Temperature-sensitive sec mutants of Escherichia coli: inhibition of protein export at the permissive temperature

A class of prUi (secY) alleles of Escherichia coli (prlA4-1 and prlA401) which specifically block the export of staphylokinase has been identified (T. lino and T. Sako, J. Biol. Chem. 263:19077-19082, 1988; T. Sako and T. lino, J. Bacteriol. 170:5389-5391, 1988). To determine more precisely the region in PriA (SecY) effective for the blockage of processing of the staphylokinase precursor, additional priA mutants which failed to support processing of the staphylokinase precursor were isolated. Two of the five mutant alleles isolated (secY121 and secY161) complemented the temperature sensitivity of a secY24 strain and had no detectable effect on the processing of endogenous secretory proteins of E. coli. In addition, a staphylokinase mutant having glycine in place of serine at position 17 in its signal sequence relieved the detrimental effect of these mutations. All of these characteristics indicate that these two alleles resemble the prlU4-1 and prlU401 alleles. On the other hand, the remaining three mutant alleles (secY47, secY105, and secY112) had no significant PrlA activity. The mutations of secY121 and secY161 were mapped very close to those of prlA4-1 and prLA401 in the presumed transmembrane segment 7 of PrlA. These results indicate that transmembrane segment 7 of PrlA plays a crucial role in the recognition of the staphylokinase signal sequence.

Section: Resultsmentioning

confidence: 99%

“…Another gene, priA (also called secY), has been identified as a mutant conditionally lethal and defective in general protein export (22,28,34) and as a suppressor for mutant lamB and malE products with defective signal sequences (4,15). PrIA (SecY) is also shown biochemically to be involved in protein translocation across the cytoplasmic membrane (16,23).…”

mentioning

confidence: 99%

Novel prlA alleles defective in supporting staphylokinase processing in Escherichia coli

Sako

1991

“…The inhibition of protein export at the permissive (low) temperature has been shown for strains containing secA51(Ts) or secY24(Ts) (21). At the low temperature, the combination of the reduced secretion capacity of the sec strains and the reduced production of the SecB might be detrimental for the cell.…”

Section: Discussionmentioning

confidence: 99%

Influence of impaired chaperone or secretion function on SecB production in Escherichia coli

Müller

1996

The efficient export of proteins through the cytoplasmic membrane of Escherichia coli requires chaperones to maintain protein precursors in a translocation-competent conformation. In addition to SecB, the major chaperone facilitating export of particular precursors, heat shock-induced chaperones DnaK-DnaJ and GroEL-GroES are also involved in this process. By use of secB-lacZ gene fusions and immunoprecipitation experiments, SecB production was studied in E. coli strains containing conditional lethal mutations in chaperone or sec genes. While the loss of heat shock chaperones resulted in an increased production of SecB, mutations in sec genes showed only minor effects on SecB synthesis. Neither the plasmid-mediated overexpression of precursors of exoproteins nor the overexpression of secB altered the synthesis of SecB. These results suggest that under conditions where chaperones become depleted, E. coli responds by raising the expression of secB. These data confirm the supposed synergy of different chaperones involved in protein export.

“…pMSY5 but not pMSY7 improved growth and protein export of strain KI330, which carries another temperature-sensitive (Ts) mutation, secY100 (17). Neither msy plasmid affected the growth or export phenotypes of secASJ(Ts) (30) and secB::Tn5 (22) mutations.…”

Section: Specificity Of Suppression Neither Of the Plasmids Pmsy5mentioning

confidence: 99%

“…Plasmid pNO1575 (18), a pBR322 derivative carrying the lac promoter and the multicloning site from pUC9, was used as a cloning vector. pNO1575H (17), a derivative of pNO1575 with a frameshift mutation in the HindIII site, was used as a control plasmid without a chromosomal insert; the mutation reduces the lac promoter-dependent elevation of the bla (1-lactamase) expression that may affect protein export in secY24 mutant cells (17).…”

Section: Methodsmentioning

confidence: 99%

Multicopy suppression: an approach to understanding intracellular functioning of the protein export system

Ueguchi

Ito

1992

Self Cite

Escherichia coli genes were cloned onto a multicopy plasmid and selected by the ability to restore growth and protein export defects caused by a temperature-sensitive secY or secA mutation. When secASI was used as the primary mutation, only clones carrying groE, which specifies the chaperonin class of heat shock protein, were obtained. Selection using secY24 yielded three major classes of genes. The first class encodes another heat shock protein, HtpG; the most frequently obtained second class encodes a neutral histonelike protein, H-NS; and the third class, msyB, encodes a 124-residue protein of which 38 residues are acidic amino acids. Possible mechanisms of suppression as well as the significance and limitations of the multicopy suppression approach are discussed.