Isolation and analysis of dominant secA mutations in Escherichia coli.
The secA gene product is an autoregulated, membrane-associated ATPase which catalyzes protein export across the Escherichia coli plasma membrane. Previous genetic selective strategies have yielded secA mutations at a limited number of sites. In order to define additional regions of the SecA protein that are important in its biological function, we mutagenized a plasmid-encoded copy of the secA gene to create small internal deletions or duplications marked by an oligonucleotide linker. The mutagenized plasmids were screened in an E. coli strain that allowed the ready detection of dominant secA mutations by their ability to derepress a secA-lacZ protein fusion when protein export is compromised. Twelve new secA mutations were found to cluster into four regions corresponding to amino acid residues 196 to 252, 352 to 367, 626 to 653, and 783 to 808. Analysis of these alleles in wild-type and secA mutant strains indicated that three of them still maintained the essential functions of SecA, albeit at a reduced level, while the remainder abolished SecA translocation activity and caused dominant protein export defects accompanied by secA depression. Three secA alleles caused dominant, conditional-lethal, cold-sensitive phenotypes and resulted in some of the strongest defects in protein export characterized to date. The abundance of dominant secA mutations strongly favors certain biochemical models defining the function of SecA in protein translocation. These new dominant secA mutants should be useful in biochemical studies designed to elucidate SecA protein's functional sites and its precise role in catalyzing protein export across the plasma membrane.
Jarosik
G P
GP
Department of Microbiology, State University of New York, Stony Brook 11794.
Oliver
D B
DB
D000251:Adenosine Triphosphatases / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005799:Genes, Dominant; D005816:Genetic Complementation Test; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009876:Operon; D010957:Plasmids; D015183:Restriction Mapping; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins