Isolation and analysis of dominant secA mutations in Escherichia coli

Abstract

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.

Escherichia coli SecA Helicase Activity Is Not Required in Vivo for Efficient Protein Translocation or Autogenous Regulation

“…Finally, strains with secretion-defective secA alleles had very high ␤-galactosidase activities, particularly after IPTG induction. This pattern is reminiscent of our previous work, where secretion-defective secA alleles caused dominant protein secretion defects and secA derepression due to subunit mixing and "poisoning" of the SecA homodimer (55).…”

Global Co-ordination of Protein Translocation by the SecA IRA1 Switch

“…1C). Six of the mutants (secAL785R, secAI789R, secAP799A, secAE802A, secAY803A, and secAE806A) were barely viable, as was partial deletion of IRA1 (secA⌬783-795) (15,30). Three mutants (secAW775A, secAF811A, and secAR792A) complemented the thermosensitive strain, albeit not as efficiently as secA.…”

“…Oligohistidinyl-tagged SecA IRA1 mutants were purified and shown, like SecA⌬783-795 (15,30), to be stable, folded, and ␣-helical (far UV CD; data not shown) and dimeric (size exclusion chromatography, blue native PAGE, sedimentation equilibrium; data not shown). We concluded that IRA1 mutant proteins do not have significantly altered structures and were characterized biochemically.…”

Identification of the Preprotein Binding Domain of SecA

“…his 10 -secA⌬351-368 was constructed by subcloning the 2.5-kb NcoI fragment from pGJ5 (13) to the corresponding site of pIMBB7, resulting in pIMBB83.…”

“…DEAD motors acquire specificity for different catalytic processes through add-on nonhomologous structural appendages (8,11). SecA has two such structures (12)(13)(14) (Fig. 1, A and B): (a) the C-domain (aa 611-832), which is fused C-terminally to the IRA2 domain of the DEAD motor and regulates its mobility and properties by "stapling" together NBD and IRA2 (14,15), and (b) a second appendage (aa 221-377) of unknown function, which forms an independent structural domain ( Fig.…”