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• W2162060803 abstract "In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitrocross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP. This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY. Here we describe a further in vitroreconstitution of SRP-mediated membrane insertion in which purified ribosome-nascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP. We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion. Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids. These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes. In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitrocross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP. This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY. Here we describe a further in vitroreconstitution of SRP-mediated membrane insertion in which purified ribosome-nascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP. We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion. Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids. These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes. Going across or integrating into the inner membrane presents two different challenges to proteins synthesized in the cytosol of a prokaryotic cell, and this is reflected by the existence of two main targeting routes. The SecB pathway is specialized for the targeting of periplasmic and outer membrane proteins. SecB is a cytosolic chaperone that binds to the mature region of a subset of preproteins (1Kumamoto C.A. Francetic O. J. Bacteriol. 1993; 175: 2184-2188Crossref PubMed Google Scholar). The SecB-preprotein complex is targeted to SecA, which is bound with high affinity to the membrane-embedded SecYEG complex (for review, see Ref.2Driessen A.J.M. Fekkes P. van der Wolk J.P.W. Curr. Opin. Microbiol. 1998; 1: 216-222Crossref PubMed Scopus (146) Google Scholar). Both in vivo and in vitro experiments indicate that the signal recognition particle (SRP) 1The abbreviations used are: SRPsignal recognition particleERendoplasmic reticulumDSSdisuccinimidyl suberateRNCribosome nascent chain complexSRNCSRP ribosome nascent chain complexGMP-PNPguanylyl-β,γ-imidodiphosphateINVinverted membrane vesicle1The abbreviations used are: SRPsignal recognition particleERendoplasmic reticulumDSSdisuccinimidyl suberateRNCribosome nascent chain complexSRNCSRP ribosome nascent chain complexGMP-PNPguanylyl-β,γ-imidodiphosphateINVinverted membrane vesicle pathway is primarily used for the targeting of integral inner membrane proteins (3Mac Farlane J. Müller M. Eur. J. Biochem. 1995; 223: 766-771Crossref Scopus (99) Google Scholar, 4de Gier J.W.L. Mansournia P. Valent Q.A. Phillips G.J. Luirink J. von Heijne G. FEBS Lett. 1996; 399: 307-309Crossref PubMed Scopus (135) Google Scholar, 5Ulbrandt N.D. Newitt J.A. Bernstein H.D. Cell. 1997; 88: 187-196Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 6Valent Q.A. de Gier J.-W.L. von Heijne G. Kendall D.A. ten Hagen-Jongman C.M. Oudega B. Luirink J. Mol. Microbiol. 1997; 25: 53-64Crossref PubMed Scopus (149) Google Scholar, 7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar). This pathway resembles SRP-mediated targeting of proteins to the membrane of the endoplasmic reticulum (ER) in eukaryotes (for review, see Ref. 8Rapoport T.A. Jungnickel B. Kutay U. Annu. Rev. Biochem. 1996; 65: 271-303Crossref PubMed Scopus (491) Google Scholar). The eukaryotic SRP interacts with nascent membrane and secreted proteins and targets them to the SRP receptor SRα at the ER membrane. The eukaryotic SRP is a complex consisting of six proteins arranged on an RNA scaffold, the 7 S RNA. Escherichia coli contains a smaller SRP composed of the P48 protein and the 4.5 S RNA, which are homologous to the eukaryotic SRP54 and the 7 S RNA, respectively (for review, see Refs. 9Luirink J. Dobberstein B. Mol. Microbiol. 1994; 11: 9-13Crossref PubMed Scopus (75) Google Scholar and 10Wolin S.L. Cell. 1994; 77: 787-790Abstract Full Text PDF PubMed Scopus (51) Google Scholar). In addition, an SRα homologue has been identified in E. coli, designated FtsY (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar, 11Luirink J. ten Hagen-Jongman C.M. van der Weijden C.C. Oudega B. High S. Dobberstein B. Kusters R. EMBO J. 1994; 13: 2289-2296Crossref PubMed Scopus (209) Google Scholar). Both P48 and FtsY (and their eukaryotic counterparts) are GTPases, and GTP binding and hydrolysis regulate the targeting cycle in a mechanism that has not yet been fully defined (12Miller J.D. Bernstein H.D. Walter P. Nature. 1994; 367: 657-659Crossref PubMed Scopus (186) Google Scholar, 13Kusters R. Lentzen G. Eppens E. van Geel A. van der Weijden C.C. Wintermeyer W. Luirink J. FEBS Lett. 1995; 372: 253-258Crossref PubMed Scopus (42) Google Scholar, 14Powers T. Walter P. Science. 1995; 269: 1422-1424Crossref PubMed Scopus (181) Google Scholar). In vivo overproduction of polytopic inner membrane proteins titrated out the limited amount of endogenous E. coli SRP (5Ulbrandt N.D. Newitt J.A. Bernstein H.D. Cell. 1997; 88: 187-196Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), whereas depletion of essential SRP components affected membrane targeting of both polytopic and bitopic inner membrane proteins (3Mac Farlane J. Müller M. Eur. J. Biochem. 1995; 223: 766-771Crossref Scopus (99) Google Scholar, 4de Gier J.W.L. Mansournia P. Valent Q.A. Phillips G.J. Luirink J. von Heijne G. FEBS Lett. 1996; 399: 307-309Crossref PubMed Scopus (135) Google Scholar). signal recognition particle endoplasmic reticulum disuccinimidyl suberate ribosome nascent chain complex SRP ribosome nascent chain complex guanylyl-β,γ-imidodiphosphate inverted membrane vesicle signal recognition particle endoplasmic reticulum disuccinimidyl suberate ribosome nascent chain complex SRP ribosome nascent chain complex guanylyl-β,γ-imidodiphosphate inverted membrane vesicle We have developed an in vitro cross-linking approach to dissect subsequent stages in SRP-mediated protein targeting. Short nascent inner membrane proteins synthesized in a membrane-free E. coli cell extract were shown to interact with the SRP via their amino-terminal hydrophobic targeting signal (6Valent Q.A. de Gier J.-W.L. von Heijne G. Kendall D.A. ten Hagen-Jongman C.M. Oudega B. Luirink J. Mol. Microbiol. 1997; 25: 53-64Crossref PubMed Scopus (149) Google Scholar). The SRP receptor FtsY, GTP, and inverted E. coli inner membrane vesicles were shown to be required for release of the SRP from the nascent chain (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar). Upon release of the SRP at the membrane, the nascent chain inserts into the membrane in the vicinity of the translocase components SecA, SecY, and SecG (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar). Hence, it seems that the SecB- and SRP-targeting pathways converge at a common membrane translocation complex. The exact composition of the translocase that is reached by the different routes may differ and has not yet been determined. Of particular interest is the cross-linking of SRP-targeted precursors to SecA, which functions as the molecular motor for translocation in the SecB pathway (2Driessen A.J.M. Fekkes P. van der Wolk J.P.W. Curr. Opin. Microbiol. 1998; 1: 216-222Crossref PubMed Scopus (146) Google Scholar). In an attempt to further define the minimal requirements for SRP-mediated targeting and membrane insertion, we have used proteoliposomes instead of inner membrane vesicles in our targeting assay. Short nascent chains of the inner membrane protein FtsQ were efficiently targeted to the E. coli SecYEG complex reconstituted in E. coli phospholipids. Hence, SecA seems dispensable for SRP-mediated protein targeting to the E. coli translocase. Before stable membrane insertion, the SRP was efficiently released from the nascent chains by FtsY in a GTP-dependent reaction. Protein-free liposomes also induced significant FtsY-mediated release of the SRP from the nascent chains indicating that the interaction of FtsY with lipids is sufficient to induce a conformational change in the SRP that lowers its affinity for the nascent chains. Restriction enzymes were from Roche Molecular Biochemicals GmbH (Mannheim, Germany). Megashortscript T7 transcription kit was from Ambion Inc. (Austin, TX). [35 S]Methionine and protein A-Sepharose were from Amersham Pharmacia Biotech (Buckinghamshire, UK). Disuccinimidyl suberate (DSS) was from Pierce.E. coli total phospholipid extract was from Avanti Polar Lipids Inc. (Pelham, AL). Aurintricarboxylic acid was from ICN Biomedicals Inc. (Aurora, OH). Nucleotides and all other chemicals were supplied by Sigma. Strain MC4100 was used to obtain translation lysate and inverted membrane vesicles (INVs) (15De Vrije T. Tommassen J. De Kruijff B. Biochim. Biophys. Acta. 1987; 900: 63-72Crossref PubMed Scopus (111) Google Scholar). Strain Top10F′ was used as host for plasmid pC4Meth108FtsQ (6Valent Q.A. de Gier J.-W.L. von Heijne G. Kendall D.A. ten Hagen-Jongman C.M. Oudega B. Luirink J. Mol. Microbiol. 1997; 25: 53-64Crossref PubMed Scopus (149) Google Scholar), and the OmpT and OmpP protease-deficient strain SF100 was used as expression host for plasmid pET349 to allow the overexpression of SecYEG (16van der Does C. Manting E.H. Kaufmann A. Lutz M. Driessen A.J.M. Biochemistry. 1998; 37: 201-210Crossref PubMed Scopus (94) Google Scholar). pC4Meth108FtsQ plasmid was linearized withHindIII and transcribed using T7 polymerase. The resulting truncated mRNAs were translated for 20 min at 25 °C in an S-135E. coli in vitro translation system to produce ribosome nascent chain complexes (RNCs) (6Valent Q.A. de Gier J.-W.L. von Heijne G. Kendall D.A. ten Hagen-Jongman C.M. Oudega B. Luirink J. Mol. Microbiol. 1997; 25: 53-64Crossref PubMed Scopus (149) Google Scholar). After 3 min of incubation, aurintricarboxylic acid (1 mm) was added to inhibit further initiation of translation resulting in homogeneous nascent chain length. Translation was stopped by adding 3 mmchloramphenicol. To allow SRP-RNC (SRNC) complex formation, 260 nm reconstituted SRP was added to the translation reaction (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar). After 5 min of incubation at 25 °C, samples were chilled on ice, and the resulting SRNC complexes were purified from the translation mixture by centrifugation through a high salt sucrose cushion (17High S. Flint N. Dobberstein B. J. Cell Biol. 1991; 113: 25-34Crossref PubMed Scopus (65) Google Scholar) and resuspended in RN buffer (100 mm KOAc, 5 mm Mg(OAc)2, 50 mm Hepes-KOH, pH 7.9). The E. coli SecYEG complex that carries a hexahistidine tag at the amino terminus of SecY was purified and reconstituted in acetone/ether-washed E. coliphospholipids as described (16van der Does C. Manting E.H. Kaufmann A. Lutz M. Driessen A.J.M. Biochemistry. 1998; 37: 201-210Crossref PubMed Scopus (94) Google Scholar). Standard proteoliposomes were obtained by the reconstitution of 200 μl of SecYEG (50 μg/ml) in 40 μl of lipids (20 mg/ml). Proteoliposomes used in the experiment described in Fig. 3 were prepared using different amounts of 0.2 mg/ml SecYEG (0–50 μl) diluted in a total volume of 200 μl of purification buffer (1.25% (w/v octylglucoside, 0.3 mg/ml phospholipids, 40% (w/v) glycerol, and 50 mm KCl in Tris-HCl, pH 7.8). These amounts of SecYEG were reconstituted with 40 μl of phospholipids, yielding (v/v) ratios of 0–1.25. Before use, proteoliposomes were sonicated 3 times for 10 s; E. coli SecA was purified as described (18Cabelli R.J. Chen L. Tai P.C. Oliver D.B. Cell. 1988; 55: 683-692Abstract Full Text PDF PubMed Scopus (173) Google Scholar). Targeting reactions were done as described previously by incubating purified SRNCs together with 1 mm FtsY, 50 μm GTP, 50 μm ATP, and either INVs (1.25 mg/ml protein) or proteoliposomes for 5 min at 25 °C (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar). Modifications to this standard reaction protocol are described in the figure legends. Cross-linking was induced with 1 mm DSS for 10 min at 25 °C and quenched at 4 °C by adding one-tenth volume of quenching buffer (1 m glycine, 100 mm NaHCO3, pH 8.5). To separate integral membrane from soluble and peripheral cross-linked complexes, samples were treated with 0.18 m Na2CO3, pH 11.3, for 15 min on ice. The membrane fractions containing integral membrane proteins were pelleted by ultracentrifugation (10 min, 110,000 × g) and resuspended in RN buffer. Supernatant fractions were trichloroacetic acid-precipitated, washed with cold acetone, and resuspended in RN buffer. Samples were immunoprecipitated as described (19Luirink J. High S. Wood H. Giner A. Tollervey D. Dobberstein B. Nature. 1992; 359: 741-743Crossref PubMed Scopus (153) Google Scholar) or mixed directly with 2× SDS-gel loading buffer before gel electrophoresis. Anti-SecA serum was added to protein A-Sepharose slurry and incubated for 1 h at room temperature. After incubation the Sepharose was washed twice with incubation buffer (30 mm Tris-HCL pH 7.5, 150 mm NaCl, 30 mm Tris-HCL pH 7.5) and once with lysate buffer (10 mm triethylammonium, 10 mmMg(OAc)2, 22 mmN(CH3)4Ac). To prepare a control lysate, protein A-Sepharose was prepared in the same way except that no anti-SecA serum was added. Both Sepharose preparations were collected by centrifugation and resuspended in MC4100 translation lysate. After 2 h of incubation at 4 °C, the Sepharose beads were spun down and the supernatants recovered. Supernatant corresponding to the incubation with anti-SecA-loaded Sepharose was referred to as SecA− lysate, and supernatant corresponding to the incubation with Sepharose only was referred to as SecA+ lysate. Both lysates were analyzed by immunoblotting using anti-SecA serum to verify the extent of depletion. Samples were analyzed on 12, 15, or 4–15% gradient SDS-polyacrylamide gels. Radiolabeled proteins were visualized by phosphor imaging using a Molecular Dynamics PhosphorImager 473 and quantified using the Imagequant software from Molecular Dynamics. FtsQ is a type II E. coli inner membrane protein, which was used as a model protein for SRP-mediated targeting throughout this study. An in vitroSRP-dependent targeting assay was developed in which short FtsQ RNCs were efficiently targeted to the translocase contained in purified INVs. Interaction of the labeled nascent chain with SecY and SecA was demonstrated by formation of covalent bonds using the bifunctional cross-linker reagent DSS (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar). In this study we further investigated this SRP-dependent targeting process. To define the essential membrane components required for targeting and membrane insertion, proteoliposomes composed of E. coliphospholipids and purified SecYEG complex were used instead of INVs. Short (108-mer) FtsQ nascent chains were produced in vitrousing a membrane-free E. coli translation lysate. Under these conditions the endogenous SRP contained in the lysate is titrated out by the preponderance of exposed hydrophobic targeting signals. SRP loading efficiency was increased by adding reconstituted E. coli SRP after the translation reaction (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar). The resulting SRP-RNCs complexes (SRNCs) were purified through a high salt sucrose cushion and incubated in the presence of FtsY, the E. colihomologue of the eukaryotic SRP receptor SRα (9Luirink J. Dobberstein B. Mol. Microbiol. 1994; 11: 9-13Crossref PubMed Scopus (75) Google Scholar) and GTP, which were shown to be essential for their proper targeting to the membranes and for efficient release of the SRP from the nascent chain (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar). As accepting membranes, we compared INVs and proteoliposomes. Each reaction contained equal amounts of SecY as judged by quantitative immunoblotting (data not shown). After the targeting reaction cross-linking of 108FtsQ was induced using the membrane-permeable bifunctional cross-linker DSS and the membranes were extracted with Na2CO3 to separate membrane integral from soluble and membrane peripheral cross-linked complexes. Either with INVs or with proteoliposomes, a 108FtsQ cross-linking adduct appeared at about 50 kDa in the Na2CO3pellet (Fig. 1, lanes 3 and5, respectively). This complex represents a membrane integral complex of the radiolabeled 108FtsQ and SecY, as demonstrated by immunoprecipitation (Fig. 1, lanes 7 and 11). The SecY adduct, which originates from the proteoliposomes, migrates slightly slower than the SecY from the INVs due to the histidine extension that is attached to the purified SecY used to reconstitute the SecYEG complex in the proteoliposomes (16van der Does C. Manting E.H. Kaufmann A. Lutz M. Driessen A.J.M. Biochemistry. 1998; 37: 201-210Crossref PubMed Scopus (94) Google Scholar). In addition, 108FtsQ was also found to be cross-linked to the other membrane integral components of the translocase, SecE and SecG (Fig. 1, lanes 8 and 9 for INVs, lanes 12 and 13for proteoliposomes). High molecular weight (∼69 kDa) cross-linked complexes in the Na2CO3 pellet of both INVs and proteoliposomes were also immunoprecipitated with antibodies directed against SecE (Fig. 1, lanes 8 and 12) and SecG (Fig. 1, lanes 9 and 13). These complexes probably represent trimeric structures consisting of 108FtsQ, SecY, and either SecE or SecG (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar). As previously reported (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar), cross-linking to SecA (∼120 kDa product) is detected in the Na2CO3 pellet samples when INVs are used in the targeting reaction (Fig. 1, lanes 3 and7). A cross-linking adduct of ∼220 kDa is also immunoprecipitated from the Na2CO3 pellet with anti-SecA serum and might correspond to cross-linked SecA dimer (Fig.1, lanes 3 and 7). Upon immunoprecipitation a weak SecA adduct is also detected in the Na2CO3pellet from proteoliposomes (Fig. 1, lane 10). Because the proteoliposomes do not contain any SecA initially, we assume that some SecA contained in the translation lysate is co-purified with the SRNCs and is incorporated into the proteoliposomes during the targeting reaction. This point will be addressed in the last paragraph under “Results.” In the Na2CO3 supernatant from the INVs adducts to the SRP component P48 and trigger factor were detected (Fig. 1,lanes 15, 19, and 20) as observed before (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar). Trigger factor may act as a general folding catalyst via its prolyl isomerase activity and was shown to interact with all nascent chains regardless of their hydrophobicity (6Valent Q.A. de Gier J.-W.L. von Heijne G. Kendall D.A. ten Hagen-Jongman C.M. Oudega B. Luirink J. Mol. Microbiol. 1997; 25: 53-64Crossref PubMed Scopus (149) Google Scholar). In contrast, hardly any P48 adduct could be detected in the Na2CO3 supernatant when proteoliposomes were used as target membranes, whereas cross-linking to trigger factor was relatively efficient (Fig. 1, lanes 17, 22, and23). This suggests an efficient release of the SRP from the FtsQ RNCs followed by an efficient targeting to the translocase as indicated by the strong cross-linking signal to SecY in the Na2CO3 pellet samples. From these data we conclude that proteoliposomes composed of E. coli phospholipids and purified SecYEG complex support the transfer of 108FtsQ in a reconstituted SRP-mediated targeting assay from the SRP to the translocase. Compared with targeting reactions that contain INVs, the release of the SRP and the association with the translocase is even more efficient when proteoliposomes are used. SRP-mediated targeting of nascent FtsQ to SecY in the INVs requires FtsY and GTP or its nonhydrolyzable analogue GMP-PNP (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar). We investigated whether release of the SRP from nascent FtsQ and association with SecY in the proteoliposomes occurs via the same mechanism by studying its dependence on FtsY and nucleotides in the targeting assay described above. As shown in Fig.2, the release of the SRP and the association with SecY did not occur in the absence of FtsY (Fig. 2,lanes 9, 10, and 11). Proteoliposomes in combination with FtsY were essential for the release of the SRP (Fig. 2, lanes 2 and 4). GTP or GMP-PNP (lanes 5 and 7) but not AMP-PNP or GDP (lanes 6 and 8) supported the transfer reaction. Consistently, mutant FtsY, FtsY A449 (13Kusters R. Lentzen G. Eppens E. van Geel A. van der Weijden C.C. Wintermeyer W. Luirink J. FEBS Lett. 1995; 372: 253-258Crossref PubMed Scopus (42) Google Scholar) that is unable to bind GTP, was not functional in the targeting assay (data not shown). Based on these results, we conclude that targeting of nascent FtsQ to proteoliposomes mechanistically resembles the targeting to INVs and thus provides a valid and attractive model for detailed studies on the mechanism of nascent chain transfer and membrane insertion. In vivo, FtsY is equally distributed between the cytosol and inner membrane (11Luirink J. ten Hagen-Jongman C.M. van der Weijden C.C. Oudega B. High S. Dobberstein B. Kusters R. EMBO J. 1994; 13: 2289-2296Crossref PubMed Scopus (209) Google Scholar). We have obtained evidence that FtsY has affinity for lipids (20de Leeuw E. Poland D. Mol O. Sinning I. ten Hagen-Jongman C.M. Oudega B. Luirink J. FEBS Lett. 1997; 416: 225-229Crossref PubMed Scopus (65) Google Scholar). 2E. de. Leeuw, K. te Kaat, C. Moser, G. Menestrina, R. Demel, B. de Kruijff, J. Luirink, and I. Sinning, manuscript submitted. To study the influence of the protein:lipid ratio on the efficiency of nascent chain transfer, proteoliposomes of different composition were prepared and tested in our targeting assay in the presence of FtsY and GMP-PNP. The efficiency of 108FtsQ targeting was monitored by quantifying the P48 cross-linking adducts in the Na2CO3 supernatant and the SecY cross-linking adducts in the Na2CO3 pellets (Fig. 3). As expected, the strongest cross-linking to P48 was observed when membranes were omitted in the targeting reaction and this signal was taken as 100%. Different proteoliposomes were made by mixing a fixed amount of E. coli phospholipids with increasing amounts of purified SecYEG complex. The targeting efficiency correlated with the SecYEG concentration in the lipid bilayer (Fig. 3). Proteoliposomes containing a SecYEG:lipid ratio of 1.25 resulted in the most efficient targeting as indicated by the weakest cross-linking to P48 and the strongest cross-linking to SecY. Surprisingly, when liposomes that do not contain any SecYEG complex were used in the targeting reaction a significant SRP release was observed (∼53%, Fig. 3). This release was completely dependent on the addition of both FtsY and GTP or GMP-PNP (not shown). Apparently, the interaction of FtsY with the lipids is sufficient to induce significant GTP-dependent release of the SRP from the nascent chain. However, this SRP release is not followed by effective membrane insertion of the 108FtsQ as judged by Na2CO3 extraction (data not shown). Apparently, the SecYEG complex is necessary and sufficient to obtain a stable integration into the lipid bilayer. SecA is a peripheral translocase component that plays a key role in the post-translational translocation process by functioning as a receptor for precursor proteins and as an ATP-driven molecular motor (2Driessen A.J.M. Fekkes P. van der Wolk J.P.W. Curr. Opin. Microbiol. 1998; 1: 216-222Crossref PubMed Scopus (146) Google Scholar). In a previous study (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar), we demonstrated that 108FtsQ RNCs targeted to INVs were in close contact not only to the “core” translocase component SecY but also to SecA (see also Fig. 1, lanes 3 and 6). It remained unclear whether SecA plays an active role in the observed SRP-mediated targeting and membrane insertion of 108FtsQ or whether the SecA cross-linking adducts were obtained just because SecA is located in the vicinity of the nascent chain as a structural subunit of theE. coli translocase (2Driessen A.J.M. Fekkes P. van der Wolk J.P.W. Curr. Opin. Microbiol. 1998; 1: 216-222Crossref PubMed Scopus (146) Google Scholar). If this last hypothesis is correct, SecA should be dispensable in the SRP-dependent targeting reaction. This could be tested by using SecYEG proteoliposomes as SecA-free target membranes. However, as shown in Fig. 1 (lane 10) weak cross-linking of SecA to 108FtsQ was observed in the Na2CO3 pellet of proteoliposomes. Apparently, SecA is co-purified with the SRNC preparation and can be incorporated into proteoliposomes during the targeting reaction. The affinity of SecA for SecY and for acidic phospholipids might underlie this transfer of SecA from the SRNCs to the proteoliposomes (2Driessen A.J.M. Fekkes P. van der Wolk J.P.W. Curr. Opin. Microbiol. 1998; 1: 216-222Crossref PubMed Scopus (146) Google Scholar). We estimated by Western blotting that about 0.3% of the SecA present in the translation lysate co-purifies with the SRNCs upon sedimentation of the ribosomes through a high salt sucrose cushion (data not shown). Two observations suggest that it is most likely that SecA is co-purified via its general affinity for ribosomes (21Cabelli R.J. Dolan K.M. Qian L. Oliver D.B. J. Biol. Chem. 1991; 266: 24420-24427Abstract Full Text PDF PubMed Google Scholar), rather than via a direct interaction with the FtsQ nascent chains. First, SecA was never found cross-linked to nascent FtsQ or any other nascent polypeptide in the absence of membranes even when added in excess (6Valent Q.A. de Gier J.-W.L. von Heijne G. Kendall D.A. ten Hagen-Jongman C.M. Oudega B. Luirink J. Mol. Microbiol. 1997; 25: 53-64Crossref PubMed Scopus (149) Google Scholar). Second, when translation reactions were either not supplemented with any mRNA or supplemented with truncated mRNA encoding nascent chains of a cytosolic protein, the same amount of SecA was co-purified with the ribosomal fraction (data not shown). To remove this residual SecA, we immunodepleted the translation lysate using anti-SecA serum (see “Experimental Procedures” for details). After this procedure no SecA could be detected by immunoblotting in the translation lysate (SecA− lysate) compared with a mock treated lysate (SecA+ lysate) (data not shown). Both lysates remained active for translation. 108FtsQ RNCs were produced using either SecA− or SecA+ lysate and loaded with reconstituted SRP. After purification the two kinds of SRNCs were used in different targeting reactions to which purified SecA was added (0, 0.1, and 0.5 μm, final concentrations). Interaction with proteoliposomes was assessed by DSS-induced cross-linking to SecY (Fig.4, A–C) and by quantification of the resistance of the RNCs to Na2CO3 extraction (Fig. 4 D). When a SecA− lysate was used for translation and when no additional SecA was added to the targeting reaction, cross-linking to SecY was observed (Fig. 4 A, lane 4) and the 108FtsQ was inserted into the membrane in a Na2CO3 resistant conformation (Fig.4 D). Thus, the FtsQ RNCs are able to reach the translocase and become inserted into the membrane in the absence of SecA. Upon addition of SecA to the targeting reaction cross-linking to SecY in the Na2CO3 pellet was slightly reduced irrespective of the translation lysate used, whereas SecA cross-linking adducts (**) appeared (Fig. 4 A, lanes 1–6and Fig. 4 B). Part of the SecA adducts were extracted by Na2CO3 (Fig. 4 A, lanes 7–12). For both translation lysates, addition of purified SecA to the targeting reaction slightly reduced the Na2CO3 resistance of the targeted RNCs (Fig.4 D) consistent with the decrease in cross-linking to SecY. To confirm that the different cross-linking adducts observed were indeed related to either SecA or to SecY, immunoprecipitations were carried out after cross-linking. The SecA+ lysate was used to generate the RNCs, and 0.5 μm SecA was added to the targeting reaction. In the Na2CO3 pellet sample the 50-kDa product (*) was indeed immunoprecipitated with anti-SecY serum (Fig. 4 C, lane 3). Both the major 120-kDa cross-linking adducts (**) and minor 220-kDa products present in the Na2CO3 pellet and supernatant were immunoprecipitated with the anti-SecA serum (Fig.4 C, lane 2). We assume that the ∼220-kDa product corresponds to cross-linked complexes between the RNCs and the dimeric form of SecA. The SecA related adducts of ∼150 and ∼45 kDa ( ) in the Na2CO3 pellet are most likely proteolytic fragments of the SecA dimer and monomer cross-linking products. From these data, we conclude that SecA is not required for efficient targeting and initial membrane insertion of FtsQ nascent chainsin vitro. Furthermore, at high SecA concentrations there is a weak competition effect between SecA and SecY for the binding of RNCs delivered to the membrane via the SRP pathway. This competition seems to reduce the 108FtsQ insertion into the membrane. We have further developed our homologous in vitrotargeting assay that supports SRP-mediated targeting and membrane insertion of nascent E. coli inner membrane proteins (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar). The assay now contains purified 108FtsQ RNCs saturated with SRP, FtsY, GTP, and proteoliposomes that consist of purified E. coliphospholipids and the core translocon complex SecYEG. In this system, the SRP was released from the nascent chain in a GTP-dependent reaction, associated with SecY and acquired a Na2CO3-resistant conformation in the proteoliposomes as efficiently as in crude INVs. Thus, we have defined the minimal requirements for an efficient in vitroSRP-dependent targeting to the core components of theE. coli translocon. Preliminary experiments on the topology of targeted 108FtsQ in both INVs and proteoliposomes using cysteine replacement coupled to chemical labeling, suggest that it has the same topology as the native protein (data not shown). No additional proteinaceous membrane factors are required in these early steps of the targeting process. Notably, the SecDF/YajC subcomplex is dispensable, and may therefore be specifically devoted for the translocation or the membrane insertion of preproteins delivered via the SecB-dependent pathway (2Driessen A.J.M. Fekkes P. van der Wolk J.P.W. Curr. Opin. Microbiol. 1998; 1: 216-222Crossref PubMed Scopus (146) Google Scholar). Apparently, for the initial insertion of the inner membrane protein FtsQ, SecA, and ATP are not required. However, as judged by protein cross-linking, SecA was in close proximity to nascent FtsQ targeted to INVs (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar) or when proteoliposomes were supplemented with SecA (this study). Probably, SecA is juxtaposed to the nascent chain due to its affinity for SecY (22Hartl F.-U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (444) Google Scholar, 23Duong F. Wickner W. EMBO J. 1997; 16: 2756-2768Crossref PubMed Scopus (226) Google Scholar) rather than playing a crucial role in the initial insertion process or in the transfer of the nascent chain to SecY. As suggested by this study, when present in excess, SecA actually interfered with the transfer to SecY. Interestingly, a point mutation in SecY has been reported that specifically impaired the integration of integral membrane proteins but not the secretion of SecB-targeted precursors. In this conditional strain, expression of SecA was up-regulated (24Newitt J.A. Bernstein H.D. J. Biol. Chem. 1998; 273: 12451-12456Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). SecA may be involved at a later stage in the insertion of inner membrane proteins especially when large periplasmic domains are translocated. The role of SecA in the complete assembly of inner membrane proteins is difficult to study in vivo and may differ for different substrate proteins (25Werner P.K. Saier Jr., M.H. Müller M. J. Biol. Chem. 1992; 267: 24523-24532Abstract Full Text PDF PubMed Google Scholar, 26Bassilana M. Gwizdek C. EMBO J. 1996; 15: 5202-5208Crossref PubMed Scopus (29) Google Scholar, 27Traxler B. Murphy C. J. Biol. Chem. 1996; 271: 12394-12400Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). It should be noted that full-length FtsQ did not integrate into SecYEG proteoliposomes either in the presence or absence of SecA (data not shown) suggesting that additional components are required for complete in vitro reconstitution of inner membrane protein biogenesis. Interestingly, liposomes that consist solely of E. coliphospholipids are still able to support the FtsY-mediated release of the SRP from the nascent chain, albeit less efficiently than SecYEG containing proteoliposomes. We have previously demonstrated that FtsY is able to associate with RNC-SRP complexes in the absence of GTP and membranes (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar). INVs and GTP-binding but not hydrolysis were required to dissociate the SRP from the nascent chain (7Valent Q.A. Scotti P.A. High S. de Gier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (244) Google Scholar). Apparently, FtsY pilots the targeting complex to the lipids without the requirement of any specific membrane receptor. This might cause a conformational change that is transferred to P48 in the SRP thus increasing the affinity of both P48 and FtsY for GTP. By analogy with the eukaryotic SRP cycle, binding of GTP to P48 may reduce its affinity for the nascent chain (28Connolly T. Gilmore R. Cell. 1989; 57: 599-610Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 29Rapiejko P.J. Gilmore R. Cell. 1997; 89: 703-713Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The nascent chain that is released at the membrane does not insert stably into the lipid bilayer in an alkali resistant conformation but requires the SecYEG complex to acquire a stable membrane association. FtsY is located in part in the cytosol and in part in the cytoplasmic membrane (11Luirink J. ten Hagen-Jongman C.M. van der Weijden C.C. Oudega B. High S. Dobberstein B. Kusters R. EMBO J. 1994; 13: 2289-2296Crossref PubMed Scopus (209) Google Scholar). It is a highly charged protein that does not contain any predicted membrane spanning segments and its mechanism of association with the membrane is obscure (20de Leeuw E. Poland D. Mol O. Sinning I. ten Hagen-Jongman C.M. Oudega B. Luirink J. FEBS Lett. 1997; 416: 225-229Crossref PubMed Scopus (65) Google Scholar). The data presented in this study indicate that in contrast to its mammalian counterpart SRα, FtsY can function in the release of the SRP in the absence of a dedicated membrane anchoring protein. This might explain the ability of FtsY to cooperate with E. coli SRP in the targeting of proteins to microsomal membranes in a heterologous in vitro targeting assay (30Powers T. Walter P. EMBO J. 1997; 16: 4880-4886Crossref PubMed Scopus (156) Google Scholar). Consistent with this suggestion, we have recently observed that FtsY interacts with lipids using a phospholipid monolayer technique.2 Through its affinity for lipids, FtsY may increase the effective concentration of SRNCs at the membrane (30Powers T. Walter P. EMBO J. 1997; 16: 4880-4886Crossref PubMed Scopus (156) Google Scholar). Still, proteoliposomes function more efficiently in the release of the SRP, which may be related to additional targeting functions of the ribosome and SRP. It is well established that eukaryotic ribosomes have affinity for Sec61p, the protein conducting channel in the ER membrane (31Kalies K.U. Görlich D. Rapoport T.A. J. Cell Biol. 1994; 126: 925-934Crossref PubMed Scopus (146) Google Scholar). Alternatively FtsY, in addition to its association with lipids, may interact directly with the SecYEG complex. However, an association of this kind could not be detected by cross-linking, immunoprecipitation or co-purification approaches. 3E. de Leeuw and J. Luirink, unpublished results. The reconstituted targeting system described in this study will be invaluable for a detailed analysis of the consecutive steps during SRP-mediated protein targeting and membrane insertion, allowing the intermolecular interactions, dynamics, and energy requirements of this process to be studied. The assay will also provide a means to identify soluble and membrane components specifically devoted to the proper assembly and folding of inner membrane proteins. Ultimately, this approach should lead to the full reconstitution of inner membrane protein biosynthesis. The purified SecYEG complex was kindly provided by C. van der Does. We thank, S. High and N. Harms for critical reading of the manuscript." @default.
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• W2162060803 title "SecA Is Not Required for Signal Recognition Particle-mediated Targeting and Initial Membrane Insertion of a Nascent Inner Membrane Protein" @default.
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