities of such phosphate-washed membranes are the highest reported to date for a mitochondrial membrane preparation (21-24 mumol of ATP hydrolyzed min-1 mg-1 in bicarbonate buffer at 37 degrees C). Deoxycholate (0.1 mg/mg of protein) extracts from these membranes a soluble, cold-stable ATPase complex which exhibits a specific activity under optimal assay conditions of 12 mumol of ATP hydrolyzed min-1 mg-1. This complex is not sedimented by centrifugation at 201000 g for 90 min, and readily passes through a 250-A Millipore filter. The ATPase activity of the soluble complex is inhibited 95% by 2.4 muM oligomycin. In addition, inhibitions of 60% or better are obtained in the presence of 1-8 muM dicyclohexylcarbodiimide, p-chloromercuribenzoate, venturicidin, and aurovertin. While a similar complex may be extracted with Triton X-100 this preparation is always lower in both specific activity and in inhibitor sensitivities than the complex extracted with deoxycholate. Detergents of the Tween and Brij series and other detergents of the Triton series are also much less effective than deoxycholate in solubilizing the oligomycin-sensitive. ATPase complex of rat liver. It is concluded that deoxycholate is superior to other detergents as an extractant of the oligomycin-sensitive ATPase complex of rat liver mitochondria, and that the complex extracted with deoxycholate possesses a closer similarity to the membrane-associated ATPase than does the complex extracted with Triton X-100. These studies document the first report of a detergent-solubilized, oligomycin-sensitive ATPase preparation from rat liver mitochondria.     

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.     

Tetradifon: an oligomycin-like inhibitor of energy-linked activities of rat liver mitochondria

Tetradifon (p-chlorophenyl-2,4,5-trichlorophenyl sulfone) at concentrations between 4.5 and 27.0 nmoles/mg mitochondrial protein provides half-maximal inhibition of the following energy-linked activities of rat liver mitochondria: ADP-stimulated respiration, DNP-stimulated ATPase activity, Mg⁺⁺-stimulated ATPase activity, and P_i-ATP exchange activity. Tetradifon has no effect on the activity of soluble ATPase purified from rat liver mitochondria. Respiration inhibited by tetradifon is restored upon addition of 2,4-dinitrophenol. It is concluded that tetradifon acts at or near the oligomycin sensitivity conferring complex located in the mitochondrial inner membrane.     

Inhibition of mitochondrial ATPase by 2,4,3',5'-tetrahydroxystilbene.

2,4,3',5'-tetrahydroxystilbene (THS) wa s found to inhibit rat liver mitochondrial adenosine triphosphatase (ATPase) activity induced by various concentrations of 2,4-dinitrophenol (DNP). The I50 was found to be 17 nmoles/mg mitochondrial protein. The maximum inhibitory effects of oligomycin and atractyloside on the DNP-activated mitochondrial ATPase activity can be enhanced by adding THS. The atractyloside-insensitive ATPase activity of Lubrol-treated rat liver mitochondria was also inhibited by low concentration of THS. The tetramethoxyderivative of THS was much less effective than the parent compound in depressing the ATPase activity of both intact and Lubrol-treated mitochondria. These observations suggest that the phenolic groups are essential for the mitochondrial actions of THS, and this compound most probably acts by a mechanism different from oligomycin on the mitochondrial ATPase complex.     

The biogenesis of rat-liver mitochondrial ATPase. Evidence that the N, N'-dicyclohexyl carbodiimide-binding protein is synthesized outside the mitochondria

1. Radioactive N,N'-dicyclohexyl carbodiimide (DCCD) is bound as effectively to the N, N'-dicyclohexyl carbodiimide- and oligomycin-sensitive ATPase complex in submitochondrial particles of normal rat liver as to the similar but partially N,N'-dicyclohexyl carbodiimide- and oligomycin-insensitive complex of thiamphenicol-treated rats. The latter complex is deficient in 3 subunits (subunit 6, 7 and 10). 2. Radioactive N,N'-dicyclohexyl carbodiimide is exclusively bound to the subunits present in the bands 8 and 11 of SDS-PAA gels of the purified ATPase complex. These subunits, most likely the dimer and monomer of the N,N'-dicyclohexyl carbodiimide-binding protein, are products of the cytoplasmic protein synthesis. 3. The results together indicate that the N,N'-dicyclohexyl carbodiimide-insensitivity of the ATPase complex formed during in vitro inhibition of mitochondrial protein synthesis, is not caused by a lack of inhibitor binding protein. The same holds for the oligomycin-insensitivity.     

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.     

Affinity labeling of yeast mitochondrial adenosine triphosphatase by reduction with [3H]borohydride.

Oligomycin-sensitive adenosine triphosphatase (ATPase) has been purified in large yields from yeast mitochondria by a procedure employing Sepharose 6B chromatography. The nature of the oligomycin binding site in this purified preparation has been studied by an affinity labeling technique in which oligomycin binding to the ATPase complex was followed by reduction of the complex with sodium [³H]borohydride. A major incorporation of label into protein with a molecular weight near 8000 was noted. This incorporation is dependent on the presence of oligomycin, is blocked by dicyclohexylcar-bodiimide, and is altered by mutations conferring oligomycin resistance to the ATPase. The evidence suggests that the low molecular weight proteolipid component of the ATPase complex is the site of oligomycin binding.     

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.     

Cation requirement for the reconstitution of oligomycin-sensitive ATPase by means of soluble F1-ATPase and F1-depleted submitochondrial particles

Bovine heart submitochondrial particles depleted of F₁ by treatment with urea (‘F₁-depleted particles’) were incubated with soluble F₁-ATPase. The binding of F₁ to the particles and the concomitant conferral of oligomycin sensitivity on the ATPase activity required the presence of cations in the incubation medium. NH⁺₄, K⁺, Rb⁺, Cs⁺, Na⁺ and Li⁺ promoted reconstitution maximally at 40–74 mM, guanidinium⁺ and Tris⁺ at 20–30 mM, and Ca²⁺ and Mg²⁺ at 3–5 mM. The particles exhibited a negative ζ-potential, as determined by microelectrophoresis, and this was neutralized by mono- and divalent cations in the same concentration range as that needed to promote F₁ binding and reconstitution of oligomycin-sensitive ATPase. It is concluded that the cations act by neutralizing negative charges on the membrane surface, mainly negatively charged phospholipids. These results are discussed in relation to earlier findings reported in the literature with F₁-depleted thylakoid membranes and with submitochondrial particles depleted of both F₁ and the coupling proteins F₆ and oligomycin sensitivity-conferring protein.     

ATP synthase complex from bovine heart mitochondria. Passive H+ conduction through F0 does not require oligomycin sensitivity-conferring protein

Oligomycin sensitivity-conferring protein (OSCP) is a water-soluble subunit of bovine heart mitochondrial H(+)-ATPase (F1-F0). In order to investigate the requirement of OSCP for passive proton conductance through mitochondrial F0, OSCP-depleted membrane preparations were obtained by extracting purified F1-F0 complexes with 4.0 M urea. The residual complexes, referred to as UF0, were found to be deficient with respect to OSCP, as well as alpha, beta, and gamma subunits of F1-ATPase, but had a full complement of coupling factor 6 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. These UF0 complexes had no intrinsic ATPase activity and were able to bind nearly the same amount of F1-ATPase in the presence of either OSCP or NH4+ ions alone, or a combination of the two. However, the preparations exhibited an absolute dependency on OSCP for conferral of oligomycin sensitivity to membrane-bound ATPase. The passive proton conductance in UF0 proteoliposomes was measured by time-resolved quenching of 9-amino-6-chloro-2-methoxyacridine or 9-aminoacridine fluorescence following a valinomycin-induced K(+)-diffusion potential. The data clearly establish that OSCP is not a necessary component of the F0 proton channel nor is its presence required for conductance blockage by the inhibitors oligomycin or dicyclohexylcarbodiimide. Furthermore, OSCP does not prevent or block passive H+ leakage. Comparisons of OSCP with the F1-F0 subunits from Escherichia coli and chloroplast lead us to suggest that mitochondrial OSCP is, both structurally and functionally, a hybrid between the beta and delta subunits of the prokaryotic systems.     

On the subunit structure of oligomycin sensitive ATPase

The subunit structure of oligomycin sensitive ATPase has been determined. In addition to the components of F₁, and the so-called oligomycin sensitivity conferring protein, there are four other polypeptides of molecular weights 55,000, 29,000, 20,000 and 10,000 which together form the intrinsic membrane portion of the enzymic complex.