In an attempt to identify amino acid residues involved in proton translocation by the F0 sector of the Escherichia coli F1F0-ATPase, 16 mutations at the carboxyl-terminal third of the a subunit have been isolated, and their phenotypes have been partially characterized. Thirteen mutations were constructed by 'cassette' mutagenesis at two highly conserved residues, a(glu196) and a(pro190). Two mutations were products of oligonucleotide-directed mutagenesis of a portion of the uncB gene cloned into an M13 vector. One mutation was isolated after in vitro mutagenesis of the entire uncB gene in a plasmid vector with hydroxylamine. Amino acid substitutions for aglu 196 (Asp, Gln, His, Asn, Lys, Ala, Ser, Pro) affect ATP-driven proton translocation and passive proton permeability by F0 to varying extents, but do not prevent growth on minimal succinate media. Amino acid substitutions of glutamine or arginine for a(pro190) affect F1F0-ATPase assembly and eliminate ATP-driven proton translocation, while the substitution of asparagine at this position does not significantly affect either assembly or proton translocation. The substitution of amino acids threonine or alanine for a(ser199) causes no detectable phenotypic change from wild type. These and other mutations are discussed in terms of the assembly, structure, and function of the a subunit. It is concluded that a(glu196) and a(pro190) are not obligate components of the proton channel, but that they affect proton translocation indirectly.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - Jan 1 1988|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology