Abstract
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.
Highlights
From the *Boston Biomedical Research Institute, Department of Cell Physiology, Medical School, Department of Biological Chemistry and Molecular Pharmacology, Boston, Massachusetts Boston, Massachusetts
We find that proteoliposomes containing the urea-F0 preparation (UFO) are still able to conduct protons, and that this conductance is blocked by oligomycin and DCCD
F0 has been shown to contain subunits that allow passive transmembrane proton conductance which may be blocked by the classical mitochondrial inhibitors oligomycin and DCCD
Summary
Boston Biomedicai’ Research Institute, Dept. of Cell Physiology, 20 Stanford St., Boston, MA 02114. To that end we have recently reported that Fs, a coupling factor presumed to be a stalk subunit of ATP synthase, is not essential for inhibitor-sensitive proton conduction through F. it is an absolute requirement for energy coupling by the intact enzyme [13]. Protein (OSCP) is another component (molecular mass = 20,967 daltons) of mitochondrial ATP synthase that copurifies with the F0 sector and, together with Fs, is considered to serve as a link between the catalytic (F1) and proton-conducting Both Fg and OSCP have been implicated in the binding of F, to the membrane the evidence concerning this aspect is controversial [15,16,17]. OSCP is not essential for binding F, to F. nor is it a necessary component of the proton-conducting pathway in mitochondrial F,,
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