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• W2139997535 abstract "Previous studies have demonstrated that signal peptides bind to the signal recognition particle (SRP) primarily via hydrophobic interactions with the 54-kDa protein subunit. The crystal structure of the conserved SRP ribonucleoprotein core, however, raised the surprising possibility that electrostatic interactions between basic amino acids in signal peptides and the phosphate backbone of SRP RNA may also play a role in signal sequence recognition. To test this possibility we examined the degree to which basic amino acids in a signal peptide influence the targeting of two Escherichia coli proteins, maltose binding protein and OmpA. Whereas both proteins are normally targeted to the inner membrane by SecB, we found that replacement of their native signal peptides with another moderately hydrophobic but unusually basic signal peptide (ΔEspP) rerouted them into the SRP pathway. Reduction in either the net positive charge or the hydrophobicity of the ΔEspP signal peptide decreased the effectiveness of SRP recognition. A high degree of hydrophobicity, however, compensated for the loss of basic residues and restored SRP binding. Taken together, the data suggest that the formation of salt bridges between SRP RNA and basic amino acids facilitates the binding of a distinct subset of signal peptides whose hydrophobicity falls slightly below a threshold level. Previous studies have demonstrated that signal peptides bind to the signal recognition particle (SRP) primarily via hydrophobic interactions with the 54-kDa protein subunit. The crystal structure of the conserved SRP ribonucleoprotein core, however, raised the surprising possibility that electrostatic interactions between basic amino acids in signal peptides and the phosphate backbone of SRP RNA may also play a role in signal sequence recognition. To test this possibility we examined the degree to which basic amino acids in a signal peptide influence the targeting of two Escherichia coli proteins, maltose binding protein and OmpA. Whereas both proteins are normally targeted to the inner membrane by SecB, we found that replacement of their native signal peptides with another moderately hydrophobic but unusually basic signal peptide (ΔEspP) rerouted them into the SRP pathway. Reduction in either the net positive charge or the hydrophobicity of the ΔEspP signal peptide decreased the effectiveness of SRP recognition. A high degree of hydrophobicity, however, compensated for the loss of basic residues and restored SRP binding. Taken together, the data suggest that the formation of salt bridges between SRP RNA and basic amino acids facilitates the binding of a distinct subset of signal peptides whose hydrophobicity falls slightly below a threshold level. The signal recognition particle (SRP) 1The abbreviations used are: SRP, signal recognition particle; ER, endoplasmic reticulum; HA, influenza hemagglutinin epitope HA.11; IM, inner membrane; IMP, IM protein; IPTG, isopropyl-β-d-thiogalactopyranoside; MBP, maltose-binding protein; TF, trigger factor; TMS, transmembrane segment. is a ribonucleoprotein complex that targets proteins to the eukaryotic endoplasmic reticulum (ER) as well as the bacterial inner membrane (IM). Although a core domain of SRP is highly conserved throughout evolution, both the size of the particle and its substrate specificity vary considerably (for review, see Ref. 1Keenan R.J. Freymann D.M. Stroud R.M. Walter P. Annu. Rev. Biochem. 2001; 70: 755-775Crossref PubMed Scopus (480) Google Scholar). Mammalian SRP is a relatively large particle comprised of six polypeptides and a 300-nucleotide RNA. In the first phase of the targeting reaction, the SRP 54-kDa subunit (SRP54) binds to both N-terminal signal sequences and transmembrane segments (TMSs) of integral membrane proteins (which often lack cleaved signal peptides) as they emerge during translation (2Krieg U.C. Walter P. Johnson A.E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8604-8608Crossref PubMed Scopus (238) Google Scholar, 3Kurzchalia T.V. Wiedmann M. Girshovich A.S. Bochkareva E.S. Bielka H. Rapoport T.A. Nature. 1986; 320: 634-636Crossref PubMed Scopus (222) Google Scholar, 4High S. Görlich D. Wiedmann M. Rapoport T.A. Dobberstein B. J. Cell Biol. 1991; 113: 35-44Crossref PubMed Scopus (72) Google Scholar). Subsequently the ribosome-nascent chain complex migrates to the ER, where an interaction between SRP54 and a membrane-bound receptor catalyzes release of the nascent chain and its insertion into a protein translocation channel (5Gilmore R. Walter P. Blobel G. J. Cell Biol. 1982; 95: 470-477Crossref PubMed Scopus (298) Google Scholar, 6Meyer D.I. Krause E. Dobberstein B. Nature. 1982; 297: 647-650Crossref PubMed Scopus (323) Google Scholar, 7Connolly T. Rapiejko P.J. Gilmore R. Science. 1991; 252: 1171-1173Crossref PubMed Scopus (160) Google Scholar). At the other extreme, Escherichia coli SRP consists of only a homolog of SRP54 (Ffh) and an ∼100 nucleotide RNA (4.5 S RNA) that is closely related to helix VIII of mammalian SRP RNA. Despite the difference in size, bacterial and mammalian SRPs share many biochemical properties (8Poritz M.A. Bernstein H.D. Strub K. Zopf D. Wilhelm H. Walter P. Science. 1990; 250: 1111-1117Crossref PubMed Scopus (210) Google Scholar, 9Miller J.D. Wilhelm H. Gierasch L. Gilmore R. Walter P. Nature. 1993; 366: 351-354Crossref PubMed Scopus (144) Google Scholar). The substrate specificity of E. coli SRP, however, is more restricted in that it targets primarily inner membrane proteins (IMPs) to the IM (10deGier, J.-W., Mansournia, P., Valent, Q. A., Phillips, G. J., Luirink, J., and von Heijne, G. (1996) FEBS Lett., 99, 307-309Google Scholar, 11Ulbrandt N.D. Newitt J.A. Bernstein H.D. Cell. 1997; 88: 187-196Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 12Koch 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 (131) Google Scholar). Most periplasmic and outer membrane proteins, which contain cleaved signal peptides, are targeted to the membrane by molecular chaperones such as SecB (13Kumamoto C.A. Beckwith J. J. Bacteriol. 1985; 163: 267-274Crossref PubMed Google Scholar). Unlike SRP, chaperones do not recognize signal sequences. Instead, they bind to the mature region of presecretory proteins late during translation or post-translationally to maintain translocation competence and to ensure that signal peptides are accessible to gate open translocation channels (14Randall L.L. Hardy S.J.S. Cell. 1986; 46: 921-928Abstract Full Text PDF PubMed Scopus (306) Google Scholar). Biochemical studies showed 20 years ago that SRP recognizes the 7-13-amino acid hydrophobic core (“H region”) that is a universal feature of signal peptides (15Walter P. Ibrahimi I. Blobel G. J. Cell Biol. 1981; 91: 545-550Crossref PubMed Scopus (393) Google Scholar). More recently, crystallographic analysis of mammalian SRP54 and its bacterial homologs revealed the presence of a large hydrophobic groove in the “M domain” that likely represents the signal peptide binding pocket (16Keenan R.J. Freymann D.M. Walter P. Stroud R.M. Cell. 1998; 94: 181-191Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 17Clemons Jr., W.M. Gowda K. Black S.D. Zwieb C. Ramakrishnan V. J. Mol. Biol. 1999; 292: 697-705Crossref PubMed Scopus (56) Google Scholar). Mammalian SRP appears to interact with signal peptides that vary widely in hydrophobicity. In bacteria and the yeast Saccharomyces cerevisiae, which also has multiple targeting pathways; however, SRP discriminates between different targeting signals that vary only slightly in hydrophobicity. In those organisms presecretory proteins that contain moderately hydrophobic signal peptides are bypassed by SRP and targeted by molecular chaperones by default. In E. coli, maltose binding protein (MBP) and OmpA are normally targeted to the IM by SecB, but increasing the net hydrophobicity of their signal peptides reroutes both proteins into the SRP pathway (18Lee H.C. Bernstein H.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3471-3476Crossref PubMed Scopus (164) Google Scholar). Furthermore, the biogenesis of M13 procoat protein, a small IMP whose insertion normally does not require any targeting factor, becomes SRP-dependent when it contains an unusually hydrophobic signal peptide (19deGier J.-W. Scotti P.A. Sääf 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). Likewise, yeast SRP binds preferentially to signal peptides that have a high hydrophobicity index (20Ng D.T. Brown J.D. Walter P. J. Cell Biol. 1996; 134: 269-278Crossref PubMed Scopus (375) Google Scholar). The data suggest that different SRP54 homologs are calibrated to bind to a different range of targeting signals. Indeed the observation that the putative binding pockets of evolutionarily distant M domains differ considerably in size and shape (16Keenan R.J. Freymann D.M. Walter P. Stroud R.M. Cell. 1998; 94: 181-191Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 17Clemons Jr., W.M. Gowda K. Black S.D. Zwieb C. Ramakrishnan V. J. Mol. Biol. 1999; 292: 697-705Crossref PubMed Scopus (56) Google Scholar) might account at least in part for the variation in substrate specificity. The recent solution of the crystal structure of the E. coli Ffh M domain bound to a fragment of 4.5 S RNA raised the unexpected possibility that SRP RNA may also play a role in substrate recognition (21Batey R.T. Rambo R.P. Lucast L. Rha B. Doudna J.A. Science. 2000; 287: 1232-1239Crossref PubMed Scopus (317) Google Scholar). The x-ray data show that a portion of the phosphate backbone of 4.5 S RNA lies adjacent to the hydrophobic groove in the Ffh M domain and appears to create an extended signal peptide binding pocket. The structure suggests that electrostatic interactions between the phosphates and basic amino acids that often reside at the N terminus (“N region”) of signal peptides and that flank TMSs might contribute to substrate recognition. Curiously, biochemical studies have not provided any evidence that SRP interacts with basic amino acids. Mutation of basic amino acids in model signal peptides does not significantly affect recognition by mammalian SRP in cell-free assays (22Szczesna-Skorupa E. Mead D.A. Kemper B. J. Biol. Chem. 1987; 262: 8896-8900Abstract Full Text PDF PubMed Google Scholar, 23Andrews D.W. Young J.C. Mirels L.F. Czarnota G.J. J. Biol. Chem. 1992; 267: 7761-7769Abstract Full Text PDF PubMed Google Scholar). By contrast, alteration of either the charge of the N region or the distance between basic amino acids and the H region can profoundly affect signal peptide cleavage, interaction with components of the translocation machinery, and translocation into ER vesicles (22Szczesna-Skorupa E. Mead D.A. Kemper B. J. Biol. Chem. 1987; 262: 8896-8900Abstract Full Text PDF PubMed Google Scholar, 23Andrews D.W. Young J.C. Mirels L.F. Czarnota G.J. J. Biol. Chem. 1992; 267: 7761-7769Abstract Full Text PDF PubMed Google Scholar, 24Nothwehr S.F. Gordon J.I. J. Biol. Chem. 1990; 265: 17202-17208Abstract Full Text PDF PubMed Google Scholar, 25Voigt S. Jungnickel B. Hartmann E. Rapoport T.A. J. Cell Biol. 1996; 134: 25-35Crossref PubMed Scopus (154) Google Scholar, 26Sakaguchi M. Tomiyoshi R. Kuroiwa T. Mihara K. Omura T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 16-19Crossref PubMed Scopus (163) Google Scholar). In E. coli, basic amino acids that flank TMSs influence IMP topology but are not required for membrane integration (27von Heijne G. Nature. 1989; 341: 456-458Crossref PubMed Scopus (431) Google Scholar). A screen for mutations in the MBP signal sequence that improve export in a secB- strain (and that probably reroute MBP into the SRP pathway) yielded changes that increase its hydrophobicity but not its net positive charge (28Collier D.N. Bassford Jr., P.J. J. Bacteriol. 1989; 171: 4640-4647Crossref PubMed Google Scholar). None of these results, however, rules out the possibility that interactions between SRP RNA and basic amino acids play a minor role in substrate recognition or that SRP RNA interacts with only a subset of signal peptides. Indeed electrostatic interactions might be expected to make a relatively small contribution to SRP recognition since signal peptides are predominantly hydrophobic and because only about a third of the putative extended binding surface is contributed by the RNA. In this study we reexamined the role of basic amino acids in the N region of signal peptides in targeting pathway selection. Our experimental strategy was based on the observation that E. coli presecretory proteins can be rerouted into the SRP pathway by increasing the hydrophobicity of their signal sequences (18Lee H.C. Bernstein H.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3471-3476Crossref PubMed Scopus (164) Google Scholar). We reasoned that if electrostatic interactions between SRP RNA and basic amino acids in signal peptides promote substrate recognition, then the presence of a highly charged signal peptide might likewise alter the targeting of presecretory proteins. Consistent with our hypothesis, we found that replacing the native signal peptides of MBP and OmpA with a moderately hydrophobic, but atypically basic signal peptide derived from the signal peptide of the E. coli autotransporter EspP directed both proteins into the SRP pathway. As expected, SRP recognition required the presence of multiple basic amino acids. Several lines of evidence indicated, however, that basic residues only promote the binding of SRP to a distinct subset of signal peptides that barely escape detection on the basis of hydrophobicity alone. Reagents, Media, and Bacterial Strains—Polyclonal rabbit antisera against MBP and influenza hemagglutinin epitope HA.11 (HA) were obtained from New England Biolabs and Covance, respectively, and a polyclonal antiserum against Ffh has been described (8Poritz M.A. Bernstein H.D. Strub K. Zopf D. Wilhelm H. Walter P. Science. 1990; 250: 1111-1117Crossref PubMed Scopus (210) Google Scholar). Selective media contained 100 μg/ml ampicillin and 30 μg/ml chloramphenicol as required. All bacterial cultures were grown at 37 °C except where indicated. The bacterial strains used in this study were MC4100 (F-araD139 Δ(argF-lac)U169 rpsL150 relA1 thi fib5301 deoC1 ptsF25 rbsR), HDB37 (MC4100 araΔ714 (29Lee H.C. Bernstein H.D. J. Biol. Chem. 2002; 277: 43527-43535Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), HDB51 (MC4100 ara+ ffh::kan λ(Para-ffh Apr) zic-4901::Tn10 (18Lee H.C. Bernstein H.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3471-3476Crossref PubMed Scopus (164) Google Scholar)), HDB52 (HDB51 secB::Tn5 (18Lee H.C. Bernstein H.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3471-3476Crossref PubMed Scopus (164) Google Scholar), HDB55 (MC4100 secB::Tn5 (29Lee H.C. Bernstein H.D. J. Biol. Chem. 2002; 277: 43527-43535Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), SKP1101 (MC4100 ara+ ffh::kan-1 pLCC29-ffh10(Ts) (30Park S.-K. Jiang F. Dalbey R.E. Phillips G.J. J. Bacteriol. 2002; 184: 2642-2653Crossref PubMed Scopus (25) Google Scholar)), and SKP1102 (SKP1101 pLCC29-ffh+ (30Park S.-K. Jiang F. Dalbey R.E. Phillips G.J. J. Bacteriol. 2002; 184: 2642-2653Crossref PubMed Scopus (25) Google Scholar)). Plasmid Construction—Plasmids pHL36, which contains an HA-tagged version of ompA under the control of a trc promoter, and pJH28 and pJH29, which contain malE under the control of a tac promoter, have been described (18Lee H.C. Bernstein H.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3471-3476Crossref PubMed Scopus (164) Google Scholar, 29Lee H.C. Bernstein H.D. J. Biol. Chem. 2002; 277: 43527-43535Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). To construct plasmid pJH46, the signal peptide of EspP was first amplified by PCR using the oligonucleotides 5′-GTTTCCCTTAAAAATGGAGCTCATATGAA-3′ (EspP1) and 5′-GATGTAGAAATTTGAAATATCCATATGTGACGC and E. coli strain EDL933 genomic DNA (ATCC) as a template. The amplified DNA was then cloned into the NdeI site of pJH29. To make plasmid pJH47, the downstream NdeI site was abolished by site-directed mutagenesis using the oligonucleotide 5′-CTTTTGCGTCACAGATGAAAATCGAAGAAGG-3′ and its complement, and a new NdeI site was introduced in the middle of the EspP signal peptide using the oligonucleotide 5′-CATCAAGAGCAACTCATATGAAAAAACACAAACGCATACTTGC-3′ and its complement. A plasmid that lacks the N terminus of the EspP signal peptide (pJH48) was then generated by resealing NdeI-digested pJH47. To construct plasmid pJH50, the EspP signal peptide was amplified with the oligonucleotides EspP1 and 5′-TTGAAATATCCATCTCGGCCGCAAAAGAATATGAGG-3′, and the amplified DNA was cloned into the NdeI and EagI sites of pHL36. Subsequently a new NdeI site was introduced into the middle of the signal peptide, and the plasmid was resealed after NdeI digestion as described above to create pJH51. All of the mutant versions of the MBP and ΔEspP signal peptides were constructed by introducing point mutations into pJH28 and pJH48. Site-directed mutagenesis was performed using the QuikChange mutagenesis kit (Stratagene). DNA encoding the first 94 amino acids of MBP and ΔEspP-MBP was amplified using the oligonucleotides 5′-GTCCGTTTAGGTGTTTTCACGAGGAATTCACCA-3′ and either 5′-TTGAGCGGATCCACCCATGCGGTCGTGTGCCCAGAA-3′ or 5′-TTGAGCGGATCCACCCATGCGGTCGTGTGCCCAGAACATAATG-3′ and either pJH28 or pJH48 as templates. The amplified DNA was then cloned into the EcoRI and BamHI sites of pGEM-4Z (Promega) to generate pJH56 and pJH57. To equalize the signal in in vitro translations, two amino acids near the C terminus of the ΔEspP-MBP 94-mer were changed to methionine during the PCR amplification. Plasmid pJH58 was constructed by transferring an NheI-Hind III fragment of pJH42 (29Lee H.C. Bernstein H.D. J. Biol. Chem. 2002; 277: 43527-43535Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) containing the tig gene into pBAD33. Protein Export Assays—For most experiments, cells were grown in M9 containing 0.2% glucose. Overnight cultures were washed and diluted into fresh medium at an optical density (OD550) of 0.025. For analysis of OmpA export in SKP1101/SKP1102 and in cells that overproduced TF, M9 supplemented with 0.2% glycerol and all the l-amino acids except methionine and cysteine was used. For Ffh depletion studies, cells were grown overnight in M9 containing 0.2% fructose and 0.2% arabinose, washed in medium lacking arabinose, and then added at OD550 = 0.005 to medium containing fructose and either arabinose or glucose (0.2%). In general, synthesis of plasmid-borne presecretory proteins was induced by the addition of 50 μm isopropyl-β-d-thiogalactopyranoside (IPTG) at OD550 = 0.2. For trigger factor (TF) overproduction studies, cultures were divided in half at OD550 = 0.2, arabinose (0.2%) was added to one portion, and incubation was continued for 30 min before IPTG addition. To analyze protein export at low temperature, cultures were shifted to 22 °C at OD550 = 0.2 and incubated for 40 min before IPTG addition. In experiments involving SKP1101/SKP1102, cultures were grown at 30 °C to OD550 = 0.1 and then incubated at 42 °C for 1.5 h before IPTG was added. In all experiments aliquots were removed from each culture 20-30 min after IPTG addition. Cells were then pulse-labeled with 30 μCi/ml Tran35S-label (Amersham Biosciences) for 30 s and incubated for various chase times. After the chase period proteins were precipitated immediately by the addition of cold 10% trichloroacetic acid. Immunoprecipitations were performed essentially as described (29Lee H.C. Bernstein H.D. J. Biol. Chem. 2002; 277: 43527-43535Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), and proteins were resolved by SDS-PAGE on 8-16% minigels (Novex). In Vitro Translation and Cross-linking—An E. coli translation extract was prepared by first rapidly chilling exponentially growing MRE600. Cells were washed and resuspended in 50 mm triethanolamine-acetic acid (pH 8.0), 50 mm KCl, 15 mm magnesium acetate, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride and passed twice through a French pressure cell at 8000 p.s.i. in a 1:1 (w/v) suspension. The cell lysate was centrifuged at 30,000 × g for 30 min, and the resulting supernatant was then incubated at 37 °C for 1 h before freezing. Truncated mRNAs were synthesized by incubating BamHI-digested pJH56 and pJH57 (100 ng/μl) and 15 units of SP6 polymerase (Promega) in 40 mm Tris-HCl (pH 7.5), 6 mm MgCl2, 2 mm spermidine, 10 mm dithiothreitol, 0.5 mm rNTPs for 1 h at 40 °C. In vitro translation reactions (50 μl) programmed with these mRNAs were performed essentially as described (31Lesley S.A. Brow M.D. Burgess R.R. J. Biol. Chem. 1991; 266: 2632-2638Abstract Full Text PDF PubMed Google Scholar), except that they were incubated at 25 °C for 20 min. Reactions were then placed on ice for 5 min and diluted with an equal volume of buffer A (35 mm triethanolamine in acetic acid (pH 8.0), 60 mm potassium acetate, 11 mm magnesium acetate, 1 mm dithiothreitol). Ribosome-nascent chain complexes were collected by centrifugation at 60,000 rpm for 30 min in a TLA100 rotor at 4 °C. The pellets were washed, resuspended in 50 μl of buffer A, and divided in half. E. coli SRP (50 nm) that had been purified as described (32Lu Y. Qi H.-Y. Hyndman J.B. Ulbrandt N.D. Teplyakov A. Tomasevic N. Bernstein H.D. EMBO J. 2001; 20: 6724-6734Crossref PubMed Scopus (36) Google Scholar) was added to one portion. Cross-linking reactions were then performed with 2 mm disuccinimidyl suberate as described (33Valent Q.A. Scotti P.A. High S. deGier J.-W.L. von Heijne G. Lentzen G. Wintermeyer W. Oudega B. Luirink J. EMBO J. 1998; 17: 2504-2512Crossref PubMed Scopus (245) Google Scholar), and proteins were precipitated with cold acetone. Half of each sample was subjected to SDS-PAGE on 14% minigels. Ffh-containing polypeptides were isolated from the other half by immunoprecipitation and resolved by SDS-PAGE on 8-16% minigels. The Highly Basic ΔEspP Signal Peptide Reroutes E. coli Presecretory Proteins into the SRP Pathway—In considering the hypothesis that basic amino acids in signal peptides play a role in targeting pathway selection, we reasoned that naturally occurring presecretory proteins that contain signal peptides with atypically charged N regions might be SRP substrates. Strikingly, the signal peptides of the serine protease autotransporters of E. coli and Shigella (“SPATEs”) are both unusually long and unusually basic. These signal peptides contain a ∼25-amino acid segment that resembles typical signal peptides as well as a ∼25-amino acid N-terminal extension of unknown function. Previous results indicate that one member of the SPATE family, Hbp, is targeted to the IM by SRP (34Sijbrandi R. Urbanus M.L. ten Hagen-Jongman C.M. Bernstein H.D. Oudega B. Otto B.R. Luirink J. J. Biol. Chem. 2003; 278: 4654-4659Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). To determine whether the basic residues found in SPATE signal peptides promote SRP recognition, we first replaced the native signal peptides of MBP and OmpA, two proteins that are normally targeted by SecB, with either the complete EspP signal peptide or a truncated version that lacks the N-terminal extension (ΔEsp). We then examined the effect of changing the signal peptide on the targeting of each protein. The EspP signal peptide was chosen as a model because its N region contains four closely spaced basic residues and a histidine, which might also be slightly charged (Fig. 1). Although the EspP signal peptide did not alter the targeting pathway of either MBP or OmpA (data not shown), we found that the ΔEspP signal peptide eliminated the SecB requirement for export. Initially MC4100 (secB+) and HDB55 (secB-) were transformed with plasmids encoding MBP or OmpA or a derivative containing the ΔEspP signal peptide ΔEspP-MBP or ΔEspP-OmpA under the control of the trc promoter. The plasmid-borne versions of OmpA were HA-tagged to distinguish them from endogenous OmpA. The synthesis of plasmid-borne proteins was induced by the addition of IPTG, and export was examined in pulse-chase labeling experiments. Radiolabeled proteins were immunoprecipitated, and export was assessed by comparing the relative amounts of precursor and mature forms of MBP or OmpA at each time point. Consistent with previous results, the wild-type proteins were exported much less efficiently in the secB- strain than in MC4100 (Fig. 2A, lanes 1-6). By contrast, ΔEspP-MBP or ΔEspP-OmpA was exported equally well in both strains (Fig. 2A, lanes 7-12). These results imply that the presence of the highly basic signal peptide reroutes the proteins from the SecB pathway to another targeting pathway or abolishes the need for a targeting factor altogether. Further investigation indicated that the ΔEspP signal peptide directs presecretory proteins into the SRP targeting pathway. To test the effect of depleting SRP on the export of proteins containing the ΔEspP signal peptide, isogenic secB+ and secB- strains in which ffh is under the control of the araBAD promoter (HDB51 and HDB52, respectively) were transformed with a plasmid encoding MBP or ΔEspP-MBP and grown in medium supplemented with arabinose. Ffh was then depleted from half of the cells by switching the carbon source to glucose, and protein export was assayed as described above. Ffh depletion did not measurably affect the export of ΔEspP-MBP in HDB51 but caused a significant export defect in the secB- strain (Fig. 2B, lane 4). The results suggest that ΔEspP-MBP is targeted by SRP in wild-type E. coli but can also be targeted effectively by molecular chaperones when the SRP pathway is impaired. Indeed given that the ΔEspP signal peptide is only moderately hydrophobic, this interpretation of the data is consistent with other results showing that SRP dependence correlates with an unusual degree of signal peptide hydrophobicity (see below and Ref. 18Lee H.C. Bernstein H.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3471-3476Crossref PubMed Scopus (164) Google Scholar). We next obtained direct evidence that SRP can interact with the ΔEspP signal peptide in chemical cross-linking experiments. Cell-free translation reactions were programmed with mRNAs that encode the first 94 amino acids of MBP or ΔEspP-MBP, radioactive nascent chains were synthesized, and the homobifunctional cross-linker disuccinimidyl suberate was added to isolated ribosome-nascent chain complexes in the presence or absence of 50 nm E. coli SRP. When ΔEspP-MBP (but not wild-type MBP) nascent chains were synthesized, a prominent radiolabeled band of ∼55 kDa (the combined molecular mass of Ffh and the nascent chain) was observed in the presence of SRP (Fig. 3A, lanes 1-4). Immunoprecipitation with an anti-Ffh antiserum confirmed that the band corresponded to a cross-linked complex of Ffh and the nascent chain (Fig. 3B, lane 4). Ffh was cross-linked to the ΔEspP signal peptide considerably less efficiently than to the highly hydrophobic MBP*1 signal peptide (data not shown), but the reason for this discrepancy is unclear. The results of a different set of experiments strongly suggested that SRP also targets ΔEspP-OmpA to the IM. Presumably because SecB targets wild-type OmpA post-translationally, a variable amount of pro-OmpA was reproducibly observed in pulse-labeled MC4100 and related secB+ strains (Figs. 2, A and C, lane 1). This effect was particularly pronounced when cells were grown at 22 °C (Fig. 2C, lane 1, top panel). Interestingly, the precursor form of ΔEspP-OmpA was not observed in pulse-labeled MC4100 (Fig. 2A, lane 7; Fig. 2C, lane 3, top panel). When a strain harboring an ffh Ts mutation (SKP1101) and an isogenic ffh+ strain (SKP1102) were shifted to 42 °C, however, the ΔEspP-OmpA precursor was observed in the mutant strain (Fig. 2C, lane 3, bottom panel). These results suggest that ΔEspP-OmpA is targeted rapidly to the IM by the co-translational SRP pathway in wild-type cells but routed by default into a slower post-translational pathway when SRP function is impaired. We obtained further evidence that SRP targets ΔEspP-OmpA to the IM in experiments in which we overproduced TF, a chaperone that binds promiscuously to nascent polypeptides early in biosynthesis. Previous work showed that TF overproduction strongly retards the export of OmpA, β-lactamase, and alkaline phosphatase (a protein that does not require a chaperone for export) but does not affect the biogenesis of proteins targeted by SRP (29Lee H.C. Bernstein H.D. J. Biol. Chem. 2002; 277: 43527-43535Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). This effect can be explained by the observation that the binding of SRP and TF to nascent polypeptides is mutually exclusive (35Ullers 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). We transformed HDB37 (MC4100 ara+) with plasmids expressing the TF gene under the control of the araBAD promoter and either OmpA or ΔEspP-OmpA. As expected, the addition of arabinose greatly delayed the export of OmpA (Fig. 2D, lanes 1-4). TF overproduction, however, only very slightly affected the export of ΔEspP-OmpA (Fig. 2D, lanes 5-8). Taken together with the results described above these data provide strong evidence that the presence of the ΔEspP signal peptide routes presecretory proteins into the SRP pathway. SRP Recognizes the ΔEspP Signal Peptide on the Basis of Both Charge and Hydrophobicity—We next wished to determine whether the basic amino acids in the N region of the ΔEspP signal peptide are re" @default.
• W2139997535 created "2016-06-24" @default.
• W2139997535 creator A5041199955 @default.
• W2139997535 creator A5049024470 @default.
• W2139997535 creator A5062522223 @default.
• W2139997535 date "2003-11-01" @default.
• W2139997535 modified "2023-09-27" @default.
• W2139997535 title "Basic Amino Acids in a Distinct Subset of Signal Peptides Promote Interaction with the Signal Recognition Particle" @default.
• W2139997535 cites W1558492944 @default.
• W2139997535 cites W1572001614 @default.
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