Mitochondrial ATP synthase (F(1)F(0)-ATPase) is regulated by an intrinsic ATPase inhibitor protein, IF(1). We previously found that six residues of the yeast IF(1) (Phe17, Arg20, Glu21, Arg22, Glu25, and Phe28) form an ATPase inhibitory site [Ichikawa, N. and Ogura, C. (2003) J. Bioenerg. Biomembr. 35, 399-407]. In the crystal structure of the F(1)/IF(1) complex [Cabezon, E. et al. (2003) Nat. Struct. Biol. 10, 744-750], the core residues of the inhibitory site interact with Arg408, Arg412 and Glu454 of the beta-subunit of F(1). In the present study, we examined the roles of the three beta residues by means of site-directed mutagenesis. A total of six yeast mutants were constructed: R408I, R408T, R412I, R412T, E454Q, and E454V. The betaArg412 and betaGlu454 mutants (R412I, R412T, E454Q, and E454V) could grow on a nonfermentable lactate medium, but the betaArg408 mutants (R408I and R408T) could not. The ATPase activity of isolated mitochondria was decreased in R412I, R412T, E454Q, and E454V mutant cells, and undetectable in R408I and R408T cells. The subunits of F(1) (alpha, beta, and gamma) were detected in mitochondria from each mutant on immunoblotting, and the F(1)F(0) complex was isolated from them. These results indicate that betaArg408 is essential not for assembly of the F(1)F(0) complex but for the catalytic activity of the enzyme. In the crystal structure of F(1), betaArg408 binds to alphaGlu399 in the alpha(DP)/beta(DP) pair and seems to be important for formation of the closed alpha(DP)/beta(DP) conformation. IF(1) seems to disrupt this alpha(DP)Glu399/beta(DP)Arg408 interaction by binding to beta(DP)Arg408, and to interfere with the change from the open alpha(DP)/beta(DP) conformation to the closed conformation that is required for catalysis by F(1)F(0)-ATPase.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|