Binding of an intrinsic ATPase inhibitor to the interface between alpha- and beta-subunits of F1FoATPase upon de-energization of mitochondria.
Yeast mitochondrial F1FoATPase has three regulatory subunit proteins: ATPase inhibitor, 9K protein, and 15K protein. Mutant yeasts lacking one or more of these protein factors were constructed by gene disruption [Ichikawa, N. et al. (1990) J. Biol. Chem. 265, 6274-6278; Yoshida, Y. et al. (1990) Eur. J. Biochem. 193, 49-53]. Dissipation of the electrochemical potential of protons of the mitochondrial inner membrane by an uncoupler or by a combination of valinomycin and potassium ions induced ATP-hydrolyzing activity of F1FoATPase in mitochondria of all the mutants, as in those of wild-type cells. However, the ATPase activity was inactivated within a minute in normal mitochondria, but was not suppressed in inhibitor-deficient mitochondria, and in mitochondria lacking either 9K or 15K protein, the inactivation of ATPase was slow and incomplete. Covalent binding of inhibitor protein to the enzyme was achieved with a zero length cross-linker, EEDQ, in uncoupled normal mitochondria, in which the inhibitor linked directly to both the alpha- and beta-subunits. This result strongly suggests that the binding site of the inhibitor protein is located at the interface between the two subunits.
Mimura
H
H
Department of Physiological Chemistry, Medical School, Osaka University.
Hashimoto
T
T
Yoshida
Y
Y
Ichikawa
N
N
Tagawa
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D009154:Mutation; D010085:Oxidative Phosphorylation; D011506:Proteins / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011804:Quinolines / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D000097795:ATPase Inhibitory Protein
D000255:Adenosine Triphosphate; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D003432:Cross-Linking Reagents; D011506:Proteins; D006180:Proton-Translocating ATPases; D011804:Quinolines; D000097795:ATPase Inhibitory Protein; C000470:EEDQ