A NEW GENETIC SELECTION IDENTIFIES ESSENTIAL RESIDUES IN SECG, A COMPONENT OF THE ESCHERICHIA-COLI PROTEIN EXPORT MACHINERY

The signal sequence of the murine serine protease inhibitor PAI-2 prom otes alkaline phosphatase export to the E.coli periplasm. However, hig h level expression of this chimeric protein interferes with cell growt h. Since most suppressors of this toxic phenotype map to secA and secY , growth arrest results from a defective interaction of the chimeric p rotein with the export machinery. We have characterized suppressors wh ich map in secG, a newly defined gene of the export machinery. All sin gle amino acid substitutions map to three adjacent codons. These secG mutants have a weak Sec phenotype, as determined by their effect on ex port mediated by wild-type and mutant signal sequences. Whilst a secG disruption allele also confers a weak Sec phenotype, it does not suppr ess the toxicity of the chimeric protein. This difference results from a selective effect of the secG suppressors on the kinetics of export mediated by the PAI-2 signal sequence. Using a malE signal sequence mu tant, which has a Malphenotype in secG mutant strains, we have isolate d extragenic Mal(+) suppressors. Most suppressors map to secY, and sev eral are allele-specific. Finally, SecG overexpression accelerates the kinetics of protein export, suggesting that there are two types of fu nctional translocation complexes: with or without SecG.