Structure-based Functional Study Reveals Multiple Roles of Transmembrane Segment IX and Loop VIII–IX in NhaA Na+/H+ Antiporter of Escherichia coli at Physiological pH
2008; Elsevier BV; Volume: 283; Issue: 23 Linguagem: Inglês
10.1074/jbc.m800482200
ISSN1083-351X
AutoresTzvi Tzubery, Abraham Rimon, Etana Padan,
Tópico(s)Ion Transport and Channel Regulation
ResumoThe three-dimensional crystal structure of Escherichia coli NhaA determined at pH 4 provided the first structural insights into the mechanism of antiport and pH regulation of a Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 6.5–8.5), many questions pertaining to the active state of NhaA have remained open including the structural and physiological roles of helix IX and its loop VIII–IX. Here we studied this NhaA segment (Glu241–Phe267) by structure-based biochemical approaches at physiological pH. Cysteine-scanning mutagenesis identified new mutations affecting the pH dependence of NhaA, suggesting their contribution to the "pH sensor." Furthermore mutation F267C reduced the H+/Na+ stoichiometry of the antiporter, and F267C/F344C inactivated the antiporter activity. Tests of accessibility to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide, a membrane-impermeant positively charged SH reagent with a width similar to the diameter of hydrated Na+, suggested that at physiological pH the cytoplasmic cation funnel is more accessible than at acidic pH. Assaying intermolecular cross-linking in situ between single Cys replacement mutants uncovered the NhaA dimer interface at the cytoplasmic side of the membrane; between Leu255 and the cytoplasm, many Cys replacements cross-link with various cross-linkers spanning different distances (10–18Å) implying a flexible interface. L255C formed intermolecular S–S bonds, cross-linked only with a 5-Å cross-linker, and when chemically modified caused an alkaline shift of 1 pH unit in the pH dependence of NhaA and a 6-fold increase in the apparent Km for Na+ of the exchange activity suggesting a rigid point in the dimer interface critical for NhaA activity and pH regulation. The three-dimensional crystal structure of Escherichia coli NhaA determined at pH 4 provided the first structural insights into the mechanism of antiport and pH regulation of a Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 6.5–8.5), many questions pertaining to the active state of NhaA have remained open including the structural and physiological roles of helix IX and its loop VIII–IX. Here we studied this NhaA segment (Glu241–Phe267) by structure-based biochemical approaches at physiological pH. Cysteine-scanning mutagenesis identified new mutations affecting the pH dependence of NhaA, suggesting their contribution to the "pH sensor." Furthermore mutation F267C reduced the H+/Na+ stoichiometry of the antiporter, and F267C/F344C inactivated the antiporter activity. Tests of accessibility to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide, a membrane-impermeant positively charged SH reagent with a width similar to the diameter of hydrated Na+, suggested that at physiological pH the cytoplasmic cation funnel is more accessible than at acidic pH. Assaying intermolecular cross-linking in situ between single Cys replacement mutants uncovered the NhaA dimer interface at the cytoplasmic side of the membrane; between Leu255 and the cytoplasm, many Cys replacements cross-link with various cross-linkers spanning different distances (10–18Å) implying a flexible interface. L255C formed intermolecular S–S bonds, cross-linked only with a 5-Å cross-linker, and when chemically modified caused an alkaline shift of 1 pH unit in the pH dependence of NhaA and a 6-fold increase in the apparent Km for Na+ of the exchange activity suggesting a rigid point in the dimer interface critical for NhaA activity and pH regulation. Regulation of intracellular pH, cellular Na+ content, and cell volume is essential for all living cells. Na+/H+ antiporters play primary roles in these crucial processes. They are integral membrane proteins, ubiquitous throughout the biological kingdom. Many Na+/H+ antiporters are tightly regulated by pH, a property that underpins their capacity to maintain pH homeostasis of the cytoplasm (1Padan E. Bibi E. Masahiro I. Krulwich T.A. Biochim. Biophys. Acta. 2005; 1717: 67-88Crossref PubMed Scopus (535) Google Scholar). NhaA, the main Na+/H+ antiporter of Escherichia coli, has eukaryotic orthologs, including human (2Brett C.L. Donowitz M. Rao R. Am. J. Physiol. 2005; 288: C223-C239Crossref PubMed Scopus (433) Google Scholar, 3Xiang M. Feng M. Muend S. Rao R. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 18677-18681Crossref PubMed Scopus (76) Google Scholar). It is an electrogenic antiporter with a stoichiometry of 2 H+/1 Na+ (1Padan E. Bibi E. Masahiro I. Krulwich T.A. Biochim. Biophys. Acta. 2005; 1717: 67-88Crossref PubMed Scopus (535) Google Scholar, 4Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar) and is strongly dependent on pH; its rate of activity changes over 3 orders of magnitude between pH 7.0 and 8.5 (1Padan E. Bibi E. Masahiro I. Krulwich T.A. Biochim. Biophys. Acta. 2005; 1717: 67-88Crossref PubMed Scopus (535) Google Scholar, 4Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar, 5Padan E. Tzubery T. Herz K. Kozachkov L. Rimon A. Galili L. Biochim. Biophys. Acta. 2004; 1658: 2-13Crossref PubMed Scopus (118) Google Scholar). NhaA is a dimer in the native membrane as revealed by genetic complementation data, biochemical pulldown experiments, intermolecular cross-linking (6Gerchman Y. Rimon A. Venturi M. Padan E. Biochemistry. 2001; 40: 3403-3412Crossref PubMed Scopus (78) Google Scholar), ESR studies (7Hilger D. Jung H. Padan E. Wegener C. Vogel K.P. Steinhoff H.J. Jeschke G. Biophys. J. 2005; 89: 1328-1338Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 8Hilger D. Polyhach Y. Padan E. Jung H. Jeschke G. Biophys. J. 2007; 93: 3675-3683Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and cryoelectron microscopy of two-dimensional crystals (9Williams K.A. Geldmacher-Kaufer U. Padan E. Schuldiner S. Kuhlbrandt W. EMBO J. 1999; 18: 3558-3563Crossref PubMed Scopus (111) Google Scholar, 10Williams K.A. Nature. 2000; 403: 112-115Crossref PubMed Scopus (203) Google Scholar). The recently determined crystal structure of NhaA monomer at pH 4 (11Hunte C. Screpanti M. Venturi M. Rimon A. Padan E. Michel H. Nature. 2005; 534: 1197-1202Crossref Scopus (521) Google Scholar) has provided the first structural insights into the mechanism of antiport and pH regulation of a Na+/H+ antiporter. NhaA consists of 12 TMSs 2The abbreviations used are: TMS, transmembrane segment; CL-NhaA, cysteineless NhaA; NEM, N-ethylmaleimide; BMH, 1,6-bismaleimidohexane; MTS-2-MTS, 1,2-ethanediyl bismethanethiosulfonate; MTSES, 2-sulfonatoethylmethanethiosulfonate; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate bromide; AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid, disodium salt; o-PDM, N,N′-o-phenylenedimaleimide; p-PDM, N,N′-p-phenylenedimaleimide; NTA, nitrilotriacetic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; L255C, NhaA Leu255 replaced with Cys (all other nhaA mutations are accordingly depicted). 2The abbreviations used are: TMS, transmembrane segment; CL-NhaA, cysteineless NhaA; NEM, N-ethylmaleimide; BMH, 1,6-bismaleimidohexane; MTS-2-MTS, 1,2-ethanediyl bismethanethiosulfonate; MTSES, 2-sulfonatoethylmethanethiosulfonate; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate bromide; AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid, disodium salt; o-PDM, N,N′-o-phenylenedimaleimide; p-PDM, N,N′-p-phenylenedimaleimide; NTA, nitrilotriacetic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; L255C, NhaA Leu255 replaced with Cys (all other nhaA mutations are accordingly depicted). with the N and C termini pointing into the cytoplasm. It represents a novel fold; TMSs IV and XI are each comprised of two short helices connected by extended chains that cross each other, in close contact, in the middle of the membrane (TMS IV/XI assembly (11Hunte C. Screpanti M. Venturi M. Rimon A. Padan E. Michel H. Nature. 2005; 534: 1197-1202Crossref Scopus (521) Google Scholar) and Fig. 1). This creates a delicately balanced electrostatic environment in the middle of the membrane. An ion funnel opens to the cytoplasm with a negatively charged orifice that is most suitable to act as a cation trap and a "pH sensor." The funnel (designated cytoplasmic) ends in the middle of the membrane in the vicinity of the putative ion binding site and the crossing of the TMS IV/XI assembly. Together these structural elements have been suggested to play a crucial role in the pH-controlled Na+/H+ exchange mechanism (11Hunte C. Screpanti M. Venturi M. Rimon A. Padan E. Michel H. Nature. 2005; 534: 1197-1202Crossref Scopus (521) Google Scholar). Based on the NhaA crystal structure obtained at pH 4, very crucial functional and structural roles have also been assigned to TMS IX and the neighboring part of loop VIII–IX in the mechanism and pH regulation of NhaA. It contains amino acid residues that may (a) participate in the pH sensor at the orifice of the cytoplasmic funnel, (b) connect TMS IX to TMS XI of the TMS IV/XI assembly, (c) line the cytoplasmic cation funnel leading to the active site, and (d) line the NhaA dimer interface. The three-dimensional structure of NhaA monomer agrees with the electron density map obtained by cryoelectron microscopy of the two-dimensional crystal, implying that it represents a native conformation in the membrane (12Screpanti E. Padan E. Rimon A. Michel H. Hunte C. J. Mol. Biol. 2006; 362: 192-202Crossref PubMed Scopus (36) Google Scholar). However, because both the two-dimensional and three-dimensional crystals were obtained at pH 4, at which NhaA is down-regulated, the structure of the active conformation(s) and the process(s) leading to it have remained open questions. Hence it is crucial to conduct experiments, both structurally and functionally oriented, at physiological pH at which NhaA is active to obtain a working model for the mechanism of activity and pH regulation of the antiporter at physiological pH. The present study examined the roles of TMS IX and the neighboring part of loop VIII–IX at physiological pH when NhaA is active. Bacterial Strains and Culture Conditions—EP432 is an E. coli K-12 derivative, which is melBLid, ΔnhaA1::kan, ΔnhaB1::cat, ΔlacZY, thr1 (13Pinner E. Kotler Y. Padan E. Schuldiner S. J. Biol. Chem. 1993; 268: 1729-1734Abstract Full Text PDF PubMed Google Scholar). TA16 is nhaA+nhaB+lacIQ (TA15lacIQ) and otherwise isogenic to EP432 (4Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar). Cells were grown in modified L broth (LBK, L broth in which Na+ is replaced by K+) (14Padan E. Maisler N. Taglicht D. Karpel R. Schuldiner S. J. Biol. Chem. 1989; 264: 20297-20302Abstract Full Text PDF PubMed Google Scholar) or in minimal medium A without sodium citrate (15Davis B.D. Mingioli E.S. J. Bacteriol. 1950; 60: 17-28Crossref PubMed Google Scholar) with 0.5% glycerol, 0.01% MgS04·7H2O, and 2.5 μg/ml thiamine. Antibiotic was 100 μg/ml ampicillin. The resistance to Na+ was tested in Na+ selective medium: LB containing 0.6 m NaCl buffered with 60 mm 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl) propane-1,3-diol; pH was adjusted with HCl to pH 7 or pH 8.3. For plates, 1.5% agar was used. Plasmids—All plasmids used have pBR322 origin of replication and carry bla (AmpR). pCL-BSTX, a pCL-GMAR100 derivative (16Tzubery T. Rimon A. Padan E. J. Biol. Chem. 2004; 279: 3265-3272Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) encodes Cys-less NhaA with a silent BstX site at codon 248 in nhaA. pCL-AXH3, a derivative of pCL-AXH2, contains a BstXI silent site at codon 248 in nhaA (17Kozachkov L. Herz K. Padan E. Biochemistry. 2007; 46: 2419-2430Crossref PubMed Scopus (42) Google Scholar). It expresses cysteineless NhaA (CL-NhaA) from the tac promoter fused at its C terminus to two factor Xa cleavage sites followed by a His6 tag. pCL-HAH4 is a derivative of pCL-HAH3 (18Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar), bearing the silent BstXI mutation at codon 248 in nhaA. It expresses from the tac promoter a variant of CL-NhaA of which segment Arg383–Val388 was replaced by a hemagglutinin epitope followed by two factor Xa cleavage sites and a His6 tag. All plasmids carrying mutations are designated by the name of the plasmid followed by the mutation. Site-directed Mutagenesis—Site-directed mutagenesis was conducted following a polymerase chain reaction-based protocol (19Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6810) Google Scholar) or site-directed mutagenesis facilitated by DpnI selection on hemimethylated DNA (20Fisher C.L. Pei G.K. BioTechniques. 1997; 23 (574): 570-571Crossref PubMed Scopus (288) Google Scholar). For Cys replacement of Lys242, His243, Gly244, Arg245, Ser246, Pro247, Ala248, Lys249, Arg250, Leu251, His253, Leu255, Trp258, and Leu262, pCL-HAH4 was used as a template. For Cys replacement of His256, Pro257, and Val259 pCL-AXH3 was used as template. For Cys replacement of Leu264, Phe267, Phe344, and Phe267/Phe344 pCL-AXH3 and pCL-BstX were used as templates. All mutations were verified by DNA sequencing of the entire gene through the ligation junctions with the vector plasmid. Isolation of Membrane Vesicles and Assay of Na+/H+ Antiporter Activity—EP432 cells transformed with the respective plasmids were grown in LBK medium, and everted membrane vesicles were prepared and used to determine the Na+/H+ antiporter activity as described previously (21Rosen B.P. Methods Enzymol. 1986; 125: 328-336Crossref PubMed Scopus (113) Google Scholar, 22Goldberg E.B. Arbel T. Chen J. Karpel R. Mackie G.A. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2615-2619Crossref PubMed Scopus (183) Google Scholar). The assay of antiport activity was based upon the measurement of Na+-induced changes in the ΔpH as measured by acridine orange, a fluorescent probe of ΔpH. The fluorescence assay was performed with 2.5 ml of reaction mixture containing 50–100 μg of membrane protein, 0.5 μm acridine orange, 150 mm KCl, 50 mm 1,3-bis[tris(hydroxymethyl)methylamino]propane, and 5 mm MgCl2, and the pH was titrated with HCl as indicated. After energization with either ATP or d-lactate (2 mm, pH 7 titrated by KOH), quenching of the fluorescence was allowed to achieve a steady state, and then Na+ was added. A reversal of the fluorescence level (dequenching) indicates that protons are exiting the vesicles in antiport with Na+. As shown previously, the end level of dequenching is a good estimate of the antiporter activity (23Schuldiner S. Fishkes H. Biochemistry. 1978; 17: 706-711Crossref PubMed Scopus (103) Google Scholar), and the concentration of the ion that gives half-maximal dequenching is a good estimate of the apparent Km for Na+ (or Li+) of the antiporter (23Schuldiner S. Fishkes H. Biochemistry. 1978; 17: 706-711Crossref PubMed Scopus (103) Google Scholar, 24Tsuboi Y. Inoue H. Nakamura N. Kanazawa H. J. Biol. Chem. 2003; 278: 21467-21473Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The concentration range of the cations tested was 0.01–100 mm at the indicated pH values, and the apparent Km values were calculated by linear regression of the Lineweaver-Burk plot. High pressure membranes were isolated as described previously (25Gerchman Y. Rimon A. Padan E. J. Biol. Chem. 1999; 274: 24617-24624Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Protein Purification—For extraction of NhaA, membranes (0.5 mg of membrane protein) were resuspended in 1.15 ml of a solution containing 4.3 mm Tris/Cl (pH 7.5), 110 mm sucrose, and 60 mm choline chloride supplemented with 20% glycerol, 1% β-dodecyl maltoside, and 0.1 m MOPS (pH 7). The suspension was incubated for 20 min at 4 °C and centrifuged (Beckman TLA 100.4; 265,000 × g for 20 min at 4 °C). For affinity purification of NhaA, the supernatant was added to 50 μl of Ni2+-NTA-agarose (Qiagen) and incubated with agitation for 1 h at 4 °C. Then beads were washed in binding and washing buffers (26Olami Y. Rimon A. Gerchman Y. Rothman A. Padan E. J. Biol. Chem. 1997; 272: 1761-1768Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The protein was eluted in 200 μl of acid elution buffer (27Rimon A. Tzubery T. Padan E. J. Biol. Chem. 2007; 282: 26810-26821Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) or by incubation for 20 min at 4 °C in 20 μl of SDS-PAGE sampling buffer supplemented with 300 mm imidazole and centrifuged (Eppendorf; 20,800 × g for 2 min at 4 °C). Protein Determination—The protein was determined according to Bradford (28Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214435) Google Scholar). Detection and Quantification of NhaA and Its Mutated Proteins in the Membrane—NhaA and its mutated derivatives in membranes were quantitated in membranes by Western analysis as described previously (29Gerchman Y. Olami Y. Rimon A. Taglicht D. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1212-1216Crossref PubMed Scopus (131) Google Scholar) using the NhaA-specific monoclonal antibody 1F6 (30Padan E. Venturi M. Michel H. Hunte C. FEBS Lett. 1998; 441: 53-58Crossref PubMed Scopus (41) Google Scholar) or by resolving the affinity-purified proteins by SDS-PAGE, staining with Coomassie Blue, and quantification of the band density by Image Gauge (Fuji) software (6Gerchman Y. Rimon A. Venturi M. Padan E. Biochemistry. 2001; 40: 3403-3412Crossref PubMed Scopus (78) Google Scholar). Accessibility of Cys Replacements of CL-NhaA to Impermeant Sulfhydryl Reagent MTSET—The accessibility assay in intact cells was performed as described previously (31Ninio S. Elbaz Y. Schuldiner S. FEBS Lett. 2004; 562: 193-196Crossref PubMed Scopus (48) Google Scholar), but the Tris buffer was replaced with 100 mm potassium phosphate and 5 mm MgSO4 (pH 7.5 or 8.5). For assay of accessibility to MTSET from the cytoplasmic side, everted membrane vesicles (500 μg of membrane protein) were incubated with 10 mm MTSET in the phosphate buffer (at pH 7.5 or 8.5) at room temperature with gentle shaking for 45 min. Then the reaction was stopped by dilution into 3 ml of TSC buffer (containing 10 mm Tris (pH 7.5), 250 mm sucrose, and 140 mm choline chloride) and centrifuged (Beckman TLA 100.4; 265,000 × g for 20 min at 4 °C). The protein was extracted, affinity-purified on Ni+2-NTA, and left on the beads. The beads were washed twice in binding buffer (26Olami Y. Rimon A. Gerchman Y. Rothman A. Padan E. J. Biol. Chem. 1997; 272: 1761-1768Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) at pH 7.4, resuspended in 100 μl of binding buffer containing 0.2 mm fluorescein-5-maleimide (Molecular Probes), and further incubated for 30 min at 25 °C to determine the Cys left free following MTSET treatment. Then the beads were washed, and the protein was eluted in a sample buffer containing 300 mm imidazole and separated by SDS-PAGE. For evaluation of the fluorescence intensity, the gels were photographed under UV light (260 nm) as described previously (18Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar). For quantifying the amount of the protein, the gels were Coomassie Blue-stained, and the density of the bands was determined. After normalization of the fluorescence intensity to the amount of protein in the band, the accessibility to MTSET was determined from the difference in the fluorescence of the reagent-treated and untreated samples (100% fluorescence = 0% modification by MTSET (18Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar)). In Situ Site-directed Intermolecular Cross-linking—Site-directed intermolecular cross-linking was conducted in situ on high pressure membrane vesicles isolated from TA16 cells overexpressing the various NhaA mutants (25Gerchman Y. Rimon A. Padan E. J. Biol. Chem. 1999; 274: 24617-24624Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Membranes (500 μg of membrane protein) were resuspended in a buffer (0.5 ml) containing 100 mm potassium phosphate (pH 7.5 or 8.5), 5 mm MgSO4, and one of the freshly prepared homobifunctional cross-linkers: 2 mm BMH (Pierce), 1 mm o-PDM (Sigma), 1 mm p-PDM (Sigma), 2 mm MTS-2-MTS (Toronto Research Chemicals), or a 4 mm concentration of the oxidizing reagent diamide (Sigma). The stock solutions of the cross-linkers were prepared in N,N-dimethylformamide so that the amount of N,N-dimethylformamide in the reaction mixture did not exceed 1%, a concentration that does not affect the antiporter activity. The reaction mixture was incubated at room temperature with gentle rotation for 45 min, and the reaction was terminated by 10 mm β-mercaptoethanol in the case of maleimides (BMH, o-PDM, and p-PDM) or by dilution in TSC buffer (in the case of MTS-2-MTS and diamide). Then the affinity-purified proteins were separated by SDS-PAGE (non-reducing conditions in the case of treatment with MTS-2-MTS and diamide) and Coomassie Blue-stained to determine the densities of the bands with mobility corresponding to that of NhaA monomers and dimers (6Gerchman Y. Rimon A. Venturi M. Padan E. Biochemistry. 2001; 40: 3403-3412Crossref PubMed Scopus (78) Google Scholar, 18Rimon A. Tzubery T. Galili L. Padan E. Biochemistry. 2002; 41: 14897-14905Crossref PubMed Scopus (25) Google Scholar). The sum of the densities of the monomers and dimers equals 100%. Measurement of accessibility to the cross-linking reagents was performed on the proteins bound to the beads as described above for MTSET. Treatment of Membranes Expressing Cys Replacement Mutations with SH Reagents for Activity Assay—Membranes (0.5 mg of membrane protein) were resuspended in a reaction mixture as described above for cross-linking but containing one of the following reagents: 1 mm NEM, 1 mm MTSES (Anatrace), 1 mm MTSET (Anatrace), 1 mm AMS (Molecular Probes), or a 4 mm concentration of the oxidizing reagent diamide. The reaction mixture was incubated with gentle shaking for 20 min at room temperature. The reaction was stopped by addition of 3 ml of TSC buffer (containing 10 mm β-mercaptoethanol in the case of maleimide reagents) and centrifugation (Beckman TLA 100.4; 265,000 × g for 20 min at 4 °C). The membranes were resuspended in TSC buffer (5–10 mg of membrane protein/ml), and the Na+/H+ antiporter activity was monitored as described above. For determination of accessibility to the SH reagents, the protein was affinity-purified, and free Cys residues were determined by fluorescein-5-maleimide labeling as described above for MTSET. Reconstitution of NhaA into Proteoliposomes and Measurement of ΔpH-driven 22Na Uptake—NhaA proteoliposomes were reconstituted and ΔpH-driven 22Na uptake was determined as described previously (4Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar, 32Rimon A. Gerchman Y. Kariv Z. Padan E. J. Biol. Chem. 1998; 273: 26470-26476Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). All experiments were repeated at least twice with practically identical results. Cys Replacement Mutations, Expression in the Membrane, and Growth Phenotypes at Physiological pH—The crystal structure of the acid pH-locked conformation of NhaA (11Hunte C. Screpanti M. Venturi M. Rimon A. Padan E. Michel H. Nature. 2005; 534: 1197-1202Crossref Scopus (521) Google Scholar) and structure-based computation (33Olkhova E. Hunte C. Screpanti E. Padan E. Michel H. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2629-2634Crossref PubMed Scopus (53) Google Scholar) combined with genetic and biochemical data (16Tzubery T. Rimon A. Padan E. J. Biol. Chem. 2004; 279: 3265-3272Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 25Gerchman Y. Rimon A. Padan E. J. Biol. Chem. 1999; 274: 24617-24624Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) have suggested that the cytoplasmic end of helix IX and the in-tandem part of loop VIII–IX contain amino acid residues involved in the pH sensor of NhaA. To identify these residues, we systematically replaced with Cys the amino acids residues (each separately) in segment Glu241–Val259 of NhaA excluding E241C, V254C (6Gerchman Y. Rimon A. Venturi M. Padan E. Biochemistry. 2001; 40: 3403-3412Crossref PubMed Scopus (78) Google Scholar, 25Gerchman Y. Rimon A. Padan E. J. Biol. Chem. 1999; 274: 24617-24624Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), and E252C (16Tzubery T. Rimon A. Padan E. J. Biol. Chem. 2004; 279: 3265-3272Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) that were isolated previously (Fig. 1). In addition, Leu262, Leu264, and Phe267 were replaced with Cys (Fig. 1). The latter two amino acids have been shown by the crystal structure to be close enough to interact with TMS XI residues and therefore, even with no experimental support, have been assigned a structural and/or functional role in the translocation machinery (Ref. 11Hunte C. Screpanti M. Venturi M. Rimon A. Padan E. Michel H. Nature. 2005; 534: 1197-1202Crossref Scopus (521) Google Scholar and Fig. 1). All mutants were constructed in CL-NhaA, a variant that is as expressed and active as the native NhaA (26Olami Y. Rimon A. Gerchman Y. Rothman A. Padan E. J. Biol. Chem. 1997; 272: 1761-1768Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The mutations were subjected to structural and functional studies at the physiological pH (pH 6.5–8.5) to reveal the physiological role of the replaced residues when NhaA is progressively activated with increasing pH (1Padan E. Bibi E. Masahiro I. Krulwich T.A. Biochim. Biophys. Acta. 2005; 1717: 67-88Crossref PubMed Scopus (535) Google Scholar, 4Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1991; 266: 11289-11294Abstract Full Text PDF PubMed Google Scholar). Challenging the results obtained at the physiological pH with the predictions based on the acid pH-locked crystal structure also allowed us to deduce whether pH-induced conformational changes occurred during the activation process. To characterize the mutations with respect to growth, expression, and antiporter activity, the mutated plasmids were transformed into EP432, an E. coli strain that lacks the two Na+-specific antiporters (NhaA and NhaB (13Pinner E. Kotler Y. Padan E. Schuldiner S. J. Biol. Chem. 1993; 268: 1729-1734Abstract Full Text PDF PubMed Google Scholar)). This strain neither grows on selective media (0.6 m NaCl at pH 7 or 8.3) nor exhibits any Na+/H+ antiporter activity in isolated everted membrane vesicles unless transformed with a plasmid encoding an active antiporter (for a review, see Ref. 1Padan E. Bibi E. Masahiro I. Krulwich T.A. Biochim. Biophys. Acta. 2005; 1717: 67-88Crossref PubMed Scopus (535) Google Scholar; see also Table 1). As compared with the level of expression of the wild type (100%), all mutants were expressed at roughly 40% of wild type level (Table 1) from the multicopy plasmids. The mutant proteins, affinity-purified on a Ni+2-NTA column, were readily detected by Coomassie Blue staining. Notably this expression level is far above that obtained from a single chromosomal gene that confers a wild type phenotype (29Gerchman Y. Olami Y. Rimon A. Taglicht D. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1212-1216Crossref PubMed Scopus (131) Google Scholar). Whereas most of the single mutants conferred upon EP432 Na+ resistance similar to that of the wild type (Table 1), a few mutants grew somewhat less efficiently on the selective media as was evident by smaller colony number and colony size at either pH (K242C) or only at alkaline pH (R250C and H253C). Most interestingly, mutant F267C grew very similarly to the wild type at neutral pH but did not grow at all at alkaline pH (Table 1).TABLE 1Cys replacements of residues in loop VIII–IX, TMS IX, and TMS XI of NhaA For characterization of the mutations, EP432 cells were transformed with the plasmids carrying the indicated mutations. The expression level was expressed as a percentage of the control cells (EP432/pCl-HAH4) encoding CL-NhaA. Growth experiments were conducted on agar plates at 37 °C with high Na+ (0.6 m) at the indicated pH values. + + +, number and size of the colonies after 48 h of incubation at neutral pH; + +, number as above but smaller sized colonies after 48 h of incubation at alkaline pH; +, both number and size of colonies were reduced; –, no growth. The apparent Km for NaCl was determined at pH 8.5 as described under "Experimental Procedures." ND, not determined.MutationExpressionGrowth, 0.6 m NaClApparent Km, Na+pH 7pH 8.3%mmE241CaThe data were obtained from Ref. 25120+ + ++ +NDK242C48++1.9H243C116+ + ++ +1.2G244C82+ + ++ +0.9R245C87+ + ++ +4.8S246C98+ + ++ +1.7P247C161+ + ++ +1.2A248C84+ + ++ +1.3K249C80+ + ++ +2.6R250C89+ + ++1.9L251C72+ + ++ +2E252C83+ + ++ +10H253C37+ + ++1.5V254CaThe data were obtained from Ref. 25110+ + ++ +NDL255C47+ + ++ +0.8H256C80+ + ++ +3.5P257C50+ + ++ +0.6W258C81+ + ++ +0.4V259C50+ + ++ +1.4L262C78+ + ++ +0.6L264C83+ + ++ +0.9F267C95+ + +–0.8F344C40+ + ++0.16F267C/F344C37––CL-NhaA100+ + ++ +0.5pBR322––a The data were obtained from Ref. 25Gerchman Y. Rimon A. Padan E. J. Biol. Chem. 1999; 274: 24617-24624Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar Open table in a new tab The Na+/H+ Antiporter Activity of the Mutants at Physiological pH—To assess the involvement of the Cys-replaced amino acid residues in the pH sensor of NhaA, we studied the antiporter activity of the mutants at th
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