Signal sequence-independent SRP-SR complex formation at the membrane suggests an alternative targeting pathway within the SRP cycle

Abstract

Protein targeting by the signal recognition particle (SRP) and the bacterial SRP receptor FtsY requires a series of closely coordinated steps that monitor the presence of a substrate, the membrane, and a vacant translocon. Although the influence of substrate binding on FtsY-SRP complex formation is well documented, the contribution of the membrane is largely unknown. In the current study, we found that negatively charged phospholipids stimulate FtsY-SRP complex formation. Phospholipids act on a conserved positively charged amphipathic helix in FtsY and induce a conformational change that strongly enhances the FtsY-lipid interaction. This membrane-bound, signal sequence-independent FtsY-SRP complex is able to recruit RNCs to the membrane and to transfer them to the Sec translocon. Significantly, the same results were also observed with an artificial FtsY-SRP fusion protein, which was tethered to the membrane via a transmembrane domain. This indicates that substrate recognition by a soluble SRP is not essential for cotranslational targeting in Escherichia coli. Our findings reveal a remarkable flexibility of SRP-dependent protein targeting, as they indicate that substrate recognition can occur either in the cytosol via ribosome-bound SRP or at the membrane via a preassembled FtsY-SRP complex.

FIGURE 1:

Anionic phospholipids stimulate FtsY-SRP complex formation. (A) In vitro synthesized FtsY and Ffh were affinity purified via metal-affinity chromatography and subsequently incubated with E. coli INV in the presence or absence of 2 mM GMP-PNP. After solubilization with DDM, the proteins were separated on a 5–15% BN-PAGE gel. (B) FtsY was incubated with INV in the presence and absence of GMP-PNP. The sample was then solubilized and incubated with preimmune serum or with the indicated antibodies and separated on a 5–10% BN-PAGE gel. (C) FtsY was incubated with either INV or buffer/liposomes together with the indicated amount of SRP. Proteins were solubilized and separated on a 5–10% BN-PAGE gel. (D) As for (C), but liposomes were prepared from synthetic lipids. PE/PG/CL (70, 25, and 5%, respectively) liposomes mimic the E. coli inner membrane lipid composition, PE/PC (65 and 35%, respectively) are zwitterionic phospholipids, which lead to the formation of neutral liposomes.

FIGURE 2:

FtsY acquires a PK-resistant conformation upon interaction with SRP and lipids. (A) FtsY was in vitro synthesized and then incubated in the absence or presence of INV or liposomes with GMP-PNP (2 mM) or INV buffer and treated with PK (0.5 mg/ml for 20 min at 25°C). Samples were precipitated with trichloroacetic acid (TCA, 5% final concentration), separated on 13% SDS–PAGE, and visualized on a phosphorimager. (B) PK resistance was tested as in (A), but after preincubation with SRP (0.1 μM) or BSA (8 μM). (C) FtsY was in vitro synthesized and purified via metal-affinity chromatography before PK resistance testing.

FIGURE 3:

A positively charged helix of FtsY is crucial for complex formation with SRP. (A) Drawing showing the domain structure of E. coli FtsY. The localization of the two lipid-binding helices and their amino acid sequences are also shown. (B) Wild-type (wt) FtsY and FtsY derivatives carrying mutations within the second lipid-binding helix were in vitro synthesized and affinity purified via a C-terminal His tag. PK resistance of the mutants was analyzed in the presence of INV as described in Figure 2. (C) As in (B), but the samples were incubated with INV, solubilized, and separated on a 5–10% BN-PAGE gel. (D). The FtsY mutant proteins were expressed in E. coli C43 (DE3) and purified via metal-affinity purification. The GTPase activity of the FtsY mutants was analyzed in a 20 μl reaction mixture containing 0.5 μM FtsY. The reaction was started by the addition of GTP (200 μM GTP and 2.5 μM [γ-³²P]-GTP). When indicated, 2 μl of liposomes (70% PE, 25% PG and 5% CL, as in Figure 1D) was added.

FIGURE 4:

An FtsY-Ffh fusion protein forms a 400-kDa complex on BN-PAGE and hydrolyzes GTP. (A) The FtsY-Ffh-fusion protein (FF-fusion) was constructed by genetically fusing the C-terminal end of the FtsY NG domain to the N-terminal end of the Ffh NG domain via a 20-amino-acid-long, flexible linker (L). Label A indicates the A domain of FtsY and label M the signal sequence–binding domain of Ffh. (B) The GTPase activity of the FF-fusion was analyzed in a 20 μl reaction mixture containing 0.05 μM FF-fusion. The reaction was started by the addition of GTP (200 μM GTP and 2.5 μM [γ-³²P]-GTP). FtsY and Ffh were also used at a final concentration of 0.05 μM each and, when indicated, 0.5 μg 4.5S RNA was added. The mean values and SD of at least three independent experiments are shown. (C) 4 μM FtsY was incubated with 1.5 μM Ffh and 0.1 mg/ml 4.5S RNA in the presence or absence of 2 mM GMP-PNP; alternatively, 1.5 μM of the FtsY-Ffh fusion protein was incubated with 0.1 mg/ml 4.5S RNA in the presence or absence of GMP-PNP and then separated on a 5–15% BN-PAGE gel. After transfer to a nitrocellulose membrane, the protein complexes were detected by α-Ffh antibodies.

FIGURE 5:

The FtsY-Ffh fusion protein is functional in vivo. (A) Plasmid-borne copies of FtsY, Ffh, or of the FF-fusion protein were expressed in the conditional Ffh mutant WAM113 or the conditional FtsY mutant strain IY28. In both strains, the respective gene is under the control of the arabinose promoter and growth requires the addition of 0.2% arabinose. Overnight cultures were grown in LB medium in the presence of arabinose to an OD₆₀₀ of 1.0, and serially diluted on LB plates containing either arabinose or fructose. The complementation did not require the presence of IPTG, which demonstrates that the basal expression level is sufficient. (B) Western blot analyses of the FF-fusion expressed in either wild-type DH5α cells or in IY28 and WAM113 cells, grown either in the presence of arabinose or fructose. Antibodies against the C-terminal His tag of the FF-fusion revealed no detectable cleavage of the fusion protein. As control, the same samples were also analyzed for the presence of the bacterial Hsp60 protein GroEL. (C) The expression level of the FF-fusion in wild-type DH5α cells was compared with the endogenous FtsY/Ffh content by Western blotting using antibodies against FtsY and Ffh. FtsY-14 corresponds to an N-terminally truncated FtsY-derivative, which lacks the first 14 amino acids. Note that FtsY has a predicted molecular mass of 56 kDa but runs at ∼100-kDa on SDS–PAGE due to its highly charged N-terminal A domain. As control, antibodies against GroEL were used. (D) For excluding the possibility that the functionality of the FF-fusion is due to proteolytic cleavage within the linker region, two FF-fusion derivatives were constructed that contained either an inactive Ffh-part (FF-fusion A144W) or an inactive FtsY part (FF-fusion A336W). The complementation assay was performed as in (A).

FIGURE 6:

The FtsY-Ffh fusion protein is capable of targeting ribosome nascent chains to the SecYEG translocon in a reconstituted in vitro transport system. (A) Coomassie staining and immune detection of SecYEG-proteoliposomes. SecYEG proteoliposomes were loaded for Coomassie staining (4 μl) and immune detection (0.2 μl). INV (3 μl) served as control. SecE and SecG have approximately the same size and are not well separated; in addition, Coomassie Brilliant Blue hardly stains SecG. (B) The SRP-dependent polytopic membrane protein MtlA consists of six transmembrane domains and a long C-terminal cytoplasmic domain, which is cleaved off by PK treatment (scissors), leaving behind a membrane-protected 30-kDa fragment (MtlA-MPF) that corresponds to the membrane-integral part of MtlA. (C) MtlA was in vitro synthesized in an E. coli transcription–translation system that does not contain FtsY, SRP, or INV (Koch et al., 1999) unless added. One-half of the reaction mixture was subsequently precipitated with TCA, whereas the second half was further digested with PK. MtlA was visualized by autoradiography after separation on a 13% SDS–polyacrylamide gel. INV correspond to E. coli inner membrane vesicles. The lipid composition of liposomes reflects the E. coli cytoplasmic membrane. SecYEG-proteoliposomes were generated as described in Material and Methods. When indicated, INV (0.5 μl), liposomes (4 μl), SecYEG proteoliposomes (4 μl), FtsY (1 μg), SRP (0.4 μg Ffh + 0.7 μg 4.5S RNA), or FF-fusion (1.5 μg FF-fusion + 0.7 μg 4.5S RNA) was added. The integration rate was calculated considering the loss of methionines for the MtlA-MPF and is the mean value of at least three independent experiments. The SD is indicated.

FIGURE 7:

The FF-fusion protein functions exclusively at the membrane. (A) The localization of the FF-fusion was analyzed in vivo by fluorescence microscopy and compared with fluorescently labeled FtsY, Ffh, and SecY. FtsY, Ffh, and SecY were C-terminally fused to either GFP or yellow fluorescent protein (YFP). For labeling of the FF-fusion, GFP was inserted into the linker region that connects FtsY and Ffh (see Figure 4A). (B) Liposomes or SecYEG-proteoliposomes were preincubated with FtsY/Ffh or FF-fusion in the presence of 4.5S RNA, isolated by centrifugation, and resuspended in buffer A (50 mM triethanolamine acetate, pH 8.0; 250 mM sucrose); for concentrations see legend to Figure 6. The resuspended liposomes/proteoliposomes were separated on a 5–20% SDS–polyacrylamide gel and stained with Coomassie Brilliant Blue. (C) The preincubated liposomes/proteoliposomes shown in (B) were used for MtlA in vitro transport assays as described in Figure 6. INV were used as control.

FIGURE 8:

A membrane-integral FtsY-Ffh-complex is functional. (A) The FF-fusion was fused to the C-terminus of the integral membrane protein TatC to generate TM-FF-fusion. (B) The functionality of the TM-FF-fusion was determined by complementation assays as described in Figure 5. (C) Expression and integrity of the TM-FF-fusion was analyzed by Western blotting using antibodies against Ffh or against the C-terminal His tag of the TM-FF-fusion. IY28 or WAM113 cells (approx. 0.5 × 10⁸ cells) grown in the absence (−) or presence (ara) of arabinose and expressing the indicated constructs were directly precipitated by TCA (10% final concentration) and separated on 5–15% SDS–PAGE. The band running below the 34-kDa marker band, which is labeled with (*), was unspecifically recognized by both antibodies and served as an internal loading control.

FIGURE 9:

Model for RNC targeting by a preformed FtsY-SRP-complex. A preassembled FtsY-SRP complex at the membrane (FtsY, red; SRP, orange) is able to recruit RNCs to the membrane and to transfer them to the SecYEG translocon (blue). It is only after contact of the FtsY-SRP complex with the translocon that the stimulation of GTPase activity induces the dissociation of the complex and allows docking of the RNCs onto the translocon. The dissociated SRP can either replenish the pool of cytosolic SRP or assemble again in a phospholipid-dependent manner with FtsY.