The F1F0-ATPase complex from bovine heart mitochondria: The molar ratio of the subunits in the stalk region linking the F-1 and F-0 domains

The F-1 globular catalytic domain and the F-o intrinsic membrane domain of the F1Fo-ATPases in bacteria, chloroplasts, and mitochondria are connected by a slender stalk. In the F1Fo complex from bovine heart mitochondria, the stalk is thought to contain subunits OSCP, d, and F-6, 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 F-6 proteins can be assembled in vitro into a water soluble complex named the ''stalk''. The stalk and F-1-ATPase together form another complex named F-1 . stalk. In this paper, the molar ratios of the OSCP, b (or b'), d, and F-6 in the stalk, F-1 . stalk, and F1Fo-ATPase complexes have been investigated by three independent methods. By quantitation of radioactivity incorporated by S-carboxymethylation with iodo-2-[(14)]acetic acid into a stalk complex containing a form of F-6 With the mutation Glu(3)-Cys, it was shown that the stalk consists of equimolar quantities of its four constituent proteins. In the stalk complex containing the natural F-6 sequence, this conclusion was confirmed both by quantitation of radioactivity incorporated by N-epsilon-acetimidation with ethyl [1-C-14]acetimidate, and by quantitative N-terminal sequence analysis of subunits. By similar N-epsilon-acetimidation experiments, it has been demonstrated that the F-1 . stalk complex contains one copy per assembly of the OSCP, b', d, and F-6 proteins and that the F1Fo-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 F-6 proteins in the F-1 . stalk and F1Fo-ATPase complexes, respectively, was confirmed by quantitative sequencing.