Glutamic acid in the inhibitory site of mitochondrial ATPase inhibitor, IF(1), participates in pH sensing in both mammals and yeast.
The mitochondrial ATPase inhibitor, IF(1), regulates the activity of F(1)F(o)-ATPase. The inhibitory activity of IF(1) is highly pH-dependent. The effective inhibition by IF(1) requires a low pH. Under basic conditions, its activity markedly declines. The importance of His49 in the pH dependence of bovine IF(1) is well-known. However, the residue is not conserved in yeast IF(1). We previously showed that Glu21 is required for the pH dependence of yeast IF(1), but the function of homologous Glu in mammalian IF(1) is not clear. In this study, we examined the requirement for Glu26 of bovine IF(1) (corresponding to Glu21 of yeast IF(1)) regarding its pH dependence by amino acid replacement. Three mutant proteins, E26A, H49K and the double mutant E26A/H49K, were overexpressed and purified. All mutants retained their inhibitory activity well at pH 8.2, although wild-type IF(1) was approximately 10-fold less active at pH 8.2 than at 6.5. A covalent cross-linking study revealed that both wild-type IF(1) and the E26A mutant formed a tetramer at pH 8.2, although H49K and E26A/H49K mutants did not. These results indicate that, in addition to His49, Glu26 participates in pH sensing in bovine IF(1), and the mechanism of pH sensing mediated by Glu26 is different from the dimer-tetramer model proposed previously.
Ando
Chiyoko
C
Department of Food and Human Health Sciences, Graduate School of Human Life Science, Osaka City University, Sumiyoshi, Osaka, Japan.
Ichikawa
Naoki
N
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D000097795:ATPase Inhibitory Protein
D017931:DNA Primers; D018698:Glutamic Acid; D011506:Proteins; D006180:Proton-Translocating ATPases; D011994:Recombinant Proteins; D000097795:ATPase Inhibitory Protein