Stability of the Escherichia coli ATP synthase F0F1 complex is dependent on interactions between gamma Gln-269 and the beta subunit loop beta Asp-301-beta Asp-305.
The role of the conserved sequence motif 301DDLTDP306 in the F0F1 ATP synthase beta subunit was assessed by mutagenic analysis in the Escherichia coli enzyme. Mutations gave variable effects on F1 sector activity, stability, and membrane binding to the F0 sector. Upon solubilization, F1 sectors of the betaD302E and betaD305E mutants (betaAsp-302 and betaAsp-305 replaced by glutamate) dissociated into subunits, while mutants with other beta305 substitutions failed to assemble. Membrane ATPase activities of beta301 and 302 mutants were 20-70% of wild type. Replacements of the gamma subunit Gln-269 had similar effects. The membrane ATPase activities of the gammaQ269E or gammaQ269D mutants were significantly lower and their F1 sectors dissociated into subunits upon solubilization. These results suggest that the beta301-305 loop and the gamma subunit region around Gln-269 form a key region for the assembly of alpha3 beta3 gamma complex. These results are consistent with the X-ray crystallographic structure of bovine F1 (J. P. Abrahams, A. G. W. Leslie, R. Lutter, and J. E. Walker (1994) Nature 370, 621-628) where the beta301DDLTD305 loop directly interacts with gammaGln-269.
Omote
H
H
Institute of Scientific and Industrial Research, Osaka University, Osaka, Ibaraki, 567-0047, Japan.
Tainaka
K
K
Fujie
K
K
Iwamoto-Kihara
A
A
Wada
Y
Y
Futai
M
M
D019943:Amino Acid Substitution / Q000235:genetics*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D016368:Frameshift Mutation; D005973:Glutamine / Q000235:genetics / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010085:Oxidative Phosphorylation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D005973:Glutamine; D001224:Aspartic Acid; D006180:Proton-Translocating ATPases