STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF SUBUNITS OF THE FO SECTOR OF THE MITOCHONDRIAL FOF1-ATP SYNTHASE

Proteolytic digestion of F1-depleted submitochondrial particles (USMP), reconstitution with isolated subunits and titration with inhibitors show that the nuclear-encoded PVP protein, previously identified as an intrinsic component of bovine heart F(o) (F(o)1) (Zanotti, F. et al. (1988) FEBS Lett. 237, 9-14), is critically involved in maintaining the proper H+ translocating configuration of this sector and its correct binding to the F1 catalytic moiety. Trypsin digestion of USMP, under conditions leading to cleavage of the carboxyl region of the PVP protein and partial inhibition of transmembrane H+ translocation, results in general loss of sensitivity of this process to F(o) inhibitors. This is restored by addition of the isolated PVP protein. Trypsin digestion of USMP causes also loss of oligomycin sensitivity of the catalytic activity of membrane reconstituted soluble F1, which can be restored by the combined addition of PVP and OSCP, or PVP and F6. Amino acid sequence analysis shows that, in USMP, modification by [C-14]N,N'-dicyclohexylcarbodiimide of subunit c of F(o) induces the formation of a dimer of this protein, which retains the C-14-labelled group. Chemical modification of cysteine-64 of subunit c results in inhibition of H+ conduction by F(o). The results indicate that proton conduction in mitochondrial F(o) depends on interaction of subunit c with the PVP protein.