Biogenesis of MalF and the MalFGK2 Maltose Transport Complex in Escherichia coli Requires YidC
2008; Elsevier BV; Volume: 283; Issue: 26 Linguagem: Inglês
10.1074/jbc.m801481200
ISSN1083-351X
AutoresSamuel Wagner, Ovidio Pop, Gert-Jan Haan, Louise Baars, Gregory M. Koningstein, Mirjam Klepsch, Pierre Genevaux, Joen Luirink, Jan‐Willem De Gier,
Tópico(s)Antibiotic Resistance in Bacteria
ResumoThe polytopic inner membrane protein MalF is a constituent of the MalFGK2 maltose transport complex in Escherichia coli. We have studied the biogenesis of MalF using a combination of in vivo and in vitro approaches. MalF is targeted via the SRP pathway to the Sec/YidC insertion site. Despite close proximity of nascent MalF to YidC during insertion, YidC is not required for the insertion of MalF into the membrane. However, YidC is required for the stability of MalF and the formation of the MalFGK2 maltose transport complex. Our data indicate that YidC supports the folding of MalF into a stable conformation before it is incorporated into the maltose transport complex. The polytopic inner membrane protein MalF is a constituent of the MalFGK2 maltose transport complex in Escherichia coli. We have studied the biogenesis of MalF using a combination of in vivo and in vitro approaches. MalF is targeted via the SRP pathway to the Sec/YidC insertion site. Despite close proximity of nascent MalF to YidC during insertion, YidC is not required for the insertion of MalF into the membrane. However, YidC is required for the stability of MalF and the formation of the MalFGK2 maltose transport complex. Our data indicate that YidC supports the folding of MalF into a stable conformation before it is incorporated into the maltose transport complex. Biogenesis of MalF and the MalFGK2 maltose transport complex in Escherichia coli requires YidC.Journal of Biological ChemistryVol. 284Issue 52PreviewVOLUME 283 (2008) PAGES 17881–17890 Full-Text PDF Open Access In the Gram-negative bacterium Escherichia coli, different inner membrane protein targeting and insertion pathways are operational (1Luirink J. von Heijne G. Houben E. de Gier J.W. Annu. Rev. Microbiol. 2005; 59: 329-355Crossref PubMed Scopus (157) Google Scholar). Although the biogenesis requirements of only a few membrane proteins have been studied thoroughly, most of them are targeted via the signal recognition particle (SRP) 5The abbreviations used are: SRP, signal recognition particle; TM, transmembrane; IMV, inverted membrane vesicle; IPTG, isopropyl 1-thio-β-d-galactopyranoside; TF, trigger factor; PSBT, P. shermanii transcarboxylase; Lep, leader peptidase. pathway to the inner membrane where they are delivered at the Sec translocon (2Luirink J. Sinning I. Biochim. Biophys. Acta. 2004; 1694: 17-35PubMed Google Scholar, 3Facey S.J. Kuhn A. Biochim. Biophys. Acta. 2004; 1694: 55-66Crossref PubMed Scopus (28) Google Scholar). The core of the Sec translocon consists of the integral membrane proteins SecY and SecE and the peripheral ATPase SecA (4Van den Berg B. Clemons Jr., W.M. Collinson I. Modis Y. Hartmann E. Harrison S.C. Rapoport T.A. Nature. 2004; 427: 36-44Crossref PubMed Scopus (995) Google Scholar, 5Veenendaal A.K. van der Does C. Driessen A.J. Biochim. Biophys. Acta. 2004; 1694: 81-95Crossref PubMed Scopus (102) Google Scholar, 6Rapoport T.A. Nature. 2007; 450: 663-669Crossref PubMed Scopus (704) Google Scholar). SecY and -E form a protein-conducting channel that can mediate both translocation and membrane insertion (4Van den Berg B. Clemons Jr., W.M. Collinson I. Modis Y. Hartmann E. Harrison S.C. Rapoport T.A. Nature. 2004; 427: 36-44Crossref PubMed Scopus (995) Google Scholar, 7Osborne A.R. Rapoport T.A. van den Berg B. Annu. Rev. Cell Dev. Biol. 2005; 21: 529-550Crossref PubMed Scopus (300) Google Scholar). It has been proposed that the translocon channel can open laterally toward the lipid bilayer thereby allowing the release of transmembrane segments (TMs) into the membrane (6Rapoport T.A. Nature. 2007; 450: 663-669Crossref PubMed Scopus (704) Google Scholar, 8Clemons Jr., W.M. Menetret J.F. Akey C.W. Rapoport T.A. Curr. Opin. Struct. Biol. 2004; 14: 390-396Crossref PubMed Scopus (50) Google Scholar). The ATPase SecA is required for the translocation of large (≥60 amino acids) periplasmic domains of inner membrane proteins (1Luirink J. von Heijne G. Houben E. de Gier J.W. Annu. Rev. Microbiol. 2005; 59: 329-355Crossref PubMed Scopus (157) Google Scholar, 9Dalbey R.E. Chen M. Biochim. Biophys. Acta. 2004; 1694: 37-53Crossref PubMed Scopus (87) Google Scholar). The inner membrane protein YidC, which is essential for viability, has been identified as a factor that assists in the integration, folding, and assembly of inner membrane proteins both in association with the Sec translocon and separately (10Kiefer D. Kuhn A. Int. Rev. Cytol. 2007; 259: 113-138Crossref PubMed Scopus (45) Google Scholar, 11Xie K. Dalbey R.E. Nat. Rev. Microbiol. 2008; 6: 234-244Crossref PubMed Scopus (90) Google Scholar). Thus far, only a handful of inner membrane proteins that insert via the YidC-only pathway have been identified. All these proteins are small and do not contain a sizable periplasmic domains and more than two TMs. It is not clear how YidC assists the biogenesis of these proteins. During the biogenesis of SRP/Sec translocon-dependent inner membrane proteins, YidC specifically interacts with the TMs of these proteins (e.g. Refs. 12Houben E.N. Scotti P.A. Valent Q.A. Brunner J. de Gier J.L. Oudega B. Luirink J. FEBS Lett. 2000; 476: 229-233Crossref PubMed Scopus (73) Google Scholar, 13Urbanus M.L. Scotti P.A. Froderberg L. Saaf A. de Gier J.W. Brunner J. Samuelson J.C. Dalbey R.E. Oudega B. Luirink J. EMBO Rep. 2001; 2: 524-529Crossref PubMed Scopus (154) Google Scholar, 14Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar, 15Beck K. Eisner G. Trescher D. Dalbey R.E. Brunner J. Müller M. EMBO Rep. 2001; 2: 709-714Crossref PubMed Scopus (139) Google Scholar). It has been suggested that YidC mediates the transfer of TMs from the Sec translocon into the lipid bilayer (1Luirink J. von Heijne G. Houben E. de Gier J.W. Annu. Rev. Microbiol. 2005; 59: 329-355Crossref PubMed Scopus (157) Google Scholar). Indeed, YidC could be co-purified with the Sec translocon, suggesting a physical connection (14Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar). A recent in vitro study using lactose permease (LacY) reveals a novel function for YidC in the co-translational folding of this inner membrane protein rather than its insertion into the membrane (16Nagamori S. Smirnova I.N. Kaback H.R. J. Cell Biol. 2004; 165: 53-62Crossref PubMed Scopus (161) Google Scholar). The observation that YidC depletion leads to the induction of the Cpx and σE envelope stress responses, which both sense protein misfolding in the cell envelope, also points to a role of YidC in the folding of inner membrane proteins (17Shimohata N. Chiba S. Saikawa N. Ito K. Akiyama Y. Genes Cells. 2002; 7: 653-662Crossref PubMed Scopus (98) Google Scholar, 18Shimohata N. Nagamori S. Akiyama Y. Kaback H.R. Ito K. J. Cell Biol. 2007; 176: 307-317Crossref PubMed Scopus (44) Google Scholar). Here, we have used the inner membrane protein MalF as a model protein to study the role of YidC in the biogenesis of polytopic inner membrane proteins, which are part of a heterooligomeric complex. MalF functions in maltose transport as a 1:1:2 complex with the integral inner membrane protein MalG and the peripheral inner membrane protein MalK (19Ehrmann M. Ehrle R. Hofmann E. Boos W. Schlosser A. Mol. Microbiol. 1998; 29: 685-694Crossref PubMed Scopus (107) Google Scholar, 20Oldham M.L. Khare D. Quiocho F.A. Davidson A.L. Chen J. Nature. 2007; 450: 515-521Crossref PubMed Scopus (421) Google Scholar). The complex belongs to the ATP-binding cassette (ABC) transporter superfamily. MalF is a 514-amino acid-long inner membrane protein containing 8 TMs, with its N and C termini facing the cytosol (21Froshauer S. Green G.N. Boyd D. McGovern K. Beckwith J. J. Mol. Biol. 1988; 200: 501-511Crossref PubMed Scopus (124) Google Scholar) (see Fig. 1). A large periplasmic domain (∼180 amino acids) is present between the third and the fourth transmembrane segments. This domain folds into a trypsin-resistant conformation when MalF is incorporated into the MalFGK2 maltose transport complex (22Traxler B. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10852-10856Crossref PubMed Scopus (35) Google Scholar). The biogenesis of MalF was studied using a combination of in vivo and in vitro approaches. MalF was found to be targeted via the SRP pathway to the Sec/YidC insertion site. Despite close proximity of nascent MalF to YidC during insertion, YidC appeared dispensable for the insertion of MalF into the membrane. However, YidC is required for the stability of MalF and the formation of the MalFGK2 maltose transport complex suggesting an important role of YidC in the assembly of oligomeric complexes in the inner membrane. Materials—Restriction endonucleases and the Expand Long Template PCR system were from Roche Molecular Biochemicals. The Megashort Script T7 transcription kit was from Ambion Inc. [35S]Methionine and protein A-Sepharose were both from GE Healthcare. T4 ligase and T4 DNA polymerase were both from Epicenter Technologies. n-Dodecyl-β-d-maltopyranoside was from Anatrace. Serva Blue G was obtained from Serva. All other chemicals were obtained from Sigma. A polyclonal antibody against the c-Myc epitope tag was obtained from Abcam. Antisera against SecY, SecE, YidC, and Ffh were from our own collection. Antisera against MalF, -G, and -K, trigger factor (TF), and L23 were kind gifts from A. Davidson, W. Wickner, and R. Brimacombe, respectively. In Vitro Cross-linking—E. coli strain MRE600 was used to prepare a lysate for translation of in vitro synthesized mRNA and suppression of UAG stop codons in the presence of (Tmd)-Phe-tRNAsup. Strain MC4100 was used to isolate inverted membrane vesicles (IMVs). Strain JM110 was used to isolate pC4Meth-derived plasmids for in vitro transcription. pC4Meth-derived plasmids for in vitro expression of truncated forms of MalF were constructed by PCR using pTAZFQ as template (23Mourez M. Hofnung M. Dassa E. EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google Scholar). These plasmids encode truncated MalF fused to a 4× methionine tag to improve labeling efficiency of nascent chains with [35S]methionine. Immunoprecipitation of nascent chains was enabled by constructing a complementary set of constructs with a C-terminal c-Myc epitope tag (EQKLI-SEEDL) fused to the truncated MalF sequence. Furthermore, amber mutations (TAG) were introduced at the indicated positions to enable sup-tRNA photo-cross-linking, as described previously (14Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar). The nucleotide sequence of all constructs was confirmed by DNA sequencing. Truncated mRNAs were prepared from HindIII- or ClaI-linearized pC4Meth-derived plasmids. In vitro translation, targeting to IMVs, photo-cross-linking, carbonate extraction (to separate membrane-bound material from non-membrane-bound material), and sample processing were performed as described previously (14Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar). Strains, Plasmids, Growth Conditions, and Assay Used in MalF Membrane Targeting/Insertion Experiments in Vivo—The 4.5 S RNA conditional strain FF283 was cultured in M9 minimal medium (for composition, see Ref. 24Froderberg L. Houben E. Samuelson J.C. Chen M.Y. Park S.K. Phillips G.J. Dalbey R. Luirink J. de Gier J.W.L. Mol. Microbiol. 2003; 47: 1015-1027Crossref PubMed Scopus (70) Google Scholar) supplemented with 1 mm IPTG as described previously (24Froderberg L. Houben E. Samuelson J.C. Chen M.Y. Park S.K. Phillips G.J. Dalbey R. Luirink J. de Gier J.W.L. Mol. Microbiol. 2003; 47: 1015-1027Crossref PubMed Scopus (70) Google Scholar). To deplete cells of 4.5 S RNA, cells were grown to mid-log phase in the absence of IPTG. The temperature-sensitive amber suppressor SecA depletion strain BA13 and the control strain DO251 were cultured in M9 minimal medium at 30 °C, as described previously (24Froderberg L. Houben E. Samuelson J.C. Chen M.Y. Park S.K. Phillips G.J. Dalbey R. Luirink J. de Gier J.W.L. Mol. Microbiol. 2003; 47: 1015-1027Crossref PubMed Scopus (70) Google Scholar). To deplete cells of SecA, they were grown to mid-log phase at 41 °C. The SecE depletion strain CM124 was cultured in M9 minimal medium supplemented with 0.2% glucose and 0.2% l-arabinose as described previously (24Froderberg L. Houben E. Samuelson J.C. Chen M.Y. Park S.K. Phillips G.J. Dalbey R. Luirink J. de Gier J.W.L. Mol. Microbiol. 2003; 47: 1015-1027Crossref PubMed Scopus (70) Google Scholar). To deplete cells of SecE, cells were grown to mid-log phase in the absence of l-arabinose. Depletion of SecA and SecE was checked by monitoring the accumulation of pro-OmpA during a short pulse-labeling step with [35S]methionine. The SecG deletion strain KN370 was cultured in M9 minimal medium to mid-log phase, and plasmid pH+ was used to complement with SecG as described previously (24Froderberg L. Houben E. Samuelson J.C. Chen M.Y. Park S.K. Phillips G.J. Dalbey R. Luirink J. de Gier J.W.L. Mol. Microbiol. 2003; 47: 1015-1027Crossref PubMed Scopus (70) Google Scholar). The YidC depletion strain FTL10 was cultured in M9 minimal medium supplemented with 0.2% glucose and 0.2% l-arabinose, as described previously (25Hatzixanthis K. Palmer T. Sargent F. Mol. Microbiol. 2003; 49: 1377-1390Crossref PubMed Scopus (103) Google Scholar). To deplete cells of YidC, cells were grown to mid-log phase in the absence of l-arabinose. MalF was expressed by arabinose induction from the pBAD24 vector (26Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3978) Google Scholar) in strains FF283, BA13, DO251, and KN370 (±pH+), by IPTG induction from the pEH1 vector (27Hashemzadeh-Bonehi L. Mehraein-Ghomi F. Mitsopoulos C. Jacob J.P. Hennessey E.S. Broome-Smith J.K. Mol. Microbiol. 1998; 30: 676-678Crossref PubMed Scopus (39) Google Scholar) in strain CM124 and by IPTG induction from the pEH3 vector (27Hashemzadeh-Bonehi L. Mehraein-Ghomi F. Mitsopoulos C. Jacob J.P. Hennessey E.S. Broome-Smith J.K. Mol. Microbiol. 1998; 30: 676-678Crossref PubMed Scopus (39) Google Scholar) in strain FTL10. Where appropriate, ampicillin (final concentration, 100 μg/ml), chloramphenicol (final concentration, 30 μg/ml), kanamycin (final concentration, 50 μg/ml), streptomycin (final concentration, 25 μg/ml), and tetracycline (final concentration, 12.5 μg/ml) were added to the medium. For all experiments cells were grown to mid-log phase. Expression of the constructs was induced for 3 min with either IPTG (final concentration, 1 mm) or l-arabinose (final concentration, 0.2%). Cells were labeled with [35S]methionine (100 μCi/ml, 1 Ci = 25 GBq) for 30 s. After labeling, cells were converted to spheroplasts. For spheroplasting, cells were collected at 14,000 rpm for 30 s in a microfuge, resuspended in ice-cold buffer (40% w/v sucrose, 33 mm Tris-HCl, pH 8.0), and incubated with lysozyme (final concentration, 5 μg/ml) and 1 mm EDTA for 15 min on ice. Aliquots of the spheroplast suspension were incubated on ice for 1 h either in the presence or absence of proteinase K (final concentration, 0.3 mg/ml). Subsequently, phenylmethylsulfonyl fluoride was added to the spheroplast suspension (final concentration, 0.33 mg/ml) to inhibit the protease. After addition of phenylmethylsulfonyl fluoride, samples were precipitated with trichloroacetic acid (final concentration, 10%), washed with acetone, resuspended in 10 mm Tris-HCl, pH 8.0/2% SDS, immunoprecipitated with antisera to MalF, OmpA (a periplasmic control), and AraB/bandX (a cytoplasmic control), washed, and analyzed by standard SDS-PAGE (24Froderberg L. Houben E. Samuelson J.C. Chen M.Y. Park S.K. Phillips G.J. Dalbey R. Luirink J. de Gier J.W.L. Mol. Microbiol. 2003; 47: 1015-1027Crossref PubMed Scopus (70) Google Scholar). Gels were scanned in a Fuji FLA-3000 phosphorimaging device and quantitated using Image Gauge (version 3.4). All pulse experiments were repeated at least three times. For the MalF biotinylation-based membrane insertion assay, the genes encoding the MalF derivatives MalF I (MalF with a PBST biotinylation domain engineered in the periplasmic loop between TM3 and -4, Fig. 1) and MalF L (MalF with a PBST biotinylation domain engineered in the cytoplasmic loop between TM4 and -5, Fig. 1) were cloned into the expression vector pASK-IBA3 (IBA GmbH, Germany) (28Jander G. Cronan J.E. Beckwith J. J. Bacteriol. 1996; 178: 3049-3058Crossref PubMed Google Scholar). The expression vectors containing the two derivatives of MalF were transformed into the conditional YidC strain FTL10. Cells were grown overnight in LB medium containing arabinose. Cultures were then washed and transferred into fresh LB medium containing arabinose or glucose to deplete cells for YidC. Expression of MalF-I and -L was induced by addition of 0.2 μg/ml anhydrotetracycline. After 4 h samples were taken and processed for SDS-PAGE and immunoblotting. Biotinylated proteins were detected using streptavidin-horseradish peroxidase (GE Healthcare). Analysis of the Accumulation Levels of the MalFGK2 Maltose Transport Complex by Blue Native PAGE—Overnight cultures of MC4100ara+ and FTL10 cells were grown in LB medium supplemented with 0.2% glycerol. To induce the expression of YidC in FTL10 during growth overnight, 0.2% arabinose was added. Cells were harvested by centrifugation and resuspended to an A660 of 0.05 in fresh LB medium supplemented with 0.2% glycerol and 0.2% maltose. It should be noted that the maltose transport complex is also expressed in the absence of maltose (29Wagner S. Baars L. Ytterberg A.J. Klussmeier A. Wagner C.S. Nord O. Nygren P.A. van Wijk K.J. de Gier J.W. Mol. Cell Proteomics. 2007; 6: 1527-1550Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). After 3 h of growth, cells were collected by centrifugation and fractionated, and IMVs were isolated as described before (29Wagner S. Baars L. Ytterberg A.J. Klussmeier A. Wagner C.S. Nord O. Nygren P.A. van Wijk K.J. de Gier J.W. Mol. Cell Proteomics. 2007; 6: 1527-1550Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). The accumulation levels of the MalFGK2 maltose transport complex were analyzed by blue native-PAGE (30Schagger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1918) Google Scholar) followed by Western blot analysis. Sample preparation and running conditions were essentially as described previously (29Wagner S. Baars L. Ytterberg A.J. Klussmeier A. Wagner C.S. Nord O. Nygren P.A. van Wijk K.J. de Gier J.W. Mol. Cell Proteomics. 2007; 6: 1527-1550Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). For blotting of blue native-PAGE gels, the cathode buffer containing 0.02% Serva Blue G was exchanged after one-third of the run to cathode buffer containing 0.002% Serva Blue G. This was done to prevent excessive binding of the dye to the polyvinylidene difluoride membrane. Blotting and decoration of blots with antisera to MalF, -G, -K, YidC, and SecY were performed as described previously (29Wagner S. Baars L. Ytterberg A.J. Klussmeier A. Wagner C.S. Nord O. Nygren P.A. van Wijk K.J. de Gier J.W. Mol. Cell Proteomics. 2007; 6: 1527-1550Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). Western Blotting Analysis—The accumulation levels of MalF, MalK, MalG, and YidC in inner membranes were monitored by immunoblot analysis. IMVs (3–5 μg of protein) were separated by standard SDS-PAGE, and immunoblotting was performed as described previously (29Wagner S. Baars L. Ytterberg A.J. Klussmeier A. Wagner C.S. Nord O. Nygren P.A. van Wijk K.J. de Gier J.W. Mol. Cell Proteomics. 2007; 6: 1527-1550Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). To verify the specificity of the MalG signal, a malG deletion mutant (SW1242) was used (see below). Construction of malG and malK Deletion Strains and FTL10 malF::Tn10—malG and malK deletion mutants were made using the Red Swap method (31Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar). In brief, primers "malG_ ECOLI_swapc_HP1" (5′-AACCTGAAAGCCACGCGAATGAAGTTTGATTAAGGGAGATAACAAAAATGTGTAGGCTGGAGCTGCTTCG-3′) and "malG_ECOLI_swapc_HP2" (5′-AGCGGCATAACATTGGCAGAACAACATCTTTAACCTTTCACACCACCTGCCATATGAATATCCTCCTTAG-3′) or "malK_ECOLI_swapc_HP1" (5′-TCATGAATGTTGCTGTCGATGACAGGTTGTTACAAAGGGAGAAGGGCATGTGTAGGCTGGAGCTGCTTCG-3′) and "malK_ECOLI_swapc_ HP2" (5′-TGACAGGCTTTGTGTGTTTTGTGGGGTGCTTAAACGCCCGGCTCCTTATGCATATGAATATCCTCCTTAG-3′) were used to amplify the chloramphenicol resistance cassette from pKD3 (31Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar). MC4100ara+ harboring plasmid pKD46 (31Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar) was grown in SOB medium (for composition, see Ref. 31Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar) supplemented with 100 μg/ml ampicillin and 0.2% l-arabinose and used to make competent cells. After electroporation with the PCR product, chloramphenicol-resistant colonies were isolated on agar plates containing 30 μg/ml chloramphenicol. Deletion of malG and malK was verified by PCR with the following primers: k1, k2 (31Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar), "malG_ECOLI_swap_U" (5′-CAATTGCCACGCTGATCTTC-3′), "malG_ECOLI_swap_D" (5′-CGTGACTCAGAGCACGAAAG-3′), "malK_ECOLI_swap_U" (5′-GGTGGAGGATTTAAGCCATC-3′), and "malK_ECOLI_swap_D" (5′-GCTACCTGTCCAACCAATAC-3′). Strains SW1242 (ΔmalG) and SW1282 (ΔmalK) were tested for defective maltose fermentation on McConkey agar plates containing 1% maltose. The malF::Tn10 (TetR) mutant allele (a kind gift of Michael Ehrmann, University of Duisburg-Essen, Germany) was moved into strain FTL10 by bacteriophage P1-mediated generalized transduction (32Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992Google Scholar). FTL10 malF::Tn10 (TetR) transductants were selected at 37 °C on LB agar plates supplemented with sodium citrate (10 mm), l-arabinose (0.1%), and tetracycline (15 μg/ml). Transductants were further tested for defective maltose fermentation on McConkey agar plates containing maltose. Monitoring the Stability of MalF upon Depletion of YidC—Overnight cultures of MC4100ara+, FTL10, SW1242, and SW1282 cells were grown in LB medium supplemented with 0.2% glycerol. To induce the expression of YidC in FTL10 during growth overnight, 0.2% arabinose was added. Cells were harvested by centrifugation and resuspended to an A660 of 0.05 in fresh LB medium supplemented with 0.2% glycerol. To induce the expression of YidC in FTL10, 0.2% arabinose was added. To induce the expression of the mal operon, 0.2% maltose was added. After 3 h of growth, cells were collected by centrifugation and resuspended to an A660 of 2.0 in 1 ml of minimal medium supplemented with all amino acids, except methionine and cysteine, and containing 0.2% glycerol and 0.2% maltose. Cells were labeled with [35S]methionine for 5 min, followed by chases of 3, 30, and 60 min, and subsequently precipitated with trichloroacetic acid. Precipitated material was processed as described above under "Strains, Plasmids, Growth Conditions, and Assay Used in MalF Membrane Targeting/Insertion Experiments in Vivo." Nascent MalF Interacts with TF, Ffh, and the Ribosomal Component L23 and Inserts into the Inner Membrane in the Vicinity of YidC and the Sec Translocon—We initially analyzed the pathway of targeting and membrane insertion of MalF using an in vitro translation/targeting/photo-cross-linking approach. Radiolabeled nascent MalF of 88 amino acids was synthesized in a wild-type cell-free E. coli extract in the presence of inverted inner membrane vesicles to allow membrane targeting and insertion on the ribosome nascent chain complex (14Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar). At this length, the ribosome nascent chain complex is expected to fully expose the first and second TMs of MalF, assuming that the ribosome covers ∼30 amino acids (33Houben E.N. Zarivach R. Oudega B. Luirink J. J. Cell Biol. 2005; 170: 27-35Crossref PubMed Scopus (47) Google Scholar). To enable the incorporation of a photoreactive probe in the middle of the first TM of MalF, a single TAG amber codon was introduced at position 26. Following translation in the presence of (Tmd)Phe-tRNAsup and UV-induced cross-linking, samples were extracted with sodium carbonate at pH 11.5 to separate membrane-integrated from untargeted material. Cross-linked partners were identified by immunoprecipitation with specific antibodies. The TAG26 mutation in 88MalFTAG26 was efficiently suppressed by (Tmd)Phe-tRNAsup (data not shown), resulting in nascent MalF of the expected molecular weight. 88MalFTAG26 was efficiently targeted to IMVs, as judged by the relative amount of carbonate-resistant material (Fig. 2, compare lanes 1 and 2). Upon UV irradiation, several specific cross-linking products could be observed in the carbonate supernatant and pellet fractions (Fig. 2, lanes 3 and 7). An adduct of ∼55 kDa could be immunoprecipitated with antiserum to Ffh, the protein component of E. coli SRP (Fig. 2, lane 5). Substantial amounts of smaller adducts were also precipitated with antiserum to Ffh, which most probably represent cross-linked degradation products of Ffh (34Ullers R.S. Houben E.N. Raine A. ten Hagen-Jongman C.M. Ehrenberg M. Brunner J. Oudega B. Harms N. Luirink J. J. Cell Biol. 2003; 161: 679-684Crossref PubMed Scopus (116) Google Scholar). A cross-linking product of ∼65 kDa could be immunoprecipitated with antiserum to trigger factor (TF), which is a cytosolic chaperone with general affinity for nascent polypeptides that resides on the ribosome close to the nascent chain exit site (35de Gier J.W. Luirink J. EMBO Rep. 2003; 4: 939-943Crossref PubMed Scopus (34) Google Scholar, 36Ferbitz L. Maier T. Patzelt H. Bukau B. Deuerling E. Ban N. Nature. 2004; 431: 590-596Crossref PubMed Scopus (305) Google Scholar) (Fig. 2, lane 4). A prominent ∼20-kDa cross-link product was precipitated with antibodies directed toward the ribosomal protein L23 (Fig. 2, lane 6), which is located near the nascent chain exit site (37Ban N. Nissen P. Hansen J. Moore P.B. Steitz T.A. Science. 2000; 289: 905-920Crossref PubMed Scopus (2813) Google Scholar). Ffh and TF have both been shown to associate with L23 (34Ullers R.S. Houben E.N. Raine A. ten Hagen-Jongman C.M. Ehrenberg M. Brunner J. Oudega B. Harms N. Luirink J. J. Cell Biol. 2003; 161: 679-684Crossref PubMed Scopus (116) Google Scholar, 36Ferbitz L. Maier T. Patzelt H. Bukau B. Deuerling E. Ban N. Nature. 2004; 431: 590-596Crossref PubMed Scopus (305) Google Scholar, 38Gu S.Q. Peske F. Wieden H.J. Rodnina M.V. Wintermeyer W. RNA (N. Y.). 2003; 9: 566-573Crossref PubMed Scopus (127) Google Scholar, 39Kramer G. Rauch T. Rist W. Vorderwulbecke S. Patzelt H. Schulze-Specking A. Ban N. Deuerling E. Bukau B. Nature. 2002; 419: 171-174Crossref PubMed Scopus (274) Google Scholar, 40Halic M. Gartmann M. Schlenker O. Mielke T. Pool M.R. Sinning I. Beckmann R. Science. 2006; 312: 745-747Crossref PubMed Scopus (115) Google Scholar). In the carbonate pellet fraction a very strong cross-linking product of ∼60 kDa becomes apparent after UV irradiation (Fig. 2, lane 7). We could immunoprecipitate this adduct with antiserum to YidC (Fig. 2, lane 8) (35de Gier J.W. Luirink J. EMBO Rep. 2003; 4: 939-943Crossref PubMed Scopus (34) Google Scholar). A less prominent band of ∼40 kDa could be immunoprecipitated with antiserum to SecY (Fig. 2, lane 9) (35de Gier J.W. Luirink J. EMBO Rep. 2003; 4: 939-943Crossref PubMed Scopus (34) Google Scholar). In summary, using this unbiased approach it was shown that, at a nascent chain length of 88 amino acids, TM1 of MalF interacts with TF, SRP, and L23 and inserts into the inner membrane in a carbonate-resistant state close to YidC and SecY. This strongly suggests that MalF is targeted via the SRP pathway to the SecY/YidC insertion site in the inner membrane. TM1 of MalF Remains Close to YidC and the Sec Translocon during Insertion of TM2 and TM3 of MalF into the Membrane, and TM2 and TM3 Insert into the Membrane in a Molecular Environment Similar to That of TM1—To study the sequence of events taking place during the biogenesis of the three N-terminal TMs of MalF, nascent chains of increasing lengths were synthesized in vitro. The shortest nascent chain, 68MalF, is expected to fully expose the first TM segment from the ribosome nascent chain complex (see Fig. 3A). At a length of 100 amino acids, 100MalF exposes TM1 and TM2. 131MalF, the longest nascent chain, exposes TM1, TM2, and TM3. By placing a TAG codon at position 26 and by exploiting the photo-cross-linking procedure described above, the molecular environment of TM1 in each of these translation intermediates was studied. C-terminal c-Myc epitope tags were introduced to enable purification of nascent chains by immunoprecipitation. Previous work has shown th
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