Title: Export of β-Lactamase Is Independent of the Signal Recognition Particle
Abstract: In Escherichia coli, three different types of proteins engage the SecY translocon of the inner bacterial membrane for translocation or insertion: 1) polytopic membrane proteins that prior to their insertion into the membrane are targeted to the translocon using the bacterial signal recognition particle (SRP) and its receptor; 2) secretory proteins that are targeted to and translocated across the SecY translocon in a SecA- and SecB-dependent reaction; and 3) membrane proteins with large periplasmic domains, requiring SRP for targeting and SecA for the translocation of the periplasmic moiety. In addition to its role as a targeting device for membrane proteins, a function of the bacterial SRP in the export of SecB-independent secretory proteins has also been postulated. In particular, β-lactamase, a hydrolytic enzyme responsible for cleavage of the β-lactam ring containing antibiotics, is considered to be recognized and targeted by SRP. To examine the role of the SRP pathway in β-lactamase targeting and export, we performed a detailed in vitro analysis. Chemical cross-linking and membrane binding assays did not reveal any significant interaction between SRP and β-lactamase nascent chains. More importantly, membrane vesicles prepared from mutants lacking a functional SRP pathway did block the integration of SRP-dependent membrane proteins but supported the export of β-lactamase in the same way as that of the SRP-independent protein OmpA. These data demonstrate that in contrast to previous results, the bacterial SRP is not involved in the export of β-lactamase and further suggest that secretory proteins of Gram-negative bacteria in general are not substrates of SRP. In Escherichia coli, three different types of proteins engage the SecY translocon of the inner bacterial membrane for translocation or insertion: 1) polytopic membrane proteins that prior to their insertion into the membrane are targeted to the translocon using the bacterial signal recognition particle (SRP) and its receptor; 2) secretory proteins that are targeted to and translocated across the SecY translocon in a SecA- and SecB-dependent reaction; and 3) membrane proteins with large periplasmic domains, requiring SRP for targeting and SecA for the translocation of the periplasmic moiety. In addition to its role as a targeting device for membrane proteins, a function of the bacterial SRP in the export of SecB-independent secretory proteins has also been postulated. In particular, β-lactamase, a hydrolytic enzyme responsible for cleavage of the β-lactam ring containing antibiotics, is considered to be recognized and targeted by SRP. To examine the role of the SRP pathway in β-lactamase targeting and export, we performed a detailed in vitro analysis. Chemical cross-linking and membrane binding assays did not reveal any significant interaction between SRP and β-lactamase nascent chains. More importantly, membrane vesicles prepared from mutants lacking a functional SRP pathway did block the integration of SRP-dependent membrane proteins but supported the export of β-lactamase in the same way as that of the SRP-independent protein OmpA. These data demonstrate that in contrast to previous results, the bacterial SRP is not involved in the export of β-lactamase and further suggest that secretory proteins of Gram-negative bacteria in general are not substrates of SRP. To target newly synthesized proteins to the SecYEG translocon of the inner bacterial membrane, Escherichia coli employs two different protein targeting routes. Secretory proteins destined for the periplasmic space or the outer membrane are recognized posttranslationally by the cytoplasmic chaperone SecB and are subsequently transferred to SecA, which translocates the preprotein across the SecYEG channel in an ATP-dependent manner (1Behrmann M. Koch H.G. Hengelage T. Wieseler B. Hoffschulte H.K. Müller M. J. Biol. Chem. 1998; 273: 13898-13904Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 2Driessen A.J. Manting E.H. van der Does C. Nat. Struct. Biol. 2001; 8: 492-498Crossref PubMed Scopus (179) Google Scholar). Inner membrane proteins on the other hand are selectively recognized by the bacterial signal recognition particle (SRP), 1The abbreviations used are: SRP, signal recognition particle; INV inner membrane vesicle; PhoA, alkaline phosphatase; Bla, β-lactamase; MtlA-MPF, membrane-protected fragment of mannitol permease; RNCs, ribosome-associated nascent chains; DSS, disuccinimidyl suberate. consisting of the protein Ffh and the 4.5 S RNA. Upon binding of SRP, ribosome-associated nascent chains of membrane proteins are cotranslationally targeted to FtsY, the bacterial homologue of the SRP receptor, and are finally inserted into the lipid bilayer through the SecYE translocon (3MacFarlane J. Müller M. Eur. J. Biochem. 1995; 233: 766-771Crossref PubMed Scopus (100) Google Scholar, 4de Gier J.W. 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 (294) Google Scholar, 6Koch H.G. Hengelage T. Neumann-Haefelin C. MacFarlane J. Hoffschulte H.K. Schimz K.L. Mechler B. Müller M. Mol. Biol. Cell. 1999; 10: 2163-2173Crossref PubMed Scopus (134) Google Scholar, 7Valent Q.A. Antonie Leeuwenhoek. 2001; 79: 17-31Crossref PubMed Scopus (13) Google Scholar). For a subset of integral membrane proteins, i.e. membrane proteins with large periplasmic domains, SRP and SecA cooperate during the integration process (8Neumann-Haefelin C. Schafer U. Müller M. Koch H.G. EMBO J. 2000; 19: 6419-6426Crossref PubMed Scopus (111) Google Scholar, 9Koch H.G. Moser M. Schimz K.L. Müller M. J. Biol. Chem. 2002; 277: 5715-5718Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 10Scotti P.A. Valent Q.A. Manting E.H. Urbanus M.L. Driessen A.J. Oudega B. Luirink J. J. Biol. Chem. 1999; 274: 29883-29888Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Targeting of these proteins to SecY is exclusively mediated by SRP and FtsY, leading to a stable binding of ribosome-associated nascent chains to the translocon. Translocation of the periplasmic moiety across the inner membrane, however, requires the activity of SecA. There is conflicting evidence as to the involvement of SRP in the targeting of a subset of secretory proteins like β-lactamase, alkaline phosphatase (PhoA), or ribose-binding protein (11Phillips G.J. Silhavy T.J. Nature. 1992; 359: 744-746Crossref PubMed Scopus (224) Google Scholar). The depletion of either FtsY or Ffh concomitantly leads to decreased translocation of these proteins, suggesting that SRP and its receptor are involved in their export across the inner bacterial membrane (11Phillips G.J. Silhavy T.J. Nature. 1992; 359: 744-746Crossref PubMed Scopus (224) Google Scholar, 12Luirink J. 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 (213) Google Scholar, 13Ribes V. Romisch K. Giner A. Dobberstein B. Tollervey D. Cell. 1990; 63: 591-600Abstract Full Text PDF PubMed Scopus (163) Google Scholar). Because these proteins are considered to be SecB-independent, it had been proposed that SRP functions as an export-specific chaperone rather than a targeting factor for β-lactamase, PhoA, and ribose-binding protein, replacing SecB during export (11Phillips G.J. Silhavy T.J. Nature. 1992; 359: 744-746Crossref PubMed Scopus (224) Google Scholar). The interpretation of in vivo Ffh and FtsY depletion experiments is, however, complicated by the observation that targeting and integration of SecY is SRP-dependent. Decreasing the cellular concentrations of either SRP or FtsY simultaneously reduces the concentration of active translocons, making it difficult to differentiate between those export defects caused primarily by SRP/FtsY depletion and those originating from diminished concentrations of SecY (6Koch H.G. Hengelage T. Neumann-Haefelin C. MacFarlane J. Hoffschulte H.K. Schimz K.L. Mechler B. Müller M. Mol. Biol. Cell. 1999; 10: 2163-2173Crossref PubMed Scopus (134) Google Scholar, 14Seluanov A. Bibi E. J. Biol. Chem. 1997; 272: 2053-2055Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In E. coli, the ability of SRP to interact with its substrate is predominantly dependent on the length and the hydrophobicity of the signal sequence (15Valent Q.A. de Gier J.W. von Heijne G. Kendall D.A. Hagen-Jongman C.M. Oudega B. Luirink J. Mol. Microbiol. 1997; 25: 53-64Crossref PubMed Scopus (149) Google Scholar, 16de Gier J.W. Scotti P.A. Saaf A. Valent Q.A. Kuhn A. Luirink J. von Heijne G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14646-14651Crossref PubMed Scopus (112) Google Scholar, 17Valent Q.A. Scotti P.A. High S. de Gier J.W. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (245) Google Scholar). Signal anchor sequences of integral membrane proteins like mannitol permease (MtlA) (18Beck K. Wu L.F. Brunner J. Müller M. EMBO J. 2000; 19: 134-143Crossref PubMed Scopus (148) Google Scholar) or FtsQ (17Valent Q.A. Scotti P.A. High S. de Gier J.W. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (245) Google Scholar) have been shown to cross-link efficiently with SRP, whereas such an interaction cannot be detected with the cleavable signal sequences of secretory proteins such as the outer membrane protein OmpA (18Beck K. Wu L.F. Brunner J. Müller M. EMBO J. 2000; 19: 134-143Crossref PubMed Scopus (148) Google Scholar). However, signal sequence mutants of the SecB-independent secretory protein PhoA can be cross-linked to SRP provided that the hydrophobicity of the signal sequence is increased by inserting multiple leucine residues (7Valent Q.A. Antonie Leeuwenhoek. 2001; 79: 17-31Crossref PubMed Scopus (13) Google Scholar, 15Valent Q.A. de Gier J.W. von Heijne G. Kendall D.A. Hagen-Jongman C.M. Oudega B. Luirink J. Mol. Microbiol. 1997; 25: 53-64Crossref PubMed Scopus (149) Google Scholar, 17Valent Q.A. Scotti P.A. High S. de Gier J.W. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (245) Google Scholar). The SecB-dependent outer membrane protein PhoE has also been shown to interact with SRP under conditions in which the hydrophobicity and the extension of the α-helical signal sequence is increased by replacing a helix-breaking glycine residue with a helix-promoting leucine residue (19Adams H. Scotti P.A. de Cock H. Luirink J. Tommassen J. Eur. J. Biochem. 2002; 269: 5564-5571Crossref PubMed Scopus (44) Google Scholar). This PhoE derivative, however, is still exported in an SRP-independent manner. SRP binding to cleavable signal sequences of secretory proteins is further influenced by trigger factor, a ribosome-associated chaperone (15Valent Q.A. de Gier J.W. von Heijne G. Kendall D.A. Hagen-Jongman C.M. Oudega B. Luirink J. Mol. Microbiol. 1997; 25: 53-64Crossref PubMed Scopus (149) Google Scholar, 17Valent Q.A. Scotti P.A. High S. de Gier J.W. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (245) Google Scholar, 18Beck K. Wu L.F. Brunner J. Müller M. EMBO J. 2000; 19: 134-143Crossref PubMed Scopus (148) Google Scholar). Binding of trigger factor to OmpA nascent chains subsequently prevents SRP binding to OmpA. In the absence of trigger factor, however, SRP can be cross-linked to the OmpA signal sequence (18Beck K. Wu L.F. Brunner J. Müller M. EMBO J. 2000; 19: 134-143Crossref PubMed Scopus (148) Google Scholar). Among the SecB-independent secretory proteins, β-lactamase has always been considered to be a bona fide SRP substrate, in particular because its signal sequence is more hydrophobic than signal sequences of other secretory proteins (7Valent Q.A. Antonie Leeuwenhoek. 2001; 79: 17-31Crossref PubMed Scopus (13) Google Scholar, 15Valent Q.A. de Gier J.W. von Heijne G. Kendall D.A. Hagen-Jongman C.M. Oudega B. Luirink J. Mol. Microbiol. 1997; 25: 53-64Crossref PubMed Scopus (149) Google Scholar). However, so far no detailed in vitro analyses have been performed to really study the involvement of SRP in the targeting pathway of β-lactamase. Our analyses using protease protection assays, flotation gradient experiments, and chemical cross-linking demonstrate that targeting and export of β-lactamase proceed independently of the SRP pathway. Bacterial Strains and Plasmids—The genotypes and sources of the E. coli strains and the plasmids used in this study are shown in Table I.Table IStrains and plasmids used in this studyStrain or plasmidRelevant genotype or descriptionRef. or sourceE. coli strainsMRE600RNase-negative40Cammack K.A. Wade H.E. Biochem. J. 1965; 96: 671-680Crossref PubMed Scopus (111) Google ScholarMC4100ΔtigaraD139 Δ(argF-lac)U169 rpsL150 relA1 fIB5301 deoC1 ptsF25 rbsR, tig::kan41Deuerling E. Schulze-Specking A. Tomoyasu T. Mogk A. Bukau B. Nature. 1999; 400: 693-696Crossref PubMed Scopus (411) Google ScholarSL119BL 21 recD F- hsdS gal OmpT-42Lesley S.A. Brow M.A. Burgess R.R. J. Biol. Chem. 1991; 266: 2632-2638Abstract Full Text PDF PubMed Google ScholarTUNER(DE3) pLysSF- ompT hsdSB (rB- mB-) gal dcm lacY1, (DE3) pLysS (CmR)NovagenTY1ompT::kan, secY20528Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google ScholarCU164secY3943Baba T. Jacq A. Brickman E. Beckwith J. Taura T. Ueguchi C. Akiyama Y. Ito K. J. Bacteriol. 1990; 172: 7005-7010Crossref PubMed Google ScholarCM124secEΔ19-111, pCM2244Traxler B. Murphy C. J. Biol. Chem. 1996; 271: 12394-12400Abstract Full Text Full Text PDF PubMed Scopus (90) Google ScholarKN533ΔuncB-C::Tn10 ΔsecG::kan29Nishiyama K. Suzuki T. Tokuda H. Cell. 1996; 85: 71-81Abstract Full Text Full Text PDF PubMed Scopus (187) Google ScholarN4156 pAra14-FtsY′polA end thy gyrA pAra14-FtsY′12Luirink J. 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 (213) Google ScholarWAM113araB-ffh+11Phillips G.J. Silhavy T.J. Nature. 1992; 359: 744-746Crossref PubMed Scopus (224) Google ScholarGN42AD202, leu::Tn10, secA3628Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google ScholarPlasmidspDMBExpression of OmpA1Behrmann M. Koch H.G. Hengelage T. Wieseler B. Hoffschulte H.K. Müller M. J. Biol. Chem. 1998; 273: 13898-13904Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholarp717MtlA-BExpression of mannitol permease18Beck K. Wu L.F. Brunner J. Müller M. EMBO J. 2000; 19: 134-143Crossref PubMed Scopus (148) Google ScholarpMomp2Expression of Momp28Neumann-Haefelin C. Schafer U. Müller M. Koch H.G. EMBO J. 2000; 19: 6419-6426Crossref PubMed Scopus (111) Google ScholarpFDX2322Source of kanamycin-cassetteJ. MacFarlanepJMSKKpBluescript SK+-derivative, in which the ampicillin gene is replaced by kanamycin resistance gene from pFDX2322J. MacFarlanepJMSKK Bla7Expression of β-lactamaseThis studypET19b-SecA36Overexpression and purification of SecA36This study Open table in a new tab Reagents—Growth media components and chemicals were obtained from Roth (Karlsruhe, FR Germany), Sigma, and Pierce. Restriction enzymes and other reagents for cloning and DNA purification were purchased from New England Biolabs (Frankfurt, FR Germany), Qiagen (Hilden, FR Germany), and Novagen (Schwalbach, Germany). Cell Growth and General Techniques—E. coli strains for plasmid isolation were cultured in Luria Broth. For the preparation of inner membrane vesicles, cells were cultured in phosphate-buffered medium as described by Müller and Blobel (20Müller M. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7421-7425Crossref PubMed Scopus (107) Google Scholar). Cell extracts for in vitro transcription and translation were prepared from cells grown either on S30 medium (20Müller M. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7421-7425Crossref PubMed Scopus (107) Google Scholar) or on S150 medium (21Müller M. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7737-7741Crossref PubMed Scopus (49) Google Scholar, 22Hoffschulte H.K. Drees B. Müller M. J. Biol. Chem. 1994; 269: 12833-12839Abstract Full Text PDF PubMed Google Scholar). When required, antibiotics were added to the culture medium at the following concentrations: 50 μg of ampicillin/ml, 25 μg of kanamycin/ml, and 10 μg of tetracycline/ml. Plasmid Construction—pJMSKK, a kanamycin-resistant pBlue-script derivative, was constructed as follows. The ampicillin resistance gene (bla) of pBluescript SK+ was excised with BspHI and the vector blunt-ended with the Klenow fragment of DNA polymerase I. The bla gene was replaced with the kanamycin cassette from pFDX2322, cut with NaeI and SpeI, and also blunt-ended with the Klenow fragment of DNA polymerase I. pJMSKK was used to construct a vector carrying the bla gene under T7 promoter control. For this, the bla gene of pBluescript SK+ was excised with BspHI, blunt-ended with the Klenow fragment of DNA polymerase I, and ligated into the EcoRV site of pJMSKK. The constructed plasmid was termed pJMSKK Bla7. For the construction of the secA36 allele under T7-RNA polymerase control, genomic DNA of the SecA36 mutant GN42 was amplified using the primer SecA1 (5′-GAGATTTTCATATGCTAATCAAATTGTTAAC-3′) and SecA2 (5′-CGCAGAATCCTCGAGCTTTTACTTCAACAG-3′) introducing an NdeI and an XhoI cleavage site. After restriction digest the PCR product was cloned into the NdeI and XhoI sites of pET19b under the control of the T7 promoter. The resulting plasmid was termed pET19b-SecA36 and coded for a SecA36 derivative carrying an N-terminal His10 tag to expedite purification. Purification of SecA, SecB, F1-ATPase, Ffh, FtsY, and SecA36 —The purification of SecA (23Helde R. Wiesler B. Wachter E. Neubuser A. Hoffschulte H.K. Hengelage T. Schimz K.L. Stuart R.A. Müller M. J. Bacteriol. 1997; 179: 4003-4012Crossref PubMed Google Scholar), SecB (22Hoffschulte H.K. Drees B. Müller M. J. Biol. Chem. 1994; 269: 12833-12839Abstract Full Text PDF PubMed Google Scholar), F1-ATPase (24Müller M. Fisher R.P. Rienhofer-Schweer A. Hoffschulte H.K. EMBO J. 1987; 6: 3855-3861Crossref PubMed Scopus (18) Google Scholar), Ffh (6Koch H.G. Hengelage T. Neumann-Haefelin C. MacFarlane J. Hoffschulte H.K. Schimz K.L. Mechler B. Müller M. Mol. Biol. Cell. 1999; 10: 2163-2173Crossref PubMed Scopus (134) Google Scholar), and FtsY (6Koch H.G. Hengelage T. Neumann-Haefelin C. MacFarlane J. Hoffschulte H.K. Schimz K.L. Mechler B. Müller M. Mol. Biol. Cell. 1999; 10: 2163-2173Crossref PubMed Scopus (134) Google Scholar) followed protocols reported previously. For the purification of His10-SecA36, pET19b-SecA36 was transformed into the E. coli strain TUNER(DE3) pLysS. Cells were grown at 37 °C overnight on Luria Broth supplemented with d-glucose (1%), washed, and subcultured on the same medium lacking glucose. At an A600 of 0.5, cells were induced with 1 mm isopropyl-β-d-thiogalactoside, incubated further until at an A600 of 1.5–1.8, and harvested. After resuspension in 50 mm triethanolamine acetate (pH 7.5), 50 mm KCH3COO (KOAc), 5 mm Mg(CH3COO)2, cells were disrupted using a French press at 8000 pounds/square inch in the presence of Complete protease inhibitor mixture (Roche Applied Science). After centrifugation at 30,000 × g, the supernatant was subjected to high speed centrifugation (150,000 × g), and SecA36 overproduction was confirmed by SDS-PAGE of the supernatant and subsequent Coomassie staining. For further purification the supernatant was buffer-exchanged in 2.5-ml steps on PD-10 columns (Amersham Biosciences), to adapt the salt conditions to 50 mm potassium phosphate (pH 7.5), and 300 mm KOAc, suitable for further purification using the TALON® IMAC System (Clontech Laboratories, Inc., Palo Alto, CA). The buffer exchanged supernatant was supplemented with Complete/EDTA protease inhibitor mixture and phenylmethylsulfonyl fluoride (0.5 mm), and 3.5 ml of this material was incubated overnight at 4 °C with 2 ml of the Talon resin. Washing was performed according to the manufacturer's manual with 50 mm potassium phosphate (pH 7.5), and 300 mm KOAc. After washing, the resin with the bound protein was filled into 2-ml gravity-flow columns (Qiagen, Hilden, Germany), and proteins were eluted stepwise with increasing imidazole concentrations (0–150 mm). SecA36 fractions were analyzed on a Coomassie-stained SDS gel, and pure SecA36 containing fractions were pooled. Protein concentrations were determined using the Pierce BCA protein assay kit. In Vitro Reactions—The composition of the transcription/translation system of E. coli and the purification of its components, the preparation of INV, the flotation gradient analysis, and proteinase K protection assay employed in this study have been described previously (1Behrmann M. Koch H.G. Hengelage T. Wieseler B. Hoffschulte H.K. Müller M. J. Biol. Chem. 1998; 273: 13898-13904Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 6Koch H.G. Hengelage T. Neumann-Haefelin C. MacFarlane J. Hoffschulte H.K. Schimz K.L. Mechler B. Müller M. Mol. Biol. Cell. 1999; 10: 2163-2173Crossref PubMed Scopus (134) Google Scholar, 8Neumann-Haefelin C. Schafer U. Müller M. Koch H.G. EMBO J. 2000; 19: 6419-6426Crossref PubMed Scopus (111) Google Scholar). Synthesis of nascent chains was achieved as described in Beck et al. (18Beck K. Wu L.F. Brunner J. Müller M. EMBO J. 2000; 19: 134-143Crossref PubMed Scopus (148) Google Scholar), in the presence of RNase H (1 unit/25 μl), 10 μg/ml 10Sa RNA antisense oligonucleotide (25Hanes J. Plückthun A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4937-4942Crossref PubMed Scopus (919) Google Scholar), and 0.5 μg/μl oligonucleotide Bla-175 (5′-GCAGGCATCGTGGTGTCACG-3′). Chemical cross-linking using DSS (Pierce) was performed as described previously (18Beck K. Wu L.F. Brunner J. Müller M. EMBO J. 2000; 19: 134-143Crossref PubMed Scopus (148) Google Scholar). The in vitro reactions for subsequent cross-linking experiments were performed in the presence of HEPES-NaOH instead of triethanolamine acetate. Immunoprecipitation was performed in 4–6-fold scaled up reactions using polyclonal rabbit antibodies against trigger factor and Ffh, covalently linked to protein A-Sepharose matrix (26Alconada A. Gartner F. Honlinger A. Kubrich M. Pfanner N. Methods Enzymol. 1995; 260: 263-286Crossref PubMed Scopus (42) Google Scholar). Sample Analysis and Quantification—All samples were analyzed on SDS-polyacrylamide gels (13, 15, or 7–17%). Radiolabeled proteins were visualized by PhosphorImaging using a Amersham Biosciences PhosphorImager and quantified using ImageQuant software from Amersham Biosciences. In Vitro Synthesis and Translocation of β-Lactamase—For analyzing its targeting to and its translocation across the E. coli membrane, the β-lactamase gene of pBluescript II was cloned under a T7-dependent promoter in a kanamycin-resistant pBluescript derivative. In vitro synthesis was performed using a highly purified E. coli cell extract. Transport of β-lactamase was analyzed using inside-out inner membrane vesicles (INV) and was compared with the transport of the SRP-dependent inner membrane protein mannitol permease (MtlA) and the SRP-independent outer membrane protein OmpA. As shown in Fig. 1, when synthesized in the absence of INV all three substrates were sensitive toward proteinase K digestion, but became protease-resistant in the presence of INV. Protease protection of both OmpA and β-lactamase in the presence of INV was accompanied by signal sequence cleavage as indicated by the presence of a protease-resistant band of lower molecular mass. The lower molecular mass of the membrane-protected fragment of MtlA (MtlA-MPF) is due to the cleavage of the large C-terminally located cytoplasmic domain. The transport efficiency into INV of β-lactamase is lower than the transport efficiency of OmpA, a phenomenon which has also been observed in vivo. A SecY Mutation Interfering with Efficient SecA Binding to the Translocon Blocks β-Lactamase Transport—Although both the SRP-dependent and the SecA-dependent protein targeting pathways converge at the SecYEG complex in the inner bacterial membrane, we have shown recently (27Koch H.G. Müller M. J. Cell Biol. 2000; 150: 689-694Crossref PubMed Scopus (60) Google Scholar) by using translocon mutants that different domains of SecY and different components of the translocon are engaged by both processes. These translocon mutants were assayed for their ability to transport β-lactamase. As shown in Fig. 2 (top panel), the translocation of β-lactamase is completely blocked in INV prepared from the secY mutants secY39 and secY205. A severe translocation defect is furthermore observed in INV from a secG deletion strain and in SecE-depleted INV. In these translocation assays using mutant INV, the effects of transport on β-lactamase are indistinguishable from those of transport on the SRP-independent secretory protein OmpA (Fig. 2, middle panel) but clearly different from those on the integration of the SRP-dependent substrate MtlA (Fig. 2, bottom panel). The integration of MtlA is not significantly influenced by the secY205 mutation and the secG deletion. The biochemical characterization of the secY205 mutant had revealed that the translocation defect originates primarily from reduced SecA binding to the mutant SecY (28Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar), and consequently, the transport of SecA-independent proteins like MtlA is not impaired by this mutation. The function of SecG is also specifically associated with the SecA function (29Nishiyama K. Suzuki T. Tokuda H. Cell. 1996; 85: 71-81Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) and does not seem to be required for SecA-independent protein transport (27Koch H.G. Müller M. J. Cell Biol. 2000; 150: 689-694Crossref PubMed Scopus (60) Google Scholar). The secY39 mutation affects both the SecA-dependent as well as the SRP-dependent protein transport. However, the effects on β-lactamase and OmpA translocation are more severe than on MtlA integration. A complete block of MtlA integration can only be observed in SecE-depleted INV (Fig. 2, middle panel). SecE depletion concomitantly leads also to reduced SecY concentrations, because in the absence of SecE, SecY is rapidly degraded by the membrane-bound protease FtsH (30Akiyama Y. Kihara A. Tokuda H. Ito K. J. Biol. Chem. 1996; 271: 31196-31201Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), resulting in a transport defect for both SecA- and SRP-dependent proteins. In summary, these data indicate that β-lactamase translocation depends on the same domains of SecY and the same components of the translocon as the translocation of the SRP-independent substrate OmpA which argues for a SecA-dependent translocation of β-lactamase. The SecA dependence was directly tested by utilizing a new assay system for SecA-dependent proteins, based on the biochemical complementation of the secY205 mutant INV with a mutant SecA (28Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar). A systematic search for secA mutations suppressing the secY205 phenotype led to the isolation of allele-specific suppressors, one of which is the secA36 mutant, carrying a single amino acid substitution within the high affinity ATP-binding site of SecA (28Matsumoto G. Yoshihisa T. Ito K. EMBO J. 1997; 16: 6384-6393Crossref PubMed Scopus (86) Google Scholar). By using PCR we amplified the secA36 allele from the mutant strain GN42 and cloned it into a T7-dependent expression vector for overexpression and purification. The purified SecA36 was then tested for its ability to suppress in vitro the translocation defect of the secY205 INV. As shown in Fig. 3 (top panel), in the absence of SecA36, no β-lactamase translocation into secY205 INV was observed; the presence of SecA36, however, enhanced β-lactamase translocation significantly (Fig. 3, top panel, compare lanes 6 and 12). Purified wild type SecA, on the other hand, was unable to overcome the SecY205 defect (data not shown). Enhanced translocation of β-lactamase in the presence of SecA36 was not only observed for secY205 INV but also for wild type INV, which is in agreement with the enhanced ATPase activity of SecA36. As a control, we also tested the effect of SecA36 on the translocation of the SecA-dependent OmpA (Fig. 3, lower panel). Like for β-lactamase, the addition of SecA36 greatly enhanced the translocation of OmpA into secY205 INV and also into wild type INV. These data demonstrate that β-lactamase, despite being a SecB-independent protein, requires SecA for translocation in the same way as the SecB-dependent protein OmpA. Ribosome-associated Nascent Chains of β-Lactamase Are Not Cotranslationally Targeted to the SecY Translocon—In principle, the observation that export of β-lactamase is catalyzed by SecA does not exclude an involvement of SRP during the targeting of β-lactamase to the SecYEG complex. SRP-dependent targeting and SecA-dependent translocation as individual steps have been identified in the export of bacterial membrane proteins with large periplasmic domains (8Neumann-Haefelin C. Schafer U. Müller M. Koch H.G. EMBO J. 2000; 19: 6419-6426Crossref PubMed Scopus (111) Google Scholar, 10Scotti P.A. Valent Q.A. Manting E.H. Urbanus M.L. Driessen A.J. Oudega B. Luirink J. J. Biol. Chem. 1999; 274: 29883-29888Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). One of the salient features of both the eukaryoti