ATP SYNTHASE FROM BOVINE HEART-MITOCHONDRIA - IDENTIFICATION BY PROTEOLYSIS OF SITES IN F-0 EXPOSED BY REMOVAL OF F1 AND THE OLIGOMYCIN-SENSITIVITY CONFERRAL PROTEIN

The exposure to trypsinolysis of subunits of F1F0-ATPase and of its F-0 domain have been compared in everted inner membrane vesicles (submitochondrial particles) made from bovine mitochondria. Treatment of submitochondrial particles with guanidine hydrochloride removed the subunits of F-1-ATPase and the oligomycin-sensitivity conferral protein (OSCP), and exposed sites that were occluded in the intact F1F0-ATPase complex. These sites were identified by purifying the subunits from the isolated F-0 and F1F0-ATPase complexes before and after proteolysis of the vesicles, and by characterizing them by N-terminal sequencing and electrospray-ionization mass spectrometry. In the stripped vesicles, subunit F-6 was completely digested away by either trypsin or chymotrypsin. Trypsin also cleaved subunit b, first at the bond arginine-166-glutamine-167, and then at the consecutive linkages, lysine-120-arginine-121 and arginine-121-histidine-122. Chymotrypsin-sensitive sites were observed after the adjacent methionines 164 and 165. Trypsin also removed amino acids 1-3 of subunit d, and minor cleavage sites were observed in subunit d between amino acids 24 and 25, in subunit g between amino acids 5 and 6, and after amino acid 40 in subunit e. The other subunits remained protected from proteolysis. In membrane-bound F1F0-ATPase, the N-terminus of subunit d was also accessible to trypsin, and subunit e was more susceptible to proteolysis than in F-0. Otherwise the F-0 subunits and the OSCP were protected. Subunits alpha and beta were cleaved by trypsin at the same sites in their N-terminal regions as in purified F-1-ATPase. The trypsinized F-0 was incapable of binding F-1-ATPase in the presence of the OSCP. These experiments and in vitro re-assembly experiments described elsewhere, that were guided by the results of the proteolysis experiments, have helped to establish a central role for subunit b in the formation of the stalk connecting the F-1 and F-0 domains of the F1F0-ATPase complex.