Escherichia coli mutants defective in the γ subunit of proton-translocating ATPase: Intracistronic mapping of the defective site and the biochemical properties of the mutants

Abstract

Various hybrid plasmids carrying a portion of the gene for the γ subunit of the H +-ATPase of Escherichia coli complemented five mutants defective in the enzyme in a genetic test, indicating that the mutants are defective in the γ subunit. Since the nucleotide sequence of genomic DNA carried on the plasmids is known, the defective site(s) of the mutants could be located within the gene for the γ subunit as follows: KF10 and NR70, KF1, and KF12 and KF13 have a mutation causing a defect(s) in amino acid residues 1 to 82, 83 to 167, and 168 to 287, respectively, of the γ subunit. The biochemical properties of all these mutants except NR70 were analyzed in terms of proton permeability of the membranes and assembly of F 1. Results suggested that KF1 and KF10 have defective F 1 without at least the α and β subunits on their membranes, whereas KF12 and KF13 have F 1's of rather similar structure to that of the wild type. Attempts were made to purify F 1 of KF12 as a single complex. Although the F 1 complex dissociated during purification, active α and β subunits of KF12 were partially purified. On the basis of these biochemical and genetic results, it is suggested that structural alterations in the primary sequence of the γ subunit corresponding to residues 1 to 167 cause more extensive defects in the assembly of F 1 than alteration in the sequence of residues 168 to 287.