Escherichia coli SecB stimulates export without maintaining export competence of ribose-binding protein signal sequence mutants.
Ribose-binding protein (RBP) is exported to the periplasm of Escherichia coli via the general export pathway. An rbsB-lacZ gene fusion was constructed and used to select mutants defective in RBP export. The spontaneous Lac+ mutants isolated in this selection contained either single-amino-acid substitutions or a deletion of the RBP signal sequence. Intact rbsB genes containing eight different point mutations in the signal sequence were reconstructed, and the effects of the mutations on RBP export were examined. Most of the mutations caused severe defects in RBP export. In addition, different suppressor mutations in SecY/PrlA protein were analyzed for their effects on the export of RBP signal sequence mutants in the presence or absence of SecB. Several RBP signal sequence mutants were efficiently suppressed, but others were not suppressed. Export of an RBP signal sequence mutant in prlA mutant strains was partially dependent on SecB, which is in contrast to the SecB independence of wild-type RBP export. However, the kinetics of export of an RBP signal sequence mutant point to a rapid loss of pre-RBP export competence, which occurs in strains containing or lacking SecB. These results suggest that SecB does not stabilize the export-competent conformation of RBP and may affect translocation by stabilizing the binding of pre-RBP at the translocation site.
Francetic
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Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Kumamoto
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CA
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D033902:Periplasmic Binding Proteins; D011498:Protein Precursors / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000069816:SEC Translocation Channels; D013489:Suppression, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D033902:Periplasmic Binding Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; C028003:RbsB protein, E coli; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; C057842:SecB protein, Bacteria; C057841:SecY protein, E coli