Mutations in three of the putative transmembrane helices of subunit a of the Escherichia coli F1F0-ATPase disrupt ATP-driven proton translocation.

Three missense mutants in subunit a of the Escherichia coli F1F0-ATPase were isolated and characterized after hydroxylamine mutagenesis of a plasmid carrying the uncB (subunit a) gene. The mutations resulted in Asp119----His, Ser152----Phe, or Gly197----Arg substitutions in subunit a. Function was not completely abolished by any of the mutations. The F0 membrane sector was assembled in all three cases as judged by restoration of dicyclohexylcarbodiimide sensitivity to the F1F0-ATPase. The H+ translocation capacity of F0 was reduced in all three mutants. ATP-driven H+-translocation was also reduced, with the response in the Gly197----Arg mutant being almost nil and that in the Asp119----His and Ser152----Phe mutants less severely affected. The substituted residues are predicted to lie in the second, third, and fourth transmembrane helices suggested in most models for subunit a. The Gly197----Arg mutation lies in a very conserved region of the protein and the substitution may disrupt a structure that is critical to function. The Asp119----His and Ser152----Phe mutations also lie in areas with sequence conservation. A further analysis of randomly generated mutants may provide more information on regions of the protein that are crucial to function. Heterodiploid transformants, carrying plasmids with either the wild-type uncB gene or mutant uncB genes in an uncB (Trp231----stop) background, were characterized biochemically. The truncated subunit a was not detected in membranes of the background strain by Western blotting, and the uncB+ plasmid complemented strain showed normal biochemistry. The uncB mutant genes were shown to cause equivalent defects in either the heterodiploid background configuration, or after incorporation into an otherwise wild-type unc operon. The subunit a (Trp231----stop) background strain was shown to bind F1-ATPase nearly normally despite lacking subunit a in its membrane.