Functional stability of the a-subunit of the F0F1-ATPase from Escherichia coli is affected by mutations in three proline residues.

Site-directed mutagenesis was used to investigate the roles of three proline residues (Pro-103, Pro-122 and Pro-143) in the a-subunit of the E. coli F0F1-ATPase. All three were found to have a role in stabilizing the a-subunit structure in that removal of the F1-ATPase from membranes prepared from each of the mutant strains resulted in the loss of passive proton translocation activity. Pro-103 is predicted to be within a transmembrane helix. Pro-122 and Pro-143 are located just outside the membrane and near two residues (Asp-124 and Arg-140) previously proposed to form a charge pair. The phenotype of mutants in which Pro-122 or Pro-143 were replaced by alanine was similar to previously isolated mutants affected in Asp-124 and Arg-140. This suggested that the main effect of the mutations was to destroy the charge pair between Asp-124 and Arg-140. Double mutants resulting from all possible combinations of these four mutations were constructed and, with the exception of P122A + D124A, had a similar phenotype to the single mutants. This is consistent with the idea that all four single changes had the same effect on a-subunit structure. In contrast, combining the P122A or P143A changes with another mutation which caused a similar phenotype (D44N) resulted in a complete loss of oxidative phosphorylation.