Mutagenesis of the alpha subunit of the F1Fo-ATPase from Escherichia coli. Mutations at Glu-196, Pro-190, and Ser-199.
In an attempt to identify amino acid residues involved in proton translocation by the Fo sector of the Escherichia coli F1Fo-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, aglu196 and apro190. Two mutations were products of oligonucleotide-directed mutagenesis of a portion of 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 aglu196 (Asp, Gln, His, Asn, Lys, Ala, Ser, Pro) affect ATP-driven proton translocation and passive proton permeability by Fo to varying extents, but do not prevent growth on minimal succinate media. Amino acid substitutions of glutamine or arginine for apro190 affect F1Fo-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 aser199 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 aglu196 and apro190 are not obligate components of the proton channel, but that they affect proton translocation indirectly.
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011392:Proline; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D012694:Serine; D013050:Spectrometry, Fluorescence
D005971:Glutamates; D046911:Macromolecular Substances; D018698:Glutamic Acid; D012694:Serine; D011392:Proline; D006180:Proton-Translocating ATPases