FUNCTIONAL STABILITY OF THE A-SUBUNIT OF THE F0F1-ATPASE FROM ESCHERICHIA-COLI IS AFFECTED BY MUTATIONS IN 3 PROLINE RESIDUES

Site-directed mutagenesis was used to investigate the roles of three p roline 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 stabilizi ng the a-subunit structure in that removal of the F1-ATPase from membr anes prepared from each of the mutant strains resulted in the loss of passive proton translocation activity. Pro-103 is predicted to be with in a transmembrane helix. Pro-122 and Pro-143 are located just outside the membrane and near two residues (Asp-124 and Arg-140) previously p roposed to form a charge pair. The phenotype of mutants in which Pro-1 22 or Pro-143 were replaced by alanine was similar to previously isola ted mutants affected in Asp-124 and Arg-140. This suggested that the m ain effect of the mutations was to destroy the charge pair between Asp -124 and Arg-140. Double mutants resulting from all possible combinati ons of these four mutations were constructed and, with the exception o f P122A + D124A, had a similar phenotype to the single mutants. This i s consistent with the idea that all four single changes had the same e ffect on a-subunit structure. In contrast, combining the P122A or P143 A changes with another mutation which caused a similar phenotype (D44N ) resulted in a complete loss of oxidative phosphorylation.