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The inner membrane of mitochondria from various strains of Saccharomyces cerevisiae has been analyzed with the patch clamp technique for comparison with the better known homologous membrane in mammals (Sorgato, M. C., and Moran, O. (1993) CRC Crit. Rev. Biochem. Mol. Biol. 18, 127-171). Differently than in mammals, the yeast inner membrane was found to harbor essentially two channels with similar anionic selectivity but otherwise different functional behavior. One had a conductance of around 45 picosiemens (in symmetrical 150 mM KCl) and an activity only marginally sensitive to voltage. The other channel was prominent for the higher outwardly rectifying current and for the dependence upon voltage of the open probability that induced rapid closure at physiological (negative) membrane potentials. Particularly interesting was the effect of ATP (Mg2+ free) added on the matrix side of the membrane. In the case of the lower conducting channel, the nucleotide caused an immediate block of activity (IC50, 0.240 mM), whereas it locked the larger conductance in the open state at both positive and negative potentials. In proteoliposomes containing both mitochondrial membranes, the small conductance was clearly evident, whereas a larger channel, cationic and without the voltage dependence typical of that in the native inner membrane, was found.

In order to assess the role of thiol groups in the Fo part of the ATP synthase in the coupling mechanism of ATP synthase, we have treated isolated Fo, extracted from beef heart Complex V with urea, with thiol reagents, primarily with diazenedicarboxylic acid bis-(dimethylamide) (diamide) but also with Cd2+ and N-ethylmaleimide. FoF1 ATP synthase was reconstituted by adding isolated F1 and the oligomycin-sensitivity-conferring-protein (OSCP) to Fo. The efficiency of reconstitution was assessed by determining the sensitivity to oligomycin of the ATP hydrolytic activity of the reconstituted enzyme. Contrary to Cd2+, incubation of diamide with Fo, before the addition of F1 and OSCP, induced a severe loss of oligomycin sensitivity, due to an inhibited binding of F1 to Fo. This effect was reversed by dithiothreitol. Conversely, if F1 and OSCP were added to Fo before diamide, no effect could be detected. These results show that F1 (and/or OSCP) protects Fo thiols from diamide and are substantiated by the finding that the oligomycin sensitivity of ATP hydrolysis activity of isolated Complex V was also unaltered by diamide. Gel electrophoresis of FoF1 ATP synthase, reconstituted with diamide-treated Fo, revealed that the loss of oligomycin sensitivity was directly correlated with diminution of band Fo 1 (or subunit b). Concomitantly a band appeared of approximately twice the molecular weight of subunit Fo 1. As this protein contains only 1 cysteine residue (Walker, J. E., Runswick, M. J., and Poulter, L. (1987) J. Mol. Biol. 197, 89-100), the effect of diamide is attributed to the formation of a disulfide bridge between two of these subunits. These results offer further evidence for the proposal, based on aminoacid sequence and structural analysis, that subunit Fo 1 of mammalian Fo is involved in the binding with F1 (Walker et al. (1987]. N-Ethylmaleimide affects oligomycin sensitivity to a lesser extent than diamide, suggesting that the mode of action of these reagents (and the structural changes induced in Fo) is different.