FtsY, the bacterial signal-recognition particle receptor, interacts functionally and physically with the SecYEG translocon

Abstract

Co-translational membrane targeting of proteins by the bacterial signal-recognition particle (SRP) requires the specific interaction of the SRP-ribosome nascent chain complex with FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRalpha-subunit of the eukaryotic SR, which is tethered to the endoplasmic-reticulum membrane by its interaction with the integral SRbeta-subunit. In contrast to SRalpha, FtsY is partly membrane associated and partly located in the cytosol. However, the mechanisms by which FtsY associates with the membrane are unclear. No gene encoding an SRbeta homologue has been found in bacterial genomes, and the presence of an FtsY-specific membrane receptor has not been shown so far. We now provide evidence for the direct interaction between FtsY and the SecY translocon. This interaction offers an explanation of how the bacterial SRP cycle is regulated in response to available translocation channels.

Figure 1

The secY40 mutation blocks the integration of SRP-dependent membrane proteins. pOmpA, MtlA, YidC and M13-coat-H5 were synthesized in vitro at 25°C for 45 min in the absence or presence of inside-out inner membrane vesicles derived from either wild-type Escherichia coli cells or the secY40 mutant strain. Translation products were precipitated either directly with trichloroacetic acid (TCA) or after incubation with 0.5 mg/ml proteinase K (Prot. K) for 20 min at 25°C. The positions of the precursor (pOmpA) and the mature form of OmpA are indicated, along with the positions of fullsize MtlA and YidC and their proteinase K-resistant membrane-protected fragments (MPF). M13-coat-H5, an M13 coat derivative lacking the signal sequence cleavage site (Kuhn & Wickner, 1985), was separated on 22% urea/SDS–PAGE, and all other samples were separated on 13% SDS–PAGE. The percentage of translocation or integration was calculated after quantification of the radioactivity of the individual protein bands using a PhosphorImager and ImageQuant software, and by calculating the ratio between the amounts present in the proteinase K-treated sample and the TCA-precipitated sample.

Figure 2

FtsY suppresses the secY40 phenotype both in vivo and in vitro. (A) MtlA was synthesized in vitro in the presence of wild-type (wt), secY40 or secY39 INV as shown in Fig 1. When indicated, purified FtsY or Ffh was added. Prot. K, proteinase K. (B) YidC was synthesized in vitro in the presence of wt or secY40 INV. Purified FtsY or FtsY-307 (a truncated FtsY derivative lacking the GTPase domain) was added when indicated. (C) SecY40 cells expressing FtsY were incubated in liquid Luria–Bertani medium at 37°C. After overnight culture, the culture was serially diluted and spotted on LB plates containing 0.2 mM isopropyl β-D-thiogalactopyranoside. Plates were incubated at either 37 or 25°C. SecY40 and wt cells carrying the empty vector were used as controls.

Figure 3

Chemical crosslinking shows the interaction between FtsY and SecY. INV containing in vitrosynthesized SecY were treated with urea to remove membrane-bound FtsY and were subsequently incubated with either buffer or cytosolic extracts derived from cells depleted of FtsY (S135-FtsY⁻) or from cells expressing FtsY (S135-FtsY⁺). Crosslinking was performed with the soluble crosslinker BS³. Crosslinked bands were identified in a sixfold scaled-up reaction mixture by immunoprecipitation using α-FtsY and α-SecY antibodies bound to protein A–Sepharose beads.

Figure 4

SecY and FtsY are co-immunoprecipitated. (A) SecY and MtlA were synthesized in vitro at 37°C for 30 min in the presence of wild-type (wt) or mutant INV. One aliquot of the reaction mixture was precipitated directly with trichloroacetic acid (TCA) and the remaining material was subjected to ultracentrifugation to collect the INV. Subsequently, the INV were solubilized with dodecyl-β-D-maltoside. As control, wt INV were treated with puromycin/EDTA (ethylenediamine tetra acetic acid) after integration of SecY and before solubilization (wt INV+puro). Immunoprecipitation was performed with protein A-coupled pre-immune serum (Pre) or antibodies, as indicated. Precipitated material was separated on SDS–PAGE and radioactively labelled bands were visualized and quantified using a PhosphorImager. The right-hand panel illustrates the quantification of the SecY data from the left-hand panel, and shows the percentage of precipitated SecY relative to the amount precipitated in wt INV, which was set to 100%. (B) FtsY was synthesized in vitro and treated as described in (A).

Figure 5

The Sec translocon co-purifies with membrane-bound FtsY. FtsY with or without His₆ tag at the carboxyl terminus was purified from Escherichia coli crude membranes (M) by solubilization and metal-affinity chromatography. After purification, samples were separated on SDS–PAGE and stained with Coomassie blue. Eluted fractions containing purified His₆-tagged FtsY (E) were further analysed for the presence of SecY and YidC by western blot analyses. As a control, the procedure was repeated with untagged FtsY. FtsY^* reflects an N-terminally truncated FtsY derivative, which is routinely observed in FtsY preparations (Luirink et al, 1994).