The core Escherichia coli signal recognition particle receptor contains only the N and G domains of FtsY

Abstract

Previous studies have proposed that the N-terminal A domain (approximately 200 amino acid residues) of the Escherichia coli signal recognition particle (SRP) receptor, FtsY, is required for membrane targeting. In contrast to this suggestion, we show that A domain-truncated versions of FtsY, harboring only domains N and G, are functional. Therefore, we propose that N and G domains constitute the core SRP receptor.

FIG. 1.

Complementation of FtsY depletion by various truncated FtsY mutants. (A) Schematic representation of the mutants. (B) The activity of the mutants was tested by monitoring the growth in arabinose-depleted 2YT medium of E. coli FJP10 transformed with the different FtsY constructs. Extracts (5 μg of protein) of noninduced E. coli FJP10 harboring vector, FtsY-NG, or FtsY-NG + 1 were analyzed by Western blotting with anti-FtsY antibodies (9).

FIG. 2.

Characterization of E. coli EA101. (A) Extracts (20 μg of proteins) of E. coli EA101 (mut) and its parental strain, BW25113 (wild type [wt]), were analyzed by Western blotting with anti-FtsY antibodies. (B, top left panel) Growth of E. coli EA101 (solid symbols) and BW25113 (open symbols) was monitored in Luria-Bertani broth at 30°C (circles), 37°C (squares), and 42°C (triangles). (B, top right panel) Growth (37°C) of E. coli EA101 (solid symbols) and BW25113 (open symbols) was monitored in 2YT (rich medium; circles) and M9 (minimal medium; squares). (B, bottom left panel) E. coli EA101 (mut) and its parental strain, BW25113 (wt), moderately (+) or highly (+++) overexpressing MdfA-6His, were disrupted, and membranes (10 μg of proteins) were analyzed by Western blotting with horseradish peroxidase-conjugated INDIA HisProbe. (B, bottom right panel) The growth of the MdfA-overexpressing cells was monitored. (C) Membrane expression of SecY was analyzed by cell fractionation and Western blotting with anti-SecY antibodies obtained from H. Tokuda (University of Tokyo) (t, total; p, ultracentrifugation pellet; s, ultracentrifugation supernatant; m, membranes isolated by flotation). (D) Cellular distribution of FtsY-NG + 1. E. coli EA101 (mut) and its parental strain, BW25113 (wt), were fractionated, and the various fractions were analyzed by Western blotting with anti-FtsY antibodies.