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• W2005963908 abstract "The Escherichia coli Tat system has unusual capacity of translocating folded proteins across the cytoplasmic membrane. The TatA protein is the most abundant known Tat component and consists of a transmembrane segment followed by an amphipathic helix and a hydrophilic C terminus. To study the operation mechanism of the Tat apparatus, we analyzed the topology of TatA. Intriguingly, alkaline phosphatase (PhoA)-positive fusions were obtained at positions Gly-38, Lys-40, Asp-51, and Thr-53, which are all located at the cytoplasmic C terminus of the TatA protein. Interestingly, replacing phoA with uidA at Thr-53 led to positive β-glucuronidase fusion, implying cytoplasmic location of the TatA C terminus. To further determine cellular localization of the TatA C terminus, we deleted the phoA gene and left 46 exogenous residues, including the tobacco etch virus (Tev) protease cleavage site (Tcs) after Thr-53, yielding TatAT53::Tcs. Unlike the PhoA and UidA fusions, which abolished the TatA function, the TatAT53::Tcs construct was able to restore the growth of tatA mutants on the minimal trimethlyamine N-oxide media. In vitro and in vivo proteolysis assay showed that the Tcs site of TatAT53::Tcs was accessible from both the periplasm and cytoplasm, indicating a dual topology of the TatA C terminus. Importantly, growth conditions seemed to influence the protein level of TatA and the cytoplasmic accessibility of the Tcs site of TatAT53::Tcs. A function-linked change of the TatA topology is suggested, and its implication in protein transport is discussed. The Escherichia coli Tat system has unusual capacity of translocating folded proteins across the cytoplasmic membrane. The TatA protein is the most abundant known Tat component and consists of a transmembrane segment followed by an amphipathic helix and a hydrophilic C terminus. To study the operation mechanism of the Tat apparatus, we analyzed the topology of TatA. Intriguingly, alkaline phosphatase (PhoA)-positive fusions were obtained at positions Gly-38, Lys-40, Asp-51, and Thr-53, which are all located at the cytoplasmic C terminus of the TatA protein. Interestingly, replacing phoA with uidA at Thr-53 led to positive β-glucuronidase fusion, implying cytoplasmic location of the TatA C terminus. To further determine cellular localization of the TatA C terminus, we deleted the phoA gene and left 46 exogenous residues, including the tobacco etch virus (Tev) protease cleavage site (Tcs) after Thr-53, yielding TatAT53::Tcs. Unlike the PhoA and UidA fusions, which abolished the TatA function, the TatAT53::Tcs construct was able to restore the growth of tatA mutants on the minimal trimethlyamine N-oxide media. In vitro and in vivo proteolysis assay showed that the Tcs site of TatAT53::Tcs was accessible from both the periplasm and cytoplasm, indicating a dual topology of the TatA C terminus. Importantly, growth conditions seemed to influence the protein level of TatA and the cytoplasmic accessibility of the Tcs site of TatAT53::Tcs. A function-linked change of the TatA topology is suggested, and its implication in protein transport is discussed. The twin arginine translocation (Tat or ΔpH) system has unusual capacity to transport folded proteins and enzyme complex across the bacterial cytoplasmic membrane or the chloroplast thylakoid membrane (1Wu L.-F. Ize B. Chanal A. Quentin Y. Fichant G. J. Mol. Microbiol. Biotechnol. 2000; 2: 179-189PubMed Google Scholar, 2Mori H. Cline K. Biochim. Biophys. Acta. 2001; 1541: 80-90Crossref PubMed Scopus (94) Google Scholar, 3Dalbey R.E. Robinson C. Trends Biochem. Sci. 1999; 24: 17-22Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Four functional Tat components, TatA, TatB, TatC, and TatE, have been identified and characterized in Escherichia coli (4Berks B.C. Sargent F. Palmer T. Mol. Microbiol. 2000; 35: 260-274Crossref PubMed Scopus (472) Google Scholar). TatA, TatB, and TatE share sequence homology at their N termini, including one transmembrane segment (TMS) 1The abbreviations used are: TMS, transmembrane segment; Kan, kanamycin; APH, amphipathic helix; Tcs, tobacco etch virus (Tev) protease cleavage site; TMAO, trimethylamine N-oxide; Amp, ampicillin; IPTG, isopropyl β-d-thiogalactoside; PhoA, alkaline phosphatase; UidA, β-glucuronidase. and an adjacent amphipathic helix (APH), whereas their C termini vary both in sequence and in length (5Chanal A. Santini C.-L. Wu L.-F. Mol. Microbiol. 1998; 30: 674-676Crossref PubMed Scopus (57) Google Scholar). Expression studies suggest that tatE may be a cryptic gene duplication of tatA (6Jack R.L. Sargent F. Berks B.C. Sawers G. Palmer T. J. Bacteriol. 2001; 183: 1801-1804Crossref PubMed Scopus (115) Google Scholar). Functional Tat translocase has been reconstituted in vitro using membrane vesicles derived from cells overproducing TatA, TatB, and TatC proteins (7Yahr T.L. Wickner W.T. EMBO J. 2001; 20: 2472-2479Crossref PubMed Scopus (138) Google Scholar, 8Alami M. Trescher D. Wu L.-F. Muller M. J. Biol. Chem. 2002; 277: 20499-20503Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). A large complex of ∼650 kDa containing TatABC has been purified from the detergent-solubilized E. coli membrane (9Bolhuis A. Mathers J.E. Thomas J.D. Barrett C.M. Robinson C. J. Biol. Chem. 2001; 276: 20213-20219Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 10de Leeuw E. Granjon T. Porcelli I. Alami M. Carr S. Muller M. Sargent F. Palmer T. Berks B. J. Mol. Biol. 2002; 322: 1135-1146Crossref PubMed Scopus (91) Google Scholar) and shown to be capable of binding a Tat signal peptide (10de Leeuw E. Granjon T. Porcelli I. Alami M. Carr S. Muller M. Sargent F. Palmer T. Berks B. J. Mol. Biol. 2002; 322: 1135-1146Crossref PubMed Scopus (91) Google Scholar). Recently, Alami et al. (11Alami M. Luke I. Deitermann S. Eisner G. Koch H.G. Brunner J. Muller M. Mol. Cell. 2003; 12: 937-946Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) have reported a hierarchy in targeting of a Tat substrate to the TatBC complex. For the primary interaction TatC is both necessary and sufficient, whereas a subsequent association with TatB likely mediates transfer from TatC to the actual Tat pore (11Alami M. Luke I. Deitermann S. Eisner G. Koch H.G. Brunner J. Muller M. Mol. Cell. 2003; 12: 937-946Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Apparently, TatB and TatC are present in a constant 1:1 stoichiometry in the E. coli TatABC complex, whereas the vast majority of the TatA protein does not co-purify with the TatBC core complex (9Bolhuis A. Mathers J.E. Thomas J.D. Barrett C.M. Robinson C. J. Biol. Chem. 2001; 276: 20213-20219Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). On the other hand a TatAB complex of ∼600 kDa with a TatA to TatB molar ratio of about 15:1 (10de Leeuw E. Granjon T. Porcelli I. Alami M. Carr S. Muller M. Sargent F. Palmer T. Berks B. J. Mol. Biol. 2002; 322: 1135-1146Crossref PubMed Scopus (91) Google Scholar, 12Sargent F. Gohlke U. De Leeuw E. Stanley N.R. Palmer T. Saibil H.R. Berks B.C. Eur. J. Biochem. 2001; 268: 3361-3367Crossref PubMed Scopus (128) Google Scholar) and TatA homo-oligomeric complexes (10de Leeuw E. Granjon T. Porcelli I. Alami M. Carr S. Muller M. Sargent F. Palmer T. Berks B. J. Mol. Biol. 2002; 322: 1135-1146Crossref PubMed Scopus (91) Google Scholar, 13Porcelli I. de Leeuw E. Wallis R. van den Brink-van der Laan E. de Kruijff B. Wallace B.A. Palmer T. Berks B.C. Biochemistry. 2002; 41: 13690-23697Crossref PubMed Scopus (96) Google Scholar) have been purified from E. coli membranes. The purification of the different Tat complexes suggests that the Tat pathway utilizes multiple, transiently interacting complexes. Consistently, it has been shown that the thylakoidal TatC-TatB complex homologue, cpTatC-Hcf106, serves as precursor receptor and the TatA homologue, Tha4, assembles with cpTatC-Hcf106 complex during the translocation step (2Mori H. Cline K. Biochim. Biophys. Acta. 2001; 1541: 80-90Crossref PubMed Scopus (94) Google Scholar, 14Mori H. Cline K. J. Cell Biol. 2002; 157: 205-210Crossref PubMed Scopus (197) Google Scholar, 15Cline K. Mori H. J. Cell Biol. 2001; 154: 719-729Crossref PubMed Scopus (244) Google Scholar). Unlike the E. coli Tat system, the thylakoidal Tha4 has not been detected in the thylakoid Tat complex in the absence of ongoing substrate binding or translocation (2Mori H. Cline K. Biochim. Biophys. Acta. 2001; 1541: 80-90Crossref PubMed Scopus (94) Google Scholar, 14Mori H. Cline K. J. Cell Biol. 2002; 157: 205-210Crossref PubMed Scopus (197) Google Scholar, 15Cline K. Mori H. J. Cell Biol. 2001; 154: 719-729Crossref PubMed Scopus (244) Google Scholar). Biochemical and expression studies show that TatA is the most abundant component of the Tat system, present at an ∼20-fold molar excess over the TatB and TatC components (6Jack R.L. Sargent F. Berks B.C. Sawers G. Palmer T. J. Bacteriol. 2001; 183: 1801-1804Crossref PubMed Scopus (115) Google Scholar, 12Sargent F. Gohlke U. De Leeuw E. Stanley N.R. Palmer T. Saibil H.R. Berks B.C. Eur. J. Biochem. 2001; 268: 3361-3367Crossref PubMed Scopus (128) Google Scholar). Minimal functional units and key residues of TatA and TatB have been determined by genetic and molecular biology approaches (16Lee P.A. Buchanan G. Stanley N.R. Berks B.C. Palmer T. J. Bacteriol. 2002; 184: 5871-5879Crossref PubMed Scopus (70) Google Scholar, 17Barrett C.M. Mathers J.E. Robinson C. FEBS Lett. 2003; 537: 42-46Crossref PubMed Scopus (34) Google Scholar, 18Hicks M.G. de Leeuw E. Porcelli I. Buchanan G. Berks B.C. Palmer T. FEBS Lett. 2003; 539: 61-67Crossref PubMed Scopus (59) Google Scholar). C-terminal truncation analysis revealed that the transmembrane and amphipathic helical regions of TatA and TatB are critical for their function but that the C-terminal domains are not essential for Tat transport activity. Using site-specific mutagenesis to probe the significance of conserved features of the related TatA/B subunits leads to the conclusion that an apparent “hinge” region between the transmembrane segment and an APH is important in both proteins (17Barrett C.M. Mathers J.E. Robinson C. FEBS Lett. 2003; 537: 42-46Crossref PubMed Scopus (34) Google Scholar, 18Hicks M.G. de Leeuw E. Porcelli I. Buchanan G. Berks B.C. Palmer T. FEBS Lett. 2003; 539: 61-67Crossref PubMed Scopus (59) Google Scholar). To gain further insight in the TatA function and the operation mechanism of the bacterial Tat translocase, we studied the topology of the Tat proteins. PhoA-positive fusions were obtained with TatA. In addition, PhoA and UidA fusions indicated that the TatA C terminus could be located both in the cytoplasm and in the periplasm. Moreover, we observed the influence of growth media on the intracellular level of TatA and TatC and on the cytoplasmic accessibility of the TatA C terminus. This finding strongly suggests dynamic topology changes of the Tat apparatus, which might be coupled to the translocation of folded proteins across the cytoplasmic membrane. Bacterial Strains, Plasmids, and Media—All E. coli strains and plasmids used in this study are listed in Table I. The bacteria were routinely grown in Luria-Bertani (LB) liquid medium, on LB plates, or in the minimal M9 medium (19Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 431-433Google Scholar) supplemented with glycerol (0.5%), ammonium molybdate (1 μm), potassium selenite (1 μm), trimethylamine N-oxide (TMAO) (0.4%), or fumarate (0.4%). Anaerobic growth was achieved normally in stoppered bottles or tubes filled to the top. As required, ampicillin (Amp) (50 μg/ml), kanamycin (Kan) (50 μg/ml), tetracycline (15 μg/ml), or isopropyl β-d-thiogalactoside (IPTG, 0.25 mm) was added. Pre-cultures were grown from single colonies and used at 100-fold dilutions for inoculation of experimental cultures.Table IStrains and plasmids used in this studyNameGenotypeReferenceStrains MC4100F′ lacΔU169 araD139 rpsL150 thi flbB5301 deoC7 ptsF25 relAlLaboratory stock JARV16as MC4100, ΔtatAE30Sargent F. Bogsch E.G. Stanley N.R. Wexler M. Robinson C. Berks B.C. Palmer T. EMBO J. 1998; 17: 3640-3650Crossref PubMed Scopus (444) Google Scholar B0Das MC4100, ΔtatB46Sargent F. Stanley N.R. Berks B.C. Palmer T. J. Biol. Chem. 1999; 274: 36073-36082Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar B1LK0as MC4100, ΔtatC47Bogsch E. Sargent F. Stanley N.R. Berks B.C. Robinson C. Palmer T. J. Biol. Chem. 1998; 273: 18003-18006Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar DADEas MC4100, ΔtatABCDE48Wexler M. Sargent F. Jack R.L. Stanley N.R. Bogsch E.G. Robinson C. Berks B.C. Palmer T. J. Biol. Chem. 2000; 275: 16717-16722Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar CC118araΔ139 Δ(ara, leu)7697 ΔlacX74 phoAΔ20 galE galK thi rpsE rpoB argEam recA1Laboratory stock EZ4rpsL Δ(add-uid-man)Laboratory stock TG1Δ(lac-pro) supE thi hsdD5/F′ traD36 proA+B+ lacI1 lacZΔM15Laboratory stockPlasmids pACYC184Stratagene pmodTapphoA+ neo+20Gouffi K. Santini C.L. Wu L.-F. FEBS Lett. 2002; 525: 65-70Crossref PubMed Scopus (42) Google Scholar pmodTinuidA+ neo+20Gouffi K. Santini C.L. Wu L.-F. FEBS Lett. 2002; 525: 65-70Crossref PubMed Scopus (42) Google Scholar pMM13tev+, derivative of pACYC18423Ehrmann M. Bolek P. Mondigler M. Boyd D. Lange R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13111-13115Crossref PubMed Scopus (42) Google Scholar pET22b+Cloning vectorNovagen p8737(tatABCD)+, derivative of pET22b+8Alami M. Trescher D. Wu L.-F. Muller M. J. Biol. Chem. 2002; 277: 20499-20503Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar pJ11tatAG38-phoA, derivative of p8737This study pG14tatAK40-phoA, derivative of p8737This study pM6tatAD51-phoA, derivative of p8737This study pG41tatAT53-phoA, derivative of p8737This study pG41DtatAT53::Tcs, (tatBCD)+, derivative of pG41This study p9910tatAT53::Tcs, deletion of Xho1-Sal1 fragment of pG41This study p9913tatAT53-uidA, derivative of p9910This study pG41K23ItatAT53-phoA, derivative of pG41, with K231 mutation in tatAThis study p9935tatAT53::Tcs, derivative of pG41DThis study Open table in a new tab Construction of tatA-phoA, tatA::Tcs and tatA-uidA Fusions—TatA-PhoA fusions were constructed by random in vitro Tn-PhoA transposition as previously reported (20Gouffi K. Santini C.L. Wu L.-F. FEBS Lett. 2002; 525: 65-70Crossref PubMed Scopus (42) Google Scholar). Briefly, the transposable element carrying the phoA-neo cassette was amplified by PCR using pmodTap as template and pmodFP (5′-attcaggctgcgcaactgt-3′) and pmodRP (5′-gtcagtgagcgaggaagcggaag-3′) as primers. The random in vitro transposition of the transposable element into p8737 was performed by using the EZ::TN transposase according to the manufacturer's instructions (Epicentre, Tebu). The transposition reaction product was then introduced by electroporation into CC118 strain. Blue (PhoA+) AmpR and KanR colonies were selected on agar plates containing 5-bromo-4-chloro-3-indolyl-phosphate (X-P, 40 μg/ml). Plasmids were prepared from these colonies, and Tn-PhoA insertion sites were determined by endonuclease digestion, PCR reaction, and DNA sequencing. The phoA-neo cassette was removed from pG41 with partial NotI digestion and self-ligation, yielding the plasmid pG41D (see Fig. 1). To construct p9913 (tatAT53-uidA), the phoA-neo cassette inserted at tatAT53 was replaced by the uidA-neo cassette by two steps of cloning. First, the NotI site in the multiple cloning sites of pG41D (tatAT53::Tcs) was removed with a XhoI-SalI double digestion and self-ligation, yielding p9910. The uidA-neo cassette was amplified by PCR using pmodTin as template and pmodFP-pmodRP as primers and cloned into the unique NotI site of p9910, resulting in p9913. Random substitution in the hinge region covering the residues from Gly-21 to Lys-24 of the TatA protein was constructed using gene splicing by an overlap extension protocol (21Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2648) Google Scholar). Briefly, two DNA fragments were amplified using pG41 as template in separate polymerase chain reactions with the oligonucleotide pairs BglIITatAup (5′-gaagatctcgatcccgcgaaattaatacgactc-3′) and TatAindown (5′-aaaaagcagtacaacgatgacggcaataatcaataa-3′) in one reaction, and TatAxup (5′-gttgtactgcttttt112322433431ctcggctcc-3′, where 1, 2, and 3 means 91.6%, respectively, of G, C, and A with 2.77% each of other 3 bases, and 4 represents 91.6% of A with 4.16% of G and 4.16% of C) and SstIITatBdw (5′-tccccgcggcctggcgtagttcatccatcg-3′) in another reaction. The two PCR products were then mixed, denatured, and re-annealed and used as template for another PCR by using BglIITatAup and SstIITatBdw as primers. The PCR products were digested by BglII and SstII, cloned into the corresponding sites of pG41, and used to transform CC118. Protein samples were prepared from a dozen of PhoA- colonies on LB-Amp plates, resolved on SDS-denaturing gels, and analyzed by immunoblot using antisera against PhoA. One of them contained intact TatAT53-PhoA fusion proteins. DNA sequencing revealed that the plasmid pG41K23I, contained an aa to tt nucleotide alteration leading to an isoleucine substitution for Lys-23. Enzyme Assays and Bioinformatics—Alkaline phosphatase and β-glucuronidase activities were assayed by the hydrolysis of p-nitrophenyl phosphate or p-nitrophenyl glucuronide, respectively. The absorption of the produced p-nitrophenol was measured at 410 nm with a Cary 50 spectrophotometer using a control without extract as the reference blank. One unit of enzyme activity is defined as the release of 1 μmol of nitrophenol/min. Specific activity is expressed by unit/mg of protein. Spheroplast preparation and trypsin sensitivity assay were performed by using the protocol described by Tian and Beckwith (22Tian H. Beckwith J. J. Bacteriol. 2002; 184: 111-118Crossref PubMed Scopus (40) Google Scholar). In vitro Tev assays were performed on spheroplasts as described by Ehrmann et al. (23Ehrmann M. Bolek P. Mondigler M. Boyd D. Lange R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13111-13115Crossref PubMed Scopus (42) Google Scholar). Briefly, cells expressing TatA containing a Tev protease cleavage site were cultivated on LB or M9 media. Harvested cells were washed once in proteolysis buffer (50 mm Tris-HCl, pH8, 0.5 mm EDTA, pH 8) and concentrated to an A600 = 1 in the same buffer. For proteolysis, 5 mm dithiothreitol and 5 μl of Tev protease (50 units) were added to 45 μl of cells and incubated at 30 °C according to the manufacturer's instructions (Invitrogen). Samples were subjected to SDS-PAGE and Western blotting by using polyclonal antibodies against TatA or PhoA. Topology prediction of the TatA protein was carried out using the ExPaSy tools available at www.expasy.ch, and secondary structure predication was performed by using the PSIPRED program at bioinf.cs.ucl.ac.uk or the HNN program at npsa-pbil.ibcp.fr. Cellular Fractionation, Electrophoresis, and Immunoblot—Spheroplasts, cytoplasmic, periplasmic, and membrane fractions were prepared by lysozyme/EDTA/cold osmo-shock and ultracentrifugation, as described previously (24Rodrigue A. Chanal A. Beck K. Muller M. Wu L.-F. J. Biol. Chem. 1999; 274: 13223-13228Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). The protein samples resolved on SDS-denaturing gels by electrophoresis and immobilized onto a polyvinylidene difluoride membrane were analyzed by immunoblot using the ECL+ method according to the manufacturer's instructions (Amersham Bioscience). The signals were quantified by using KODAK 1D image analysis software. Detection of Cellular Location of the TatA C Terminus by Using PhoA and UidA Fusions—Using an in vitro random Tn-PhoA transposition approach with p8737 ((tatABCD)+) as a target, a dozen of colonies showing a positive PhoA activity were obtained (20Gouffi K. Santini C.L. Wu L.-F. FEBS Lett. 2002; 525: 65-70Crossref PubMed Scopus (42) Google Scholar). Further mapping by endonuclease restriction enzyme digestion, polymerase chain reaction, and DNA-sequencing analysis revealed that four were in TatA after glycine 38 (TatAG38), lysine 40 (TatAK40), aspartate 51 (TatAD51), and threonine 53 (TatAT53) (Fig. 1B). The PhoA moiety gains its active form only if it is translocated into the periplasm and is, thus, widely used as a marker for studying membrane protein topology (25Manoil C. Mekalanos J.J. Beckwith J. J. Bacteriol. 1990; 172: 515-518Crossref PubMed Google Scholar, 26von Heijne G. J. Mol. Biol. 1992; 225: 487-494Crossref PubMed Scopus (1404) Google Scholar). Strikingly, the four TatA-PhoA fusions occurred all at the C terminus of TatA, which has been reported to be located in the cytoplasm (13Porcelli I. de Leeuw E. Wallis R. van den Brink-van der Laan E. de Kruijff B. Wallace B.A. Palmer T. Berks B.C. Biochemistry. 2002; 41: 13690-23697Crossref PubMed Scopus (96) Google Scholar). Therefore, we performed a series of experiments to check such a periplasmic location of the TatA C terminus. Enzyme assay showed that the specific activities of PhoA expressed from the TatAG38-PhoA, TatAK40-PhoA, TatAD51-PhoA, and TatAT53-PhoA fusions in CC118 (ΔphoA) were 433, 291, 71, and 62, respectively. These values were significantly higher than 1.1, a value obtained with the control plasmid pET22b+ in CC118. Furthermore, immunoblot analysis revealed that PhoA fusions were completely degraded when the spheroplasts were treated with trypsin (Fig. 2A1). Therefore, the TatA-PhoA fusions were indeed exposed to the periplasm. In the following experiments we chose to study TatAT53-PhoA and its derivatives alone for two reasons. First, they gave the strongest signal by immunoblot analysis using anti-TatA antisera. Second, Thr-53 is located downstream of the amphipathic helix that is essential for the TatA function (16Lee P.A. Buchanan G. Stanley N.R. Berks B.C. Palmer T. J. Bacteriol. 2002; 184: 5871-5879Crossref PubMed Scopus (70) Google Scholar). The E. coli TatA protein is anchored in the cytoplasmic membrane via the amino-proximal transmembrane segment (13Porcelli I. de Leeuw E. Wallis R. van den Brink-van der Laan E. de Kruijff B. Wallace B.A. Palmer T. Berks B.C. Biochemistry. 2002; 41: 13690-23697Crossref PubMed Scopus (96) Google Scholar, 27De Leeuw E. Porcelli I. Sargent F. Palmer T. Berks B.C. FEBS Lett. 2001; 506: 143-148Crossref PubMed Scopus (71) Google Scholar). To obtain a PhoA positive phenotype, the APH must adopt a membrane-span topology to place the TatA C terminus into the periplasm (see Fig. 4C2, see “Discussion”). The hinge region should be crucial for such topology and for the PhoA positive phenotype. To verify this hypothesis we introduced by PCR random substitution in the hinge region of TatA-PhoA fusion expressed from pG41. Altered plasmid pG41 was introduced into CC118 and the transformants obtained were screened for PhoA- phenotype. Crude extracts were prepared from six PhoA- colonies. TatAT53-PhoA fusion protein was detected by immunoblot in one of the strains (data not shown). DNA sequencing analysis revealed that the corresponding plasmid, pG41K23I, encoded TatAT53-PhoA fusion with a substitution of isoleucine for the lysine at position 23 in the hinge region of TatA (see “Experimental Procedures”). This mutation abolished the PhoA activity but did not affect the stability of the TatAT53-PhoA fusion protein, indicating that the PhoA moiety is blocked in the cytoplasm. This finding suggests that the lysine 23 is essential for the membrane span topology of the TatA amphipathic helix. Interestingly, it has been recently reported that changing residues in the hinge region of TatA leads to the defect in TMAO reductase export (17Barrett C.M. Mathers J.E. Robinson C. FEBS Lett. 2003; 537: 42-46Crossref PubMed Scopus (34) Google Scholar, 18Hicks M.G. de Leeuw E. Porcelli I. Buchanan G. Berks B.C. Palmer T. FEBS Lett. 2003; 539: 61-67Crossref PubMed Scopus (59) Google Scholar). To investigate further the topology of TatA, we replaced phoA by uidA and constructed the TatAT53-UidA fusion. The uidA gene encodes the β-glucuronidase, which is active in the cytoplasm and has been generally used to identify cytoplasmic located segment of membrane proteins (23Ehrmann M. Bolek P. Mondigler M. Boyd D. Lange R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13111-13115Crossref PubMed Scopus (42) Google Scholar). The EZ4/p9913 (uidA-/tatAT53-uidA+) displayed a β-glucuronidase activity of 86 units, which was 30-fold higher than the basic level observed for the same mutant carrying the control plasmid p8737. Unlike the TatAT53-PhoA fusion, the TatAT53-UidA fusion was resistant to trypsin digestion in the spheroplast-trypsin sensitivity assay (Fig. 2A2 compared with 2A1). These results indicate a cytoplasmic location of the TatA C terminus of the TatAT53-UidA fusion. The PhoA- and UidA-positive phenotypes indicated that the TatA C terminus might exist in two different topologies. The requirement of the other Tat proteins for the periplasmic location of the PhoA moiety of the TatAT53-PhoA fusion was analyzed by measuring the PhoA activity in tat mutants. The ΔtatB, ΔtatC, and ΔtatABCDE mutations resulted in about a 3-fold decrease of the PhoA activity (Table II). Immunoblot analysis revealed that the amount of the TatAT53-PhoA decreased in the tat mutants. In contrast, these mutations slightly increased the β-glucuronidase activity. Together these results would suggest that the presence of the TatB and TatC proteins would be more favorable for a periplasmic location of the TatA C terminus.Table IITatA-PhoA and TatA-UidA fusion activity in MC4100 and its tat mutant derivatives The bacteria were inoculated in LB-Amp media and incubated at 37 °C overnight. PhoA and UidA activities were defined as the release of 1 μmol of nitrophenol/min/mg of protein. Data are the average of at least three independent cultures with S.D.PlasmidsWild typeΔtatBΔtatCΔtatABCDEAlkaline phosphatase activities p8737 ((tatABCD)+)3.41.72.92.9 pG41 (tatAT53-phoA)49.1 ± 8.013.1 ± 5.114.3 ± 7.413.1 ± 1.7β-Gluronidase activities p8737 ((tatABCD)+)14.38.625.711.4 p9913 (tatAT53-uidA)78.3 ± 10.393.7 ± 10.9121.1 ± 14.9109.7 ± 16.6 Open table in a new tab Cellular Location of the C Terminus of Functional TatA::Tcs Chimera—The PhoA and UidA fusions indicated a dual topology of the TatA C terminus. Because both TatA-PhoA and TatA-UidA lost the TatA function (data not shown), we constructed another TatA derivative to analyze the topology of TatA under physiological conditions. The phoA and neo genes of pG41 were deleted by NotI digestion (Fig. 1C), and a fragment of 138 bp was left at the Tn5 insertion site (23Ehrmann M. Bolek P. Mondigler M. Boyd D. Lange R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13111-13115Crossref PubMed Scopus (42) Google Scholar). The encoded 46 amino acids (see “Experimental Procedures”), containing a tobacco etch virus (Tev) protease cleavage site (Tcs), were thus inserted in-frame after Thr-53 (Fig. 1D), yielding pG41D encoding TatAT53::Tcs. The synthesis and cellular location of the TatAT53::Tcs were analyzed by immunoblot. The anti-TatA polyclonal antisera recognized TatA and, more weakly, an additional nonspecific polypeptide in the urea-washed membrane fractions prepared from the wild type strain carrying the plasmid p8737 ((tatABC)+) (Fig. 2B, lane 1). As anticipated, the TatA protein was absent from the ΔtatAE mutant, and the plasmid pG41D specifically directed the synthesis of a polypeptide bigger than TatA and with the size expected for TatAT53::Tcs in this mutant (Fig. 2B, lane 2). Both TatA and TatAT53::Tcs were detected in the membrane fractions of the ΔtatB and ΔtatC mutants carrying the plasmid pG41D (tatAT53::Tcs-(tatBC)+) (Fig. 2B, lanes 3 and 4). Importantly, the plasmid pG41D (tatAT53::Tcs-(tatBC)+) was able to restore the growth defect of the ΔtatAE, ΔtatB, ΔtatC, and ΔtatABCDE mutants (Fig. 3A1) on minimal TMAO/glycerol liquid media. Therefore, TatAT53::Tcs preserves the TatA function, and the insertion of 138-bp in the tatA gene had no effect on the synthesis of the downstream TatB and TatC proteins, which was confirmed by immunoblot (data not shown). The cellular accessibility of the Tcs site within the physiologically active TatAT53::Tcs and of those located at the junction between TatAT53 and PhoA/UidA in the non-functional TatA-PhoA and TatA-UidA fusions was assessed by proteolysis and immunoblot analysis of cells grown in rich LB media. As anticipated, upon the treatment of the spheroplasts with exogenously provided Tev protease, the TatAT53-PhoA fusion was reduced to the size corresponding to the PhoA protein, implying the separation of the PhoA moieties from TatAT53 (Fig. 2C1, lane 3, compared with lane 2). Interestingly, when the spheroplasts were subjected to an in vitro Tev treatment the intensity of the TatAT53::Tcs band was significantly reduced (Fig. 2C2, lanes 3 versus 2). Notably, the nonspecific band in the Tev-treated fraction was stronger than that in the non-treated fraction. Therefore, the decrease of the TatAT53::Tcs intensity in the Tev-treated fraction could not be due to an uneven loading of the samples. Unlike the TatAT53-PhoA fusion, TatAT53::Tcs was not completely digested by the in vitro Tev treatment. These results would suggest that not all Tev cleavage sites in the TatAT53::Tcs are accessible from the periplasm and that the PhoA moiety of the TatA-PhoA fusion blocks the C terminus in the periplasm. To analyze the cytoplasmic accessibility of the Tev cleavage sites, we co-introduced pMM13 plasmid carryi" @default.
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• W2005963908 title "Dual Topology of the Escherichia coli TatA Protein" @default.
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• W2005963908 doi "https://doi.org/10.1074/jbc.m313187200" @default.
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