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Journal of Biochemistry Advance Access originally published online on August 7, 2008
Journal of Biochemistry 2008 144(4):547-553; doi:10.1093/jb/mvn100
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© 2008 The Japanese Biochemical Society.

Glutamic Acid in the Inhibitory Site of Mitochondrial ATPase Inhibitor, IF1, Participates in pH Sensing in Both Mammals and Yeast

Chiyoko Ando and Naoki Ichikawa*

Department of Food and Human Health Sciences, Graduate School of Human Life Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585, Japan

*To whom correspondence should be addressed. Tel/Fax: +81-6-6605-2870, E-mail: ichikawa{at}life.osaka-cu.ac.jp

Received June 26, 2008; Accepted August 2, 2008


  Abstract

The mitochondrial ATPase inhibitor, IF1, regulates the activity of F1Fo-ATPase. The inhibitory activity of IF1 is highly pH-dependent. The effective inhibition by IF1 requires a low pH. Under basic conditions, its activity markedly declines. The importance of His49 in the pH dependence of bovine IF1 is well-known. However, the residue is not conserved in yeast IF1. We previously showed that Glu21 is required for the pH dependence of yeast IF1, but the function of homologous Glu in mammalian IF1 is not clear. In this study, we examined the requirement for Glu26 of bovine IF1 (corresponding to Glu21 of yeast IF1) 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 IF1 was ~10-fold less active at pH 8.2 than at 6.5. A covalent cross-linking study revealed that both wild-type IF1 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 IF1, and the mechanism of pH sensing mediated by Glu26 is different from the dimer–tetramer model proposed previously.

Key Words: ATP synthase (F1Fo-ATPase), IF1 (ATPase inhibitor), mitochondria, pH sensing, regulation

Abbreviations: F1 or F1-ATPase, catalytic part of ATP synthase; F1F0 or F1F0 -ATPase, mitochondrial ATP synthase; I50, amount of IF1 required to inhibit 50% of F1F0-ATPase; IF1, regulatory subunit of ATP synthase (mitochondrial ATPase inhibitor protein)


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