Interaction of Regulatory Subunits with the F1 Sector of ATP Synthase in Mitochondria

Abstract

Mitochondrial ATP synthase has two stabilizing factors, 9K protein and 15K protein. The 9K protein was found to bind directly to F1-ATPase in the same way as ATPase inhibitor does. When 9K protein and the inhibitor were added simultaneously to F1-ATPase, they bound to the enzyme competitively, while when added successively with several minutes between their additions only the one added first bound tightly and it was scarcely replaced by the other added later. The F1-ATPase-9K protein complex retained about 60% of the ATP-hydrolyzing activity of the free enzyme and this activity was not inhibited by addition of the inhibitor. These characteristic binding features of the inhibitor and 9K protein were also observed with membrane-bound F1F0-ATPase unless the other stabilizing factor, 15K protein, was present. In the presence of 15K protein both the inhibitor and 9K protein could bind to F1F0-ATPase simultaneously but only one of the two ligands interacted directly with the F1 sector; exchange with the other ligand could not be achieved by the enzyme alone. These results indicated the existence of an assembly for regulating the activity of mitochondrial ATP synthase consisting of an inhibitor and stabilizing factors; the inhibitor and stabilizing 9K protein are counterparts in regulation of the enzyme activity, their bindings resulting in inactive and active F1, respectively.