Inhibition of the Uncoupler-Induced Mitochondrial ATP-Hydrolysis by the Cooperative Work of the ATPase Inhibitor, 9K Protein and 15K Protein

Abstract

Yeast mitochondrial F1Fo-ATPase has three regulatory proteins, ATPase inhibitor, 9K protein and 15K protein. Binding of ATPase inhibitor to the enzyme is facilitated and stabilized by cooperative work of 9K protein and 15K protein [Hashimoto, T. et al.(1986) J. Biochem. 99, 251–256]. In the present study, we constructed mutant yeasts lacking in ATPase inhibitor, 9K protein or 15K protein. ATP-synthesizing activities of mitochondria from these mutant yeasts were similar to that of wild-type mitochondria. An uncoupler, CCCP, induced ATP hydrolysis in ATPase inhibitor-deficient mitochondria but not in normal mitochondria. Mitochondria from 9K protein-deficient and 15K protein-deficient cells also exhibited the uncoupler-induced ATP hydrolysis, although their rates were smaller than that of the inhibitor-deficient mitochondria. Submitochondrial particles from the 15K protein-deficient cells exhibited low ATPase activity. However, its ATPase activity gradually increased during the incubation in diluted buffer, while that of wild-type cells remained low. These observations strongly suggest that the ATPase inhibitor acts only to inactivate the ATP-hydrolyzing activity of F1Fo-ATPase when the membrane potential disappeared, and that 15K protein and 9K protein act to reinforce this action of the inhibitor protein.