Efficient reconstitution of mitochondrial energy-transfer reactions from depleted membranes and F 1-ATPase as a function of the amount of bound oligomycin sensitivity-conferring protein (OSCP)

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(1) Pig heart mitochondrial membranes depleted of F 1 and OSCP by various treatments were analyzed for their content in α and β subunits of F 1 and in OSCP using monoclonal antibodies. Membrane treatments and conditions of rebinding of F 1 and OSCP were optimized to reconstitute efficient NADH- and ATP-dependent proton fluxes, ATP synthesis and oligomycin-sensitive ATPase activity. (2) F 1 and OSCP can be rebound independently to depleted membranes but to avoid unspecific binding of F 1 to depleted membranes (ASUA) which is not efficient for ATP synthesis, F 1 must be rebound before the addition of OSCP. (3) The rebinding of OSCP to depleted membranes reconstituted with F 1 inhibits the ATPase activity of rebound F 1, 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 F 1 sensitive to uncouplers. (4) The rebinding of OSCP alone or F 1 alone, does not modify the NADH-dependent proton flux, while the rebinding of both F 1 and OSCP controls this flux, inducing an inhibition of the rate of NADH oxidation. Similarly, oligomycin, which seals the F 0 channel even in the absence of F 1 and OSCP, inhibits the rate of NADH oxidation. (5) OSCP is required to adjust the fitting of F 1 to F 0 for a correct channelling of protons efficient for ATP synthesis. (6) 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 F 1 in the F 0-F 1 complex.