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 (submit ochondrial particles) made from bovine mitochondria. Treatment of subm itochondrial particles with guanidine hydrochloride removed the subuni ts of F-1-ATPase and the oligomycin-sensitivity conferral protein (OSC P), and exposed sites that were occluded in the intact F1F0-ATPase com plex. These sites were identified by purifying the subunits from the i solated 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, s ubunit F-6 was completely digested away by either trypsin or chymotryp sin. Trypsin also cleaved subunit b, first at the bond arginine-166-gl utamine-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 rem oved amino acids 1-3 of subunit d, and minor cleavage sites were obser ved in subunit d between amino acids 24 and 25, in subunit g between a mino acids 5 and 6, and after amino acid 40 in subunit e. The other su bunits remained protected from proteolysis. In membrane-bound F1F0-ATP ase, the N-terminus of subunit d was also accessible to trypsin, and s ubunit e was more susceptible to proteolysis than in F-0. Otherwise th e F-0 subunits and the OSCP were protected. Subunits alpha and beta we re 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 cen tral role for subunit b in the formation of the stalk connecting the F -1 and F-0 domains of the F1F0-ATPase complex.