Identification and Characterization of the Na+/H+ Antiporter NhaS3 from the Thylakoid Membrane of Synechocystis sp. PCC 6803
2009; Elsevier BV; Volume: 284; Issue: 24 Linguagem: Inglês
10.1074/jbc.m109.001875
ISSN1083-351X
AutoresKenta Tsunekawa, Toshiaki Shijuku, Mitsuo Hayashimoto, Yoichi KOJIMA, Kiyoshi Onai, Megumi Morishita, Masahiro Ishiura, Teruo Kuroda, Tatsunosuke Nakamura, Hiroshi Kobayashi, Mayuko Sato, Kiminori Toyooka, Ken Matsuoka, Tatsuo Omata, Nobuyuki Uozumi,
Tópico(s)Plant Stress Responses and Tolerance
ResumoNa+/H+ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na+/H+ antiporters, nhaS1–5 and sll0556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na+/H+ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na+ concentrations. NhaS3 had no K+/H+ exchange activity itself but enhanced K+ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO2 to high CO2 conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO2 consumption caused by the ceasing of O2-evolving photosynthesis. This is the first report of a Na+/H+ antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H+, Na+, and K+ in supporting the conversion of photosynthetic products and in the supply of energy in the dark. Na+/H+ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na+/H+ antiporters, nhaS1–5 and sll0556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na+/H+ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na+ concentrations. NhaS3 had no K+/H+ exchange activity itself but enhanced K+ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO2 to high CO2 conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO2 consumption caused by the ceasing of O2-evolving photosynthesis. This is the first report of a Na+/H+ antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H+, Na+, and K+ in supporting the conversion of photosynthetic products and in the supply of energy in the dark. Na+/H+ antiporters are integral membrane proteins that transport Na+ and H+ in opposite directions across the membrane and that occur in virtually all cell types. These transporters play an important role in the regulation of cytosolic pH and Na+ concentrations and influence proton or sodium motive force across the membrane (1Padan E. Schuldiner S. Biochim. Biophys. Acta. 1994; 1185: 129-151Crossref PubMed Scopus (146) Google Scholar, 2Padan E. Schuldiner S. Biochim. Biophys. Acta. 1994; 1187: 206-210Crossref PubMed Scopus (16) Google Scholar). In Escherichia coli, three Na+/H+ antiporters (NhaA, NhaB, and ChaA) have been described in detail. Of these, NhaA is the functionally best characterized transporter. The crystal structure of NhaA has been resolved (3Hunte C. Screpanti E. Venturi M. Rimon A. Padan E. Michel H. Nature. 2005; 435: 1197-1202Crossref PubMed Scopus (539) Google Scholar). In addition, mutants of nhaA, nhaB, and chaA as well as the triple mutant have been generated (4Ohyama T. Igarashi K. Kobayashi H. J. Bacteriol. 1994; 176: 4311-4315Crossref PubMed Google Scholar). The triple mutant was shown to be hypersensitive to extracellular Na+. The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains six genes encoding Na+/H+ antiporters (NhaS1–5 and sll0556). NhaS1 (slr1727) has also been designated SynNhaP (5Hamada A. Hibino T. Nakamura T. Takabe T. Plant Physiol. 2001; 125: 437-446Crossref PubMed Scopus (73) Google Scholar, 6Inaba M. Sakamoto A. Murata N. J. Bacteriol. 2001; 183: 1376-1384Crossref PubMed Scopus (76) Google Scholar). Null mutants of nhaS1, nhaS2, nhaS4, and nhaS5 have been generated; however, a null mutant of nhaS3 could not be obtained, indicating that it is an essential gene (6Inaba M. Sakamoto A. Murata N. J. Bacteriol. 2001; 183: 1376-1384Crossref PubMed Scopus (76) Google Scholar, 7Mikkat S. Milkowski C. Hagemann M. Plant Cell Environ. 2000; 23: 549-559Crossref Scopus (24) Google Scholar, 8Elanskaya I.V. Karandashova I.V. Bogachev A.V. Hagemann M. Biochemistry. 2002; 67: 432-440PubMed Google Scholar). By heterologous expression in E. coli, Na+/H+ exchange activities could be shown for NhaS1–5 (5Hamada A. Hibino T. Nakamura T. Takabe T. Plant Physiol. 2001; 125: 437-446Crossref PubMed Scopus (73) Google Scholar, 6Inaba M. Sakamoto A. Murata N. J. Bacteriol. 2001; 183: 1376-1384Crossref PubMed Scopus (76) Google Scholar). Inactivation of nhaS1 and nhaS2 results in retardation of growth of Synechocystis (5Hamada A. Hibino T. Nakamura T. Takabe T. Plant Physiol. 2001; 125: 437-446Crossref PubMed Scopus (73) Google Scholar, 6Inaba M. Sakamoto A. Murata N. J. Bacteriol. 2001; 183: 1376-1384Crossref PubMed Scopus (76) Google Scholar). It has been reported that in these mutants the concentration of Na+ in cytosol and intrathylakoid space (lumen) increases and impairs the photosynthetic and/or respiratory activity of the cell (9Allakhverdiev S.I. Nishiyama Y. Suzuki I. Tasaka Y. Murata N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5862-5867Crossref PubMed Scopus (179) Google Scholar, 10Allakhverdiev S.I. Sakamoto A. Nishiyama Y. Inaba M. Murata N. Plant Physiol. 2000; 123: 1047-1056Crossref PubMed Scopus (478) Google Scholar). Therefore the Na+ extrusion by Synechocystis Na+/H+ antiporters similar to E. coli NhaA, NhaB, and ChaA is essential for the adaptation to salinity stress. In contrast to the case in E. coli, Na+ is an essential element for the growth of some cyanobacteria (11Miller A.G. Turpin D.H. Canvin D.T. J. Bacteriol. 1984; 159: 100-106Crossref PubMed Google Scholar, 12Kaplan A. Scherer S. Lerner M. Plant Physiol. 1989; 89: 1220-1225Crossref PubMed Google Scholar). Interestingly, the Na+/H+ antiporter homolog NhaS4 was identified as an uptake system for Na+ from the medium during a screen for mutations in Synechocystis that result in lack of growth at low Na+ concentrations (7Mikkat S. Milkowski C. Hagemann M. Plant Cell Environ. 2000; 23: 549-559Crossref Scopus (24) Google Scholar). The requirement of a Na+ uptake antiporter for cell growth is consistent with the physiology of Synechocystis. Specifically, photoautotrophic bacteria like cyanobacteria share some components (plastoquinone, cytochrome b6f, and c6) of the thylakoid membrane for electron transport for both photophosphorylation and respiratory oxidative phosphorylation. Na+/H+ antiporters therefore may coordinate both H+ and Na+ gradients across the plasma and thylakoid membranes to adapt to daily environmental changes (11Miller A.G. Turpin D.H. Canvin D.T. J. Bacteriol. 1984; 159: 100-106Crossref PubMed Google Scholar). It remains to be determined whether the six Na+/H+ antiporters are localized to the plasma membrane or to the thylakoid membrane in Synechocystis. Information on the membrane localization will also provide information on the physiological role in Synechocystis. In this study, we explored the membrane localization of NhaS3, the role of specific amino acid residues for its function, and the effect of CO2 concentration and circadian rhythms on the expression pattern of nhaS3 to gain insight into the physiological role of NhaS3 in Synechocystis. Synechocystis cells were grown at 30 °C in BG11 medium (13Rippka R. Deruelles J. Waterbury J.B. Herdman M. Stanier R.Y. J. Gen. Microbiol. 1979; 111: 1-61Crossref Google Scholar) containing 20 mm TES-KOH 3The abbreviation used is: TES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. (pH 8.0) and bubbled with either 2% CO2 in air (v/v) or air alone (0.035% v/v CO2). Solid medium contained BG11 buffered at pH 8.0, 1.5% agar, and 0.3% sodium thiosulfate. Continuous illumination was provided by fluorescent lamps (50 μmol of photons m−2 s−1; 400–700 nm). To test the activation of expression of the nhaS3 promoter-luciferase fusions (see below), cells grown under low CO2 conditions (0.035% v/v CO2) were collected by centrifugation at 5000 × g for 10 min at 30 °C, washed with fresh growth medium to remove dissolved CO2, and inoculated into fresh medium that was aerated with air containing 2% (v/v) CO2. Control cells were kept at 0.035% (v/v CO2). 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. For heterologous expression in E. coli, the nhaS3 (sll0689) gene was isolated from chromosomal DNA by PCR using KpnI site-containing forward primer 5′-ATAGGTACCAGGAGGGAAAAGAATGTTTATGAACCCAT-3′ and SalI site-containing reverse primer 5′-AAAGTCGACCTAATCTGGGGTGGGAAC-3′. The KpnI-SalI DNA fragment was ligated into the corresponding sites in pPAB404 (14Buurman E.T. Kim K.T. Epstein W. J. Biol. Chem. 1995; 270: 6678-6685Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and the resulting plasmid was used to transform E. coli strain LB2003, which lacks the three K+ uptake systems (15Stumpe S. Bakker E.P. Arch. Microbiol. 1997; 167: 126-136Crossref PubMed Scopus (104) Google Scholar), or E. coli strain TO114, which lacks the three Na+ extrusion type Na+/H+ antiporters (4Ohyama T. Igarashi K. Kobayashi H. J. Bacteriol. 1994; 176: 4311-4315Crossref PubMed Google Scholar). Growth tests of the transformed strains were carried out as described previously (16Matsuda N. Kobayashi H. Katoh H. Ogawa T. Futatsugi L. Nakamura T. Bakker E.P. Uozumi N. J. Biol. Chem. 2004; 279: 54952-54962Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 17Uozumi N. Nakamura T. Schroeder J.I. Muto S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9773-9778Crossref PubMed Scopus (83) Google Scholar). For replacement of NhaS3 with E. coli NhaA in Synechocystis, the E. coli nhaA gene was cloned behind the iron-transporter promoter in a plasmid containing the spectinomycin resistance gene (18Katoh H. Hagino N. Grossman A.R. Ogawa T. J. Bacteriol. 2001; 183: 2779-2784Crossref PubMed Scopus (164) Google Scholar) and inserted by homologous recombination into targeting site 4 4K. Onai and M. Ishiura, unpublished observations. of the chromosomal DNA of Synechocystis (16Matsuda N. Kobayashi H. Katoh H. Ogawa T. Futatsugi L. Nakamura T. Bakker E.P. Uozumi N. J. Biol. Chem. 2004; 279: 54952-54962Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). For disruption of nhaS3, the kanamycin resistance gene was amplified by PCR using HpaI site-containing forward primer 5′-CAGTGTTAACAAAGCCACGTTGTGTCTC-3′ and HpaI site-containing reverse primer 5′-CAGTGTTAACGCGCTGAGGTCTGCCTCG-3′. The HpaI-digested DNA fragment was inserted into the EcoRV site in the middle of the nhaS3 gene of the nhaA-expressing strain. For generation of the NhaS3 knockdown strain, the same HpaI-digested DNA fragment was inserted into the EcoRV site in the middle of the nhaS3 gene of wild type Synechocystis. For generation of nhaS3-overexpressing Synechocystis, the full-length coding sequence of NhaS3 was amplified by PCR using NdeI site-containing forward primer 5′-CGGCATATGATGTTTATGAACCCATTG-3′ and SalI site-containing reverse primer 5′-AAGTCGACCTAATCTGGGGTGGGAAC-3′. The NdeI-SalI DNA fragment was ligated into the corresponding sites in p68TS4OxCm4 and inserted by homologous recombination into targeting site 4 of the chromosomal DNA of Synechocystis (16Matsuda N. Kobayashi H. Katoh H. Ogawa T. Futatsugi L. Nakamura T. Bakker E.P. Uozumi N. J. Biol. Chem. 2004; 279: 54952-54962Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). This vector contains the inducible trc promoter. Expression of nhaS3 was induced by the addition of 0.25 mm isopropyl β-d-thiogalactopyranoside to the medium. Thylakoid and plasma membranes were prepared from Synechocystis cells as described previously (19Norling B. Zak E. Andersson B. Pakrasi H. FEBS Lett. 1998; 436: 189-192Crossref PubMed Scopus (107) Google Scholar). An anti-NhaS3 antibody was raised against synthetic peptides with the two sequences NH2-LAEINRLSSNEGQI-COOH and NH2-KKEEAPEKPVPTPD-COOH (Operon Biotechnologies, Japan). Polyclonal antibodies raised against the plasma membrane nitrate transporter NrtA (20Omata T. Plant Cell Physiol. 1995; 36: 207-213Crossref PubMed Scopus (84) Google Scholar) or against the thylakoid membrane proteins NdhD3 and NdhF3 (21Zhang P. Battchikova N. Jansen T. Appel J. Ogawa T. Aro E.M. Plant Cell. 2004; 16: 3326-3340Crossref PubMed Scopus (189) Google Scholar) were used to identify they Synechocystis plasma membrane, or thylakoid membrane fractions, respectively. The proteins were separated by SDS-PAGE on 10 or 12% polyacrylamide gels and then transferred to polyvinylidene fluoride membranes. The membranes were incubated for 1 h with primary antibody (1:1000 in blocking buffer) and were then incubated for 30 min with the secondary antibody with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000; Amersham Biosciences) and subsequently subjected to chemiluminescence detection (ECL; Amersham Biosciences). Synechocystis cells grown to an optical density of about 1.2 in BG11 medium were frozen and then fixed with anhydrous acetone containing 1% glutaraldehyde at −80 °C. The samples were warmed and embedded in LR White resin as described (22Toyooka K. Moriyasu Y. Goto Y. Takeuchi M. Fukuda H. Matsuoka K. Autophagy. 2006; 2: 96-106Crossref PubMed Scopus (93) Google Scholar). Ultra-thin sections were first labeled with the IgG fraction of NhaS3 antiserum (1:20) in Tris-buffered saline and then with 12-nm colloidal gold particles coupled to goat anti-rabbit IgG. IgG fractions were purified from the NhaS3 or preimmune serum using the MelonTM Gel IgG Spin Purification Kit (Pierce). The sections were stained with uranyl acetate and examined with a 1010EX transmission electron microscope (JEOL) at 80 kV as described (22Toyooka K. Moriyasu Y. Goto Y. Takeuchi M. Fukuda H. Matsuoka K. Autophagy. 2006; 2: 96-106Crossref PubMed Scopus (93) Google Scholar). A 1000-bp nhaS3 promoter sequence was fused to the bacterial luciferase gene set luxAB at the BglII and NdeI sites of p68TS1ΩLuxAB(+)PLNK,4 and the construct was inserted into the TS1 region in Synechocystis chromosomal DNA (23Kucho K. Aoki K. Itoh S. Ishiura M. Genes Genet. Syst. 2005; 80: 19-23Crossref PubMed Scopus (7) Google Scholar). Bioluminescence from cells grown on the solid BG11 medium was measured as described previously (24Onai K. Morishita M. Itoh S. Okamoto K. Ishiura M. J. Bacteriol. 2004; 186: 4972-4977Crossref PubMed Scopus (45) Google Scholar, 25Okamoto K. Onai K. Furusawa T. Ishiura M. Plant Cell Environ. 2005; 28: 1305-1315Crossref Scopus (15) Google Scholar, 26Okamoto K. Onai K. Ishiura M. Anal. Biochem. 2005; 340: 193-200Crossref PubMed Scopus (38) Google Scholar). The selected cells were cultured in liquid BG11 medium at 30 °C under 91 μmol of white light illumination m−2 s−1 with bubbling of air and stirring. The optical density of the culture at 730 nm was maintained at ∼0.35 by dilution with fresh BG11 medium. To entrain the circadian clock, the culture was placed in darkness for 12 h and then kept under constant light conditions. Bioluminescence was measured every hour. The preparation of E. coli membrane vesicles was carried out as described previously (27Kuroda T. Shimamoto T. Inaba K. Tsuda M. Tsuchiya T. J. Biochem. 1994; 116: 1030-1038Crossref PubMed Scopus (41) Google Scholar). Na+/H+ antiporter activity was measured by the acridine orange fluorescence quenching method (27Kuroda T. Shimamoto T. Inaba K. Tsuda M. Tsuchiya T. J. Biochem. 1994; 116: 1030-1038Crossref PubMed Scopus (41) Google Scholar) at 25 °C in an assay mixture (2 ml) in the buffer (10 mm Tris-HCl (pH 7.2), 140 mm choline chloride, 5 mm MgSO4, 6 mm 2-mercaptoethanol, and 10% glycerol) supplemented with 1 μm acridine orange. The membrane vesicles equal to 50 μg of protein were added to the assay mixture. Tris-dl-lactate (2 mm) was added to initiate fluorescence quenching caused by respiration. Changes in fluorescence were monitored after the addition of 5 mm NaCl. Then 20 ml of 5% Triton X-100 were added to dissipate the pH gradient across the membrane. Fluorescence emission was monitored at 530 nm with excitation at 495 nm. K+ uptake by E. coli was measured at described previously (16Matsuda N. Kobayashi H. Katoh H. Ogawa T. Futatsugi L. Nakamura T. Bakker E.P. Uozumi N. J. Biol. Chem. 2004; 279: 54952-54962Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The cells expressing nhaS3 or containing the empty vector were incubated with 1 mm KCl in HEPES-NaOH, pH 7.5, containing 10 mm glucose, and aliquots were withdrawn at the times indicated in Fig. 3. The cellular K+ content was determined by flame photometry. Previously it had been reported that NhaS3 is an essential gene in Synechocystis 6803 (6Inaba M. Sakamoto A. Murata N. J. Bacteriol. 2001; 183: 1376-1384Crossref PubMed Scopus (76) Google Scholar, 8Elanskaya I.V. Karandashova I.V. Bogachev A.V. Hagemann M. Biochemistry. 2002; 67: 432-440PubMed Google Scholar). Likewise our attempts to construct a null mutant of nhaS3 were also not successful (see Fig. 5). In contrast, in E. coli even disruption of all three Na+/H+ antiporter genes is not lethal. Based on these observations we hypothesized that NhaS3 may have additional unknown characteristics. To test this hypothesis, we tried to replace nhaS3 in Synechocystis with the E. coli nhaA gene encoding a plasma membrane Na+/H+ antiporter (28Goldberg 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 (190) Google Scholar, 29Karpel R. Olami Y. Taglicht D. Schuldiner S. Padan E. J. Biol. Chem. 1988; 263: 10408-10414Abstract Full Text PDF PubMed Google Scholar). As a first step nhaA was introduced into the Synechocystis genome (Fig. 1A). Next, nhaS3 was disrupted in this background by insertion of a kanamycin resistance cassette, and recovery of a fully segregated mutant of nhaS3 expressing E. coli nhaA was attempted. PCR analysis of kanamycin-resistant cells showed that nhaS3 was not fully disrupted by the kanamycin resistance gene because of the incomplete segregation of chromosomes (Fig. 1B). These results indicate that nhaA cannot replace nhaS3 functionally.FIGURE 1Introduction of E. coli nhaA into Synechocystis. A, E. coli nhaA under control of the iron-inducible promoter was introduced at a site between slr0370 and sll0337 of Synechocystis chromosome (left panel). During growth in BG11 medium, nhaA is constitutively expressed. Correct insertion of the expression construct was confirmed by PCR on genomic DNA using specific primers a and b; the results are shown in the right panel. WT, wild type; +nhaA, nhaA-expressing cells; Spe, spectinomycin resistance gene. B, disruption of nhaS3 in the strain expressing E. coli nhaA was performed by insertion of the kanamycin resistance gene (Km) into nhaS3. Correct integration of the kanamycin gene was tested by PCR on genomic DNA using specific primers c and d. The results for four independent clones and WT are shown in the right panel. Note that nhaA could not replace nhaS3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The finding that E. coli nhaA cannot substitute for nhaS3 (Fig. 1) suggested that NhaS3 might differ from E. coli NhaA with respect to its activity and/or subcellular localization. We therefore performed immunolocalization experiments using anti-NhaS3 antibodies to determine the subcellular localization of NhaS3. Membrane fractions of thylakoid and plasma membranes were prepared by aqueous polymer two-phase partitioning followed by sucrose density gradient centrifugation (19Norling B. Zak E. Andersson B. Pakrasi H. FEBS Lett. 1998; 436: 189-192Crossref PubMed Scopus (107) Google Scholar). As shown in Fig. 2A, a single protein band of the corresponding molecular mass of NhaS3 was found in the thylakoid membrane fraction, which was identified by the presence of the thylakoid membrane marker proteins NdhD3 and NdhF3 (21Zhang P. Battchikova N. Jansen T. Appel J. Ogawa T. Aro E.M. Plant Cell. 2004; 16: 3326-3340Crossref PubMed Scopus (189) Google Scholar, 30Ohkawa H. Price G.D. Badger M.R. Ogawa T. J. Bacteriol. 2000; 182: 2591-2596Crossref PubMed Scopus (93) Google Scholar). Only a weak signal for NhaS3 was found in the plasma membrane fraction, which was identified by the presence of the nitrate transporter NrtA (20Omata T. Plant Cell Physiol. 1995; 36: 207-213Crossref PubMed Scopus (84) Google Scholar). This indicated that NhaS3 was localized in the thylakoid membrane in Synechocystis. In addition, the subcellular localization of the NhaS3 protein was analyzed by immunogold labeling followed by electron microscopy. A cross-section of wild type Synechocystis cells grown under standard conditions (see "Experimental Procedures") showed gold particles decorating the thylakoid membrane when the IgG fraction of NhaS3 antiserum was used (Fig. 2, B and C). Only a small amount of the label was found on the plasma membrane or in other locations. Control experiments with IgG fraction of preimmune serum did not show any significant labeling (data not shown). These results indicate that NhaS3 is associated with the thylakoid membrane fraction (Fig. 2A). The activity of NhaS3 may be influenced by the proton gradient formed through respiration or photosynthesis across the thylakoid membrane. In E. coli, three Na+/H+ antiporters, NhaA, NhaB, and ChaA, show different pH dependence from each other and also have different physiological roles (31Shijuku T. Saito H. Kakegawa T. Kobayashi H. Biochim. Biophys. Acta. 2001; 1506: 212-217Crossref PubMed Scopus (9) Google Scholar, 32Shijuku T. Yamashino T. Ohashi H. Saito H. Kakegawa T. Ohta M. Kobayashi H. Biochim. Biophys. Acta. 2002; 1556: 142-148Crossref PubMed Scopus (19) Google Scholar). In Synechocystis it has been reported that NhaS3 has antiporter activity at pH 8.5 (6Inaba M. Sakamoto A. Murata N. J. Bacteriol. 2001; 183: 1376-1384Crossref PubMed Scopus (76) Google Scholar), but its detailed properties remain to be studied. To measure the pH dependence of NhaS3, E. coli strain TO114, which possesses low Na+/H+ exchange activity, was used as a host (4Ohyama T. Igarashi K. Kobayashi H. J. Bacteriol. 1994; 176: 4311-4315Crossref PubMed Google Scholar). Inverted membrane vesicles were prepared from E. coli TO114 cells transformed with plasmids encoding NhaS3 or NhaA or the empty plasmid pPAB404. Transport activities were assessed by measuring the dequenching of acridine orange fluorescence upon the addition of 5 mm NaCl at different pH values (Fig. 3A). E. coli NhaA had a peak of activity at pH 8.0 and 8.5, which is consistent with the pH profile of NhaA previously reported (33Inoue H. Sakurai T. Ujike S. Tsuchiya T. Murakami H. Kanazawa H. FEBS Lett. 1999; 443: 11-16Crossref PubMed Scopus (26) Google Scholar). In contrast, NhaS3 showed a similar level of activity across the entire pH spectrum tested (pH 6.5–9.0). This indicates that NhaS3 activity is pH-independent. Na+/H+ antiporter activities affect K+ transport across the membrane and the balance of the cytosolic Na+/K+ concentration ratio (34Serrano R. Rodriguez-Navarro A. Curr. Opin. Cell Biol. 2001; 13: 399-404Crossref PubMed Scopus (234) Google Scholar, 35Shi H. Quintero F.J. Pardo J.M. Zhu J.K. Plant Cell. 2002; 14: 465-477Crossref PubMed Scopus (1016) Google Scholar, 36Venema K. Belver A. Marin-Manzano M.C. Rodríguez-Rosales M.P. Donaire J.P. J. Biol. Chem. 2003; 278: 22453-22459Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 37Yamaguchi T. Apse M.P. Shi H. Blumwald E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12510-12515Crossref PubMed Scopus (148) Google Scholar). To test the influence of NhaS3 on K+ uptake, it was expressed in an E. coli strain, LB2003, lacking three major K+ uptake systems and unable to grow at low K+ concentrations (15Stumpe S. Bakker E.P. Arch. Microbiol. 1997; 167: 126-136Crossref PubMed Scopus (104) Google Scholar). NhaS3 restored growth of the LB2003 strain at low K+ concentrations (10 mm) (Fig. 3B). Under the same conditions, expression of the Synechocystis K+ uptake system, KtrABE (16Matsuda N. Kobayashi H. Katoh H. Ogawa T. Futatsugi L. Nakamura T. Bakker E.P. Uozumi N. J. Biol. Chem. 2004; 279: 54952-54962Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), which was used as a positive control, rescued the mutant. No growth was observed with the empty vector alone. Although the initial net K+ uptake of the cells expressing NhaS3 was higher than that of the cells containing the empty vector (Fig. 3C), NhaS3 did not show K+/H+ exchange activity (Fig. 3D). The measurement was performed under conditions where NhaS3 showed Li+/H+ as well as Na+/H+ antiporter activities, but no Mg2+/H+ antiporter activity (Fig. 3D). These data indicate that NhaS3 either functions as a K+ uptake transporter or that Na+ extrusion mediated by NhaS3 may indirectly increase the influx of K+ into the cells. NhaS3 may contribute to maintaining the balance of the cytosolic Na+/K+ ratio. In the NhaA antiporter protein negatively charged residues in the hydrophobic transmembrane domains play a crucial role for Na+ or H+ electrostatic interaction and ion transport function (3Hunte C. Screpanti E. Venturi M. Rimon A. Padan E. Michel H. Nature. 2005; 435: 1197-1202Crossref PubMed Scopus (539) Google Scholar). Two aspartates, corresponding to Asp217 and Asp218 in NhaS3, are proposed to be the ion binding site in NhaS3 (3Hunte C. Screpanti E. Venturi M. Rimon A. Padan E. Michel H. Nature. 2005; 435: 1197-1202Crossref PubMed Scopus (539) Google Scholar). Another negatively charged residue, glutamate (Glu402), is located in the eleventh hydrophobic domain of NhaS3 but is not conserved in the other Synechocystis Na+/H+ antiporters or in E. coli NhaA (supplemental Fig. S1). To test the role of these charged residues in the transport function of NhaS3, mutant versions of NhaS3 were generated. In these mutant proteins single negatively charged residues (Asp or Glu) were replaced by either a neutral nonpolar residue (alanine) or a neutral polar residue (asparagine or glutamine). The mutant proteins were then expressed in E. coli, and their antiporter activity was determined by fluorescence dequenching in membrane vesicles (Fig. 4A). Amino acid substitutions at Asp217, Asp218, and Glu402 abolished the transport activities of NhaS3 regardless of the replacing amino acid. The protein expression levels of these NhaS3 variants that caused loss of transport activity were verified by Western blotting analysis (Fig. 4B). There was no difference in the amount of protein present, indicating that the loss of activity was not caused by a lack of protein. The lower bands seen on the blots are most likely degradation products of the full-length protein (upper band). Based on data available for NhaA (3Hunte C. Screpanti E. Venturi M. Rimon A. Padan E. Michel H. Nature. 2005; 435: 1197-1202Crossref PubMed Scopus (539) Google Scholar), Asp217, Asp218, and Glu402 are likely to be part of the structure for translocation of Na+/H+. Replacement of Asp99, Glu114, Glu121, and Glu359 caused a marked decrease in the activities only in the case of one of the substituted amino acids (Fig. 4B). In these cases the replacement of the residues affected the transport activity, but the results suggest that they may not be essential for binding or translocation of cations. These results show that the conserved negative residues Asp217 and Asp218 in NhaS3 have the same function as their counterparts in NhaA, which provides further evidence that NhaS3 functions as a Na+/H+ antiporter. Based on the above data on NhaS3-mediated Na+ transport activities, we examined whether NhaS3 contributes to the cellular ion homeostasis in response to environmental stress. It was tested whether the amount of NhaS3 affects the stress tolerance of Synechocystis. An nhaS3 knockdown strain was obtained by insertion of kanamycin resistance gene into nhaS3 (Fig. 5A). The decrease in NhaS3 protein was confirmed by Western blotting (Fig. 5C). A Synechocystis strain overexpressing nhaS3 was generated by integration of a copy of nhaS3 under control of the inducible trc promoter (Fig. 5B). Overexpression of nhaS3 and the increased presence of the NhaS3 protein was confirmed by Western blot (Fig. 5C). Both knockdown cells and cells overexpressing nhaS3 were grown under conditions of salinity stress and hyperosmotic stress (Fig. 5, D and E). The nhaS3 knockdown strain grew less well on solid or liquid medium supplemented with either 500 mm NaCl or 500 mm sorbitol when compared with the growth of wild type or overexpressor. This suggests that NhaS3 contributes to the osmoadaptation of Synechocystis. Overexpression of NhaS3 did not increase the salt or osmotolerance of the cells. In wild type cells the amo
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