Location of protein(s) involved in oligomycin-induced inhibition of mitochondrial adenosinetriphosphatase near the outer surface of the inner membrane

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Abstract
Mitoplasts, that is, mitochondria freed from their outer membranes, were prepared from pig heart. Sonication induced an inversion of these mitoplasts, giving inside-out vesicles. Added cytochrome c could be bound much better to mitoplasts than to sonicated vesicles; addition of trypsin increased adenosinetriphosphatase (ATPase) (ATP phosphohydrolase; EC 3.6.1.3) activity of sonicated vesicles without significantly affecting that of the mitoplasts. Since the site of fixation of cytochrome c was located on the outer side of the inner mitochondrial membrane and since the protein inhibitor of the mitochondrial ATPase was present on the inner face of the inner membrane and was very sensitive to trypsin, it could be concluded that mitoplasts were mainly oriented as normal mitochondria while sonicated vesicles were mainly inverted. Trypsin treatment could abolish the oligomycin sensitivity of ATPase activity of either mitoplasts or sonicated vesicles. Trypsin induced the solubilization of the soluble F1-ATPase of sonicated vesicles while the ATPase activity remained with the mitoplasts after trypsin action. Therefore, trypsin destroyed the oligomycin effect by rupturing the liaison between F1 and the membrane in sonicated vesicles. The effect of trypsin on mitoplasts must be attributed to the hydrolysis of a protein located near the outer surface of the inner membrane that was at least structurally involved in the oligomycin sensitivity of the ATPase complex.