A Mutation in the Escherichia coli secY Gene That Produces Distinct Effects on Inner Membrane Protein Insertion and Protein Export

John A Newitt,Harris D Bernstein

doi:10.1074/jbc.273.20.12451

Abstract

E. coli strains that contain the secY40 mutation are cold-sensitive, but protein export defects have not been observed even at the nonpermissive temperature. Here we describe experiments designed to explain the conditional phenotype associated with this allele. We found that combining the secY40 mutation with defects in the signal recognition particle targeting pathway led to synthetic lethality. Since the signal recognition particle is required for the insertion of inner membrane proteins (IMPs) into the cytoplasmic membrane but not for protein export, this observation prompted us to examine the effect of the secY40 mutation on IMP biogenesis. The membrane insertion of all IMPs that we tested was impaired at both permissive and nonpermissive temperatures in secY40 cells grown in either rich or minimal medium. The magnitude of the insertion defects was greatest in cells grown at low temperature in rich medium, conditions in which the growth defect was most pronounced. Consistent with previous reports, we could not detect protein export defects in secY40 cells grown in minimal medium. Upon growth in rich medium, only slight protein export defects were observed. Taken together, these results suggest that the impairment of IMP insertion causes the cold sensitivity of secY40 strains. Furthermore, these results provide the first evidence that the protein export and membrane protein insertion functions of the translocon are genetically separable.

Highlights

E. coli strains that contain the secY40 mutation are cold-sensitive, but protein export defects have not been observed even at the nonpermissive temperature
Slight signal recognition particle (SRP) deficiencies can be tolerated by wild-type E. coli, but a 5– 8-fold decrease in the intracellular Ffh concentration becomes growth-limiting [29], presumably because the biogenesis of inner membrane proteins (IMPs) is reduced below a critical level
Assessment of the viability of secY40 strains that have reduced SRP activity should provide an indication as to whether the mutation impedes IMP insertion. In support of this notion, we found that the secY39 mutation, which has been shown to inhibit IMP insertion [16], is lethal in strains carrying SRP deficiencies

Summary

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol 273, No 20, Issue of May 15, pp. 12451–12456, 1998 Printed in U.S.A. A Mutation in the Escherichia coli secY Gene That Produces Distinct Effects on Inner Membrane Protein Insertion and Protein Export*. Upon growth in rich medium, only slight protein export defects were observed Taken together, these results suggest that the impairment of IMP insertion causes the cold sensitivity of secY40 strains. Several studies using conditional alleles of secY isolated in screens for protein export mutants have suggested that the insertion of model inner membrane proteins (IMPs) is SecY-dependent (10 –13). Consistent with these results, depletion of SecE was recently shown to block the insertion of the maltose transporter MalF [14]. The mutation gives rise to a moderate increase in secA synthe-

TABLE I Bacterial strains

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION