The F1F0-ATPase complex from bovine heart mitochondria: the molar ratio of the subunits in the stalk region linking the F1 and F0 domains.
The F1 globular catalytic domain and the F0 intrinsic membrane domain of the F1F0-ATPases in bacteria, chloroplasts, and mitochondria are connected by a slender stalk. In the F1F0 complex from bovine heart mitochondria, the stalk is thought to contain subunits OSCP, d, and F6, and the globular part of the membrane bound subunit b, referred to as b'. It has been shown previously that the OSCP, b', d, and F6 proteins can be assembled in vitro into a water soluble complex named the "stalk". The stalk and F1-ATPase together form another complex named F1.stalk. In this paper, the molar ratios of the OSCP, b (or b'), d, and F6 in the stalk, F.stalk, and F1F0-ATPase complexes have been investigated by three independent methods. By quantitation of radioactivity incorporated by S-carboxymethylation with iodo-2-[14C]acetic acid into a stalk complex containing a form of F6 with the mutation Glu3-Cys, it was shown that the stalk consists of equimolar quantities of its four constituent proteins. In the stalk complex containing the natural F6 sequence, this conclusion was confirmed both by quantitation of radioactivity incorporated by Nepsilon-acetimidation with ethyl [1-14C]acetimidate, and by quantitative N-terminal sequence analysis of subunits. By similar Nepsilon-acetimidation experiments, it has been demonstrated that the F1.stalk complex contains one copy per assembly of the OSCP, b', d, and F6 proteins and that the F1F0-ATPase contains one copy per enzyme complex of subunits OSCP, b, and d. The presence of one copy per complex of the OSCP, b' (or b), d, and F6 proteins in the F1.stalk and F1F0-ATPase complexes, respectively, was confirmed by quantitative sequencing.
Collinson
I R
IR
MRC Laboratory of Molecular Biology, Cambridge, U.K.
Skehel
J M
JM
Fearnley
I M
IM
Runswick
M J
MJ
Walker
J E
JE
D000107:Acetylation; D000251:Adenosine Triphosphatases / Q000032:analysis / Q000737:chemistry; D000818:Animals; D002352:Carrier Proteins; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007096:Imidoesters; D007461:Iodoacetates; D019807:Iodoacetic Acid; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry; D008929:Mitochondria, Heart / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases; D009943:Organophosphorus Compounds; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D017421:Sequence Analysis
C043517:2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide; D002352:Carrier Proteins; D007096:Imidoesters; D007461:Iodoacetates; D008565:Membrane Proteins; D009943:Organophosphorus Compounds; C014539:ethyl acetimidate; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C020439:oligomycin sensitivity-conferring protein; D019807:Iodoacetic Acid