A defined mutation in the protein export gene within the spc ribosomal protein operon of Escherichia coli: isolation and characterization of a new temperature-sensitive secY mutant.

Abstract

We describe the properties of a temperature-sensitive mutant, ts24, of Escherichia coli. The mutant has a conditional defect in export of periplasmic and outer membrane proteins. At 42°C, precursor forms of these proteins accumulate within the cell where they are protected from digestion by extemally added trypsin. The accumulated precursors are secreted and processed very slowly at 42°C. The mutation is complemented by expression of the wild-type secY(orprlA) gene, which has been cloned into a plasmid vector from the promoter-distal part of the spc ribosomal protein operon. The mutant has a single base change in the middle of the secY gene, which would result in the replacement of a glycine residue by aspartic acid in the protein product. These results demonstrate that the gene secY (priA) is essential for protein translocation across the E. coli cytoplasmic membrane.

Complementation of the protein transport defect of an Escherichia coli sec Y mutant (sec Y24) by Bacillus subtilis sec Y homologue

“…In Escherichia coli, several genes whose products are necessary for protein translocation have been defined [2-61. One of them, the secY (prlA) gene product is an essential component of the E. coli cytoplasmic membrane with multiple spanning membrane domains [7], and it plays an apparent pivotal role in protein translocation across the cytoplasmic membrane of E. coli [2,8]. In order to analyse the protein secretion system in Bacillus subtilis, we have identified the secY homologue of B. subtilis within the promoterdistal portion of the spc-CY ribosomal protein operon [9].…”

“…The isogenic derivatives of E. coli MC4100, strains K1297 (secY24) and K1298 (secY') which carry la@, lacZ* and pro+ were constructed by introducing F' la& lacZ+pro+ factor into IQ85 and IQ86 [2], respectively, and served as recipients for complementation analysis. The mutant (secY24) is temperature-sensitive.…”

“…On minimal agar plates, it can grow below 40°C but not at 42°C. The pulselabeling analysis indicates that the mutant (secY24) has a defect in the conversion of precursors of periplasmic and outer membrane proteins to mature forms at 42°C; precursors were located inside the cell [2].…”

“…pKY3 was a derivative of pN01575 carrying the E. cofi secY gene and most of the X reading frame. The E. coli secY in pKY3 is transcribed from the lac promoter [2]. For the controlled expression of the B. subtilis secY in E. coli, a series of pKY3-based plasmids, pTUE804, pTUE806 and pTUE808 were constructed as illustrated in Fig.…”

Nucleotide sequence of the secY gene from Lactococcus lactis and identification of conserved regions by comparison of four SecY proteins

“…The mutants that have been obtained of the E. coli secY gene are either temperature sensitive (Ts) [22,24], cold sensitive (Cs) [25 1, or they have altered interactions with the signal sequences (SS) [23,26]. Only some of the E. co/i mutations correspond to conserved residues: one Cs-mutation (secYdO), one Ts-mutation (secA100) and three SS-mutations (@A#-2, secY121, secYl6l) have affected residues conserved in the 4 sequences (Fig.…”