ATP synthase from bovine heart mitochondria: identification by proteolysis of sites in F0 exposed by removal of F1 and the oligomycin-sensitivity conferral protein.
The exposure to trypsinolysis of subunits of F1F0-ATPase and of its F0 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 F1-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 F0 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 F6 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 F0. Otherwise the F0 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 F1-ATPase. The trypsinized F0 was incapable of binding F1-ATPase in the presence of the OSCP. These experiments and in vitro re-assembly experiments described elsewehere, 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 F1 and F0 domains of the F1F0-ATPase complex.
Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.
I R
IR
Collinson
I M
IM
Fearnley
J M
JM
Skehel
M J
MJ
Runswick
J E
JE
Walker
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases; D009840:Oligomycins / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D002352:Carrier Proteins; D006146:Guanidines; D008565:Membrane Proteins; D009840:Oligomycins; D002918:Chymotrypsin; D014357:Trypsin; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C020439:oligomycin sensitivity-conferring protein; D019791:Guanidine