CHARACTERIZATION OF COLD-SENSITIVE SECY MUTANTS OF ESCHERICHIA-COLI

Mutations which cause poor growth at a low temperature, which affect aspects of protein secretion, and which map in or around secY (prlA) were characterized. The prlA1012 mutant, previously shown to suppress a secA mutation, proved to have a wild-type secY gene, indicating that this mutation cannot be taken as genetic evidence for the secA-secY interaction. Two cold-sensitive mutants, the secY39 and secY40 mutants, which had been selected by their ability to enhance secA expression, contained single-amino-acid alterations in the same cytoplasmic domain of the SecY protein. Protein export in vivo was partially slowed down by the secY39 mutation at 37 to 39°C, and the retardation was immediately and strikingly enhanced upon exposure to nonpermissive temperatures (15 to 23°C). The rate of posttranslational translocation of the precursor to the OmpA protein (pro-OmpA protein) into wild-type membrane vesicles in vitro was only slightly affected by reaction temperatures ranging from 37 to 15°C, and about 65% of OmpA was eventually sequestered at both peratures. Membrane vesicles from the secY39 mutant were much less active in supporting pro-OmpA translocation even at 37°C, at which about 20% sequestration was attained. At 15°C, the activity of the mutant membrane decreased further. The rapid temperature response in vivo and the impaired in vitro translocation activity at low temperatures with the secY39 mutant support the notion that SecY, a membrane-embedded secretion factor, participates in protein translocation across the bacterial cytoplasmic membrane.