Membrane binding of the bacterial signal recognition particle receptor involves two distinct binding sites

Abstract

Cotranslational protein targeting in bacteria is mediated by the signal recognition particle (SRP) and FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRalpha subunit of eukaryotes, which is tethered to the membrane via its interaction with the membrane-integral SRbeta subunit. Despite the lack of a membrane-anchoring subunit, 30% of FtsY in Escherichia coli are found stably associated with the cytoplasmic membrane. However, the mechanisms that are involved in this membrane association are only poorly understood. Our data indicate that membrane association of FtsY involves two distinct binding sites and that binding to both sites is stabilized by blocking its GTPase activity. Binding to the first site requires only the NG-domain of FtsY and confers protease protection to FtsY. Importantly, the SecY translocon provides the second binding site, to which FtsY binds to form a carbonate-resistant 400-kD FtsY-SecY translocon complex. This interaction is stabilized by the N-terminal A-domain of FtsY, which probably serves as a transient lipid anchor.

Figure 1.

FtsY acquires a carbonate-resistant conformation in the presence of INVs and GMP-PNP. (A) Wild-type (wt) FtsY was in vitro synthesized and incubated with buffer or membranes in the presence or absence of nucleotides (final concentration, 2 mM) and a nucleotide-regenerating system (Koch et al., 1999). Subsequently, the samples were extracted with Na₂CO₃ and ultracentrifuged. Soluble material (S) was neutralized with glacial acetic acid and TCA precipitated. Carbonate-resistant material (P) was directly dissolved in SDS loading buffer. For quantification, the amount of soluble and resistant material was set as 100% and the material present in the individual fractions was quantified. Several independent experiments were performed, and a representative gel is shown. (B) Carbonate resistance of wild-type and mutant FtsY. Analysis was performed as in A. Asterisks indicate that although the calculated molecular mass of FtsY is 54 kD, it migrates in SDS-PAGE as a 90-kD band (Fig. 5).

Figure 2.

Carbonate resistance of FtsY requires the SecY translocon. (A) In vitro–synthesized FtsY was incubated with wild-type (wt) INVs or INVs derived from secY mutants (secY40 and secY205) and a SecE-depletion mutant (CM124). After incubation in the presence or absence of GMP-PNP, samples were carbonate extracted and quantified as described in Fig. 1. A representative gel is shown, and the standard deviation is indicated. The asterisk indicates that although the calculated molecular mass of FtsY is 54 kD, it migrates in SDS-PAGE as a 90-kD band.

Figure 3.

In the presence of GMP-PNP, FtsY assembles into a 400-kD membrane complex. (A) In vitro–synthesized FtsY was purified and subsequently incubated with membranes in the absence or presence of GMP-PNP. The samples were either directly solubilized with 0.2% (wt/vol) n-dodecylmaltoside or only after carbonate extraction. The solubilized material was separated on a 5–13% BN-PAGE, and radioactively labeled samples were visualized by phosphorimaging. Indicated are the positions of the 400- and 200-kD FtsY complexes. FtsY and FtsY* represent as-yet-uncharacterized FtsY variants (see Results). (B) The formation of the 400-kD complex was analyzed in a time-course experiment. Purified FtsY was incubated with INVs, and after the addition of GMP-PNP, samples were withdrawn at the indicated time points and separated on BN-PAGE as in A. The amount of radioactively labeled material present in the 200-kD complex at 0 min was set as 100%. (C) BN-PAGE analyses of FtsY bound to either wild-type (wt) membranes or SecE-depleted INVs (CM124) in the presence or absence of GMP-PNP. (D) Detection of the SecYEG complex in wild-type membranes by immune detection. 100 μg INV was solubilized with 0.2% n-dodecylmaltoside and separated on BN-PAGE. After Western transfer, the membrane was decorated with the indicated polyclonal antibodies.

Figure 4.

The 400-kD complex represents an FtsY–SecYEG complex. (A) A large-scale FtsY in vitro synthesis was incubated with membranes in the absence or presence of GMP-PNP. After solubilization, the solubilized material was purified via metal affinity chromatography. (top) A small portion of the eluted material was separated on SDS-PAGE, and the radiolabeled FtsY was detected via phosphorimaging (L, 1% of load; W, 5% of final wash; E, 5% of eluted material). (bottom) The remaining material was separated on SDS-PAGE and transferred to nitrocellulose membranes for subsequent immune detection (L, 2% of load; W, 10% of final wash; E, 90% of eluted material). As control, nontagged FtsY was subjected to the same purification procedure (lanes 1–6). (B) 5% of the eluted material shown in lane 9 (without GMP-PNP) and lane 12 (with GMP-PNP) was separated on BN-PAGE, and FtsY complexes were detected by phosphorimaging.

Figure 5.

A protease-resistant conformation of FtsY is observed in the presence of membranes and GMP-PNP. (A) In vitro–synthesized FtsY was incubated with buffer or INVs in the presence of absence of GMP-PNP. Half of the reaction was directly TCA precipitated and the other half only after treatment with 0.5 mg/ml proteinase K for 20 min at 25°C. Indicated are full-size FtsY and its proteinase K–protected fragments. Immunoprecipitation (IP) experiments were performed with antibodies covalently bound to protein A–Sepharose beads. For proteinase K–treated samples, the protease inhibitor PMSF was added before the addition of the antibody beads. The asterisk indicates unspecific cleavage products of FtsY, which were observed only after immunoprecipitations. (B) Proteinase K protection was analyzed as in A in the presence of wild-type (WT), SecE-depleted (CM124), and secY40 INVs. The percentage of protease protection was calculated by quantifying the ratio of radioactivity present in the proteinase K–treated sample and the directly TCA precipitated sample. The protease-protected fragment of FtsY comprises the major parts of the NG-domain (Fig. 7), and the percentage of protease protection was corrected for the loss of methionine residues as a result of cleaving off the A-domain (see Fig 6 for the position of methionine residues in FtsY). However, as the exact boundaries of the 33-kD fragment are not known, the values are estimates only.

Figure 6.

The A-domain of FtsY is required for carbonate-resistant interaction with the membrane. (A) Wild-type (wt) FtsY and FtsY(NG+1), lacking the A-domain, were in vitro synthesized and incubated with INV or buffer in the presence or absence of GMP-PNP. Treatment of the samples and quantification was performed as described in Fig. 1. The asterisk indicates that although the calculated molecular mass of FtsY is 54 kD, it migrates in SDS-PAGE as a 90-kD band. S, soluble material; P, carbonate-resistant material.

Figure 7.

The A-domain is not essential for the proteinase K–resistant interaction of FtsY with the membrane. (A) Proteinase K protection of full-size FtsY and FtsY(NG+1) was analyzed as in Fig. 5. (B) Carbonate resistance of full-size FtsY was analyzed after a proteinase K treatment (lanes 3 and 4). As control, carbonate extraction without prior proteinase K treatment is shown (lanes 1 and 2). S, soluble material; P, carbonate-resistant material.

Figure 8.

FtsY(NG+1) is less active than full-size FtsY. (A) The activity of FtsY and FtsY(NG+1) was analyzed in an in vitro complementation assay using INVs derived from the secY40 mutant (Angelini et al., 2005). MtlA was in vitro synthesized in the presence of wild-type (WT) or secY40 INV. When indicated, 1 μg of purified FtsY or FtsY(NG+1) was added. Translation products were precipitated either directly with TCA or only after incubation with proteinase K as described in Fig. 5. The percentage of integration was calculated as in Fig. 5, and the values were corrected for the loss of methionine by cleaving off the 30-kD cytoplasmic domain of MtlA. (B) Integration of MtlA into secY40 INVs was tested as in A but with increasing concentrations of FtsY and FtsY(NG+1).