Characterization of a Tobacco TPK-type K+ Channel as a Novel Tonoplast K+ Channel Using Yeast Tonoplasts
2007; Elsevier BV; Volume: 283; Issue: 4 Linguagem: Inglês
10.1074/jbc.m708213200
ISSN1083-351X
AutoresShin Hamamoto, Junichiro Marui, Ken Matsuoka, Kyohei Higashi, Kazuei Igarashi, Tsuyoshi Nakagawa, Teruo Kuroda, Yasuo Mori, Yoshiyuki Murata, Yoichi Nakanishi, Masayoshi Maeshima, Isamu Yabe, Nobuyuki Uozumi,
Tópico(s)Polyamine Metabolism and Applications
ResumoThe tonoplast K+ membrane transport system plays a crucial role in maintaining K+ homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K+ channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased ∼2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K+ over Na+. NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca2+, but a cytosolic concentration of 45 μm Ca2+ resulted in a 2-fold increase in the amplitude of the K+ current. Acidification of the cytosol to pH 5.5 also markedly increased NtTPK1-mediated K+ currents. These results show that NtTPK1 is a novel tonoplast K+ channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK. The tonoplast K+ membrane transport system plays a crucial role in maintaining K+ homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K+ channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased ∼2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K+ over Na+. NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca2+, but a cytosolic concentration of 45 μm Ca2+ resulted in a 2-fold increase in the amplitude of the K+ current. Acidification of the cytosol to pH 5.5 also markedly increased NtTPK1-mediated K+ currents. These results show that NtTPK1 is a novel tonoplast K+ channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK. Plants take up potassium (K+) from the soil and plant cells accumulate K+ to regulate the membrane potential and turgor pressure. The cytoplasmic K+ concentration is tightly controlled at ∼100 mm (1Walker D.J. Leigh R.A. Miller A.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10510-10514Crossref PubMed Scopus (336) Google Scholar). Vacuoles are major subcellular reservoirs for controlling K+ homeostasis in plant cells (1Walker D.J. Leigh R.A. Miller A.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10510-10514Crossref PubMed Scopus (336) Google Scholar). During cell expansion, for instance during stomata opening or cell growth, tonoplast transport system moves K+ into the vacuole, whereas, under conditions of salinity stress, K+ is replaced by Na+ (2Pandey S. Zhang W. Assmann S.M. FEBS Lett. 2007; 581: 2325-2336Crossref PubMed Scopus (177) Google Scholar, 3Apse M.P. Blumwald E. FEBS Lett. 2007; 581: 2247-2254Crossref PubMed Scopus (408) Google Scholar, 4Gierth M. Maser P. FEBS Lett. 2007; 581: 2348-2356Crossref PubMed Scopus (312) Google Scholar, 5Lebaudy A. Very A.A. Sentenac H. FEBS Lett. 2007; 581: 2357-2366Crossref PubMed Scopus (251) Google Scholar). Several kinds of genes encoding K+ channels and K+ transporters have been identified in the Arabidopsis thaliana genome, and their function and tissue and cellular distribution have been extensively studied. They consist of two families, the Shaker-type channels, with six hydrophobic transmembrane domains and a single pore domain, and the two-pore K+ channel (TPK) 2The abbreviations used are: TPKtwo-pore K+ channelRACErapid amplification of cDNA endsMES4-morpholineethanesulfonic acidGUSβ-glucuronidase. family, with four transmembrane and two pore domains. Six different genes encoding TPK-type channels are present in A. thaliana. AtTPK4 is targeted to the plasma membrane (6Becker D. Geiger D. Dunkel M. Roller A. Bertl A. Latz A. Carpaneto A. Dietrich P. Roelfsema M.R. Voelker C. Schmidt D. Mueller-Roeber B. Czempinski K. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15621-15626Crossref PubMed Scopus (126) Google Scholar), while the other five, AtTPK1, AtTPK2, AtTPK3, AtTPK5, and AtKCO3, are localized in the vacuolar membrane (7Voelker C. Schmidt D. Mueller-Roeber B. Czempinski K. Plant J. 2006; 48: 296-306Crossref PubMed Scopus (127) Google Scholar). AtTPK1 and AtTPK4 have been functionally characterized. AtTPK4 shows a voltage-independent K+ profile in Xenopus laevis ooctyes and in yeast, and the K+ current is inhibited by extracellular Ca2+ and reduced by shifting the cytosolic pH from 7.5 to 6.3, but is not affected by the external pH (6Becker D. Geiger D. Dunkel M. Roller A. Bertl A. Latz A. Carpaneto A. Dietrich P. Roelfsema M.R. Voelker C. Schmidt D. Mueller-Roeber B. Czempinski K. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15621-15626Crossref PubMed Scopus (126) Google Scholar). AtTPK1 has different properties to AtTPK4 (7Voelker C. Schmidt D. Mueller-Roeber B. Czempinski K. Plant J. 2006; 48: 296-306Crossref PubMed Scopus (127) Google Scholar, 8Bihler H. Eing C. Hebeisen S. Roller A. Czempinski K. Bertl A. Plant Physiol. 2005; 139: 417-424Crossref PubMed Scopus (68) Google Scholar). In the yeast and plant tonoplast membrane, cytosolic Ca2+ enhances AtTPK1 activity, and the optimum cytosolic pH is 6.5 (6Becker D. Geiger D. Dunkel M. Roller A. Bertl A. Latz A. Carpaneto A. Dietrich P. Roelfsema M.R. Voelker C. Schmidt D. Mueller-Roeber B. Czempinski K. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15621-15626Crossref PubMed Scopus (126) Google Scholar, 9Gobert A. Isayenkov S. Voelker C. Czempinski K. Maathuis F.J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 10726-10731Crossref PubMed Scopus (201) Google Scholar, 10Latz A. Becker D. Hekman M. Muller T. Beyhl D. Marten I. Eing C. Fischer A. Dunkel M. Bertl A. Rapp U.R. Hedrich R. Plant J. 2007; 52: 449-459Crossref PubMed Scopus (120) Google Scholar). two-pore K+ channel rapid amplification of cDNA ends 4-morpholineethanesulfonic acid β-glucuronidase. Stomata opening and closing is closely associated with membrane transport of K+. In a study on guard cells, three kinds of tonoplast channels, SV, FV, and VK, were distinguished by their K+ current profiles (11Ward J.M. Pei Z.M. Schroeder J.I. Plant Cell. 1995; 7: 833-844Crossref PubMed Scopus (274) Google Scholar). On the basis of patch clamp recording, the SV channel was recently shown to be AtTPC1 in Arabidopsis (12Peiter E. Maathuis F.J. Mills L.N. Knight H. Pelloux J. Hetherington A.M. Sanders D. Nature. 2005; 434: 404-408Crossref PubMed Scopus (421) Google Scholar), and the VK channel was reported to be AtTPK1 (9Gobert A. Isayenkov S. Voelker C. Czempinski K. Maathuis F.J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 10726-10731Crossref PubMed Scopus (201) Google Scholar, 13Ward J.M. Schroeder J.I. Plant Cell. 1994; 6: 669-683Crossref PubMed Scopus (10) Google Scholar). Further studies on FV, detailed data on SV and VK, and the identification of other TPK-type channels are needed to understand vacuolar K+ transport. To gain insights into the biophysical properties of the TPK-type channels, we isolated the homologous genes from BY-2 cultured tobacco cells and Nicotiana tabacum cv. SR1. The protein sequences revealed a novel feature of the K+ filter in these channels. We also examined promoter activity and the intracellular localization of the channel and performed accurate electrophysiological measurements of NtTPK1 using the vacuolar membrane from enlarged mutant yeast cells with very low background currents, unlike the plant vacuolar membrane. The results showed that NtTPK1 was a novel tonoplast channel with characteristics different from those of previously characterized tonoplast channels. Plant Material—Tobacco BY-2 (N. tabacum cv. Bright Yellow 2) cells were grown in BY-2 growth medium (modified Linsmaier and Skoog medium) (14Nagata T. Nemoto Y. Hasezawa S. Int. Rev. Cytol. 1992; 132: 1-30Crossref Scopus (1027) Google Scholar) at 26 °C in the dark with constant shaking and were maintained by subculturing 1 ml of BY-2 cells into 100 ml of fresh medium every 7 days. Cloning of NtTPK1 Full-length cDNAs from N. tabacum cv. SR1 and BY-2 Cells—A search of the EST data base of tobacco BY-2 cells for sequence homology with the known TPK family (Transcriptome Analysis of BY-2 revealed a 684-bp partial clone which was a homologue of A. thaliana TPK2 (known as KCO2). Total RNA was isolated from N. tabacum cv. SR1 and BY-2 cells using a standard guanidine thiocyanate method (15Goodall G.J. Wiebauer K. Filipowicz W. Methods Enzymol. 1990; 181: 148-161Crossref PubMed Scopus (149) Google Scholar). To obtain the 5′-end of the NtTPK1 cDNA, 5′-RACE was performed as described previously (16Frohman M.A. Methods Enzymol. 1993; 218: 340-356Crossref PubMed Scopus (465) Google Scholar). Polyadenylated RNA was reverse-transcribed using three different NtTPK1-specific antisense primers, 5′-GGATGCAAAGATACGACCGG-3′, 5′-CGCCATACCCCACAGTGGTAAC-3′, and 5′-CAAAATGCGTAACCGG-3′, and RACE amplification primers. First strand cDNA was synthesized using SuperScript™ III Reverse Transcriptase (Invitrogen) and was used as template DNA for the isolation of full-length NtTPK1 cDNA by PCR reactions using primer 5′-ATGGAGAAAGAGCCTCTTC-3′, starting at the start codon, and primer 5′-TTAATGGTGGTGGCTTTCC-3′, starting at the first stop codon. Expression of NtTPK1 in Saccharomyces cerevisiae and Escherichia coli NtTPK1 was amplified by PCR using the EcoRI site-containing sense primer 5′-CAGGAATTCATGGAGAAAGAGCCTCTTC-3′ and the SalI site-containing antisense primer 5′-CAGGTCGACTTAATGGTGGTGGCTTTCC-3′. The PCR fragment was then digested with EcoRI and SalI and ligated into the corresponding sites of the yeast expression vector pKT10 (17Nakanishi Y. Saijo T. Wada Y. Maeshima M. J. Biol. Chem. 2001; 276: 7654-7660Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 18Tanaka K. Nakafuku M. Tamanoi F. Kaziro Y. Matsumoto K. Toh-e A. Mol. Cell. Biol. 1990; 10: 4303-4313Crossref PubMed Scopus (241) Google Scholar). NtTPK1 was ligated into the BamHI and PstI cloning sites in pPAB404 (19Buurman E.T. Kim K.T. Epstein W. J. Biol. Chem. 1995; 270: 6678-6685Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and the resulting plasmid introduced into E. coli strain LB2003, which lacks the three K+ uptake systems Trk, Kup, and Kdp (20Stumpe S. Bakker E.P. Arch. Microbiol. 1997; 167: 126-136Crossref PubMed Scopus (100) Google Scholar). Growth tests of the plasmid-containing E. coli LB2003 at different K+ concentrations were carried out as described previously (21Uozumi N. Nakamura T. Schroeder J.I. Muto S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9773-9778Crossref PubMed Scopus (82) Google Scholar, 22Gambale F. Uozumi N. J. Membr. Biol. 2006; 210: 1-19Crossref PubMed Scopus (88) Google Scholar, 23Uozumi N. Am. J. Physiol. Cell. Physiol. 2001; 281: C733-C739Crossref PubMed Google Scholar). Generation of NtTPK1-overexpressing Tobacco BY-2 Transgenic Lines—Full-length cDNA for NtTPK1 was amplified by PCR using primers 5′-CACCATGGAGAAAGAGCCTCTTC-3′ and 5′-TTAATGGTGGTGGCTTTCC-3′ and subcloned into the pENTR/D TOPO vector (Invitrogen). The cDNA was then transferred into the GATEWAY destination vector pGWB2 containing the CAMV 35S promoter by an LR Clonase reaction (Invitrogen) (24Nakagawa T. Kurose T. Hino T. Tanaka K. Kawamukai M. Niwa Y. Toyooka K. Matsuoka K. Jinbo T. Kimura T. J. Biosci. Bioeng. 2007; 104: 34-41Crossref PubMed Scopus (1210) Google Scholar). The [35S]NtTPK1 construct was introduced into A. tumefaciens EHA101 by electroporation (25Mersereau M. Pazour G.J. Das A. Gene. 1990; 90: 149-151Crossref PubMed Scopus (137) Google Scholar). Stable transformation of BY-2 cells mediated by A. tumefaciens (strain EHA101) was carried out as described by Matsuoka and Nakamura (26Matsuoka K. Nakamura K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 834-838Crossref PubMed Scopus (205) Google Scholar). Cloning of the NtTPK1 Promoter Region for Measurement of Promoter GUS Activity—Tobacco genomic DNA was extracted from tobacco leaves by the CTAB method (27McGarvey P. Kaper J.M. BioTechniques. 1991; 11: 428-432PubMed Google Scholar) and used as the template for the PCR. To isolate the NtTPK1 promoter region, thermal asymmetric interlaced (TAIL) PCR was performed (28Liu Y.G. Whittier R.F. Genomics. 1995; 25: 674-681Crossref PubMed Scopus (1015) Google Scholar). Three nested primers hybridizing to the NtTPK1 cDNA were used: the 1st PCR primer, 5′-GTCAAGAGCAGTGGCAGAAGCTTC-3′; 2nd PCR primer, 5′-CAGAGGTACCAAAGATAAGGCGTTC-3′; and 3rd PCR primer, 5′-TGTTCTGGTGCTGACATAAGG-3′. To confirm the DNA sequence of the NtTPK1 promoter region in the genomic DNA, three upstream primers, 5′-CGTTTATATCGTGTCAACCTTTG-3′,5′-GCGTTCTTGAATCCAAACGAC-3′, and 5′-GCTGGGAGAGTCCTATACCGC-3′, were used with the downstream primer 5′-TATGGGGTTTCGCCGGAAAACGG-3′. The promoter sequence was subcloned into the pENTR/D TOPO vector and transferred to the GATEWAY destination vector pGWB3 (24Nakagawa T. Kurose T. Hino T. Tanaka K. Kawamukai M. Niwa Y. Toyooka K. Matsuoka K. Jinbo T. Kimura T. J. Biosci. Bioeng. 2007; 104: 34-41Crossref PubMed Scopus (1210) Google Scholar). pGWB3 vector harboring the promoter sequence was used to transform BY-2 cells as described above. To measure promoter activity, fluorimetric determination of GUS activity was performed as reported previously (29Jefferson R.A. Kavanagh T.A. Bevan M.W. EMBO J. 1987; 6: 3901-3907Crossref PubMed Scopus (8352) Google Scholar), using 4-methylumbelliferyl-β-d-glucuronide as substrate. BY-2 cells grown in BY-2 growth medium for 5 days were transferred for 10 min to BY-2 growth medium containing 250 mm NaCl or 500 mm mannitol, harvested by centrifugation, rapidly frozen in liquid nitrogen, and stored at –80 °C until analyzed. Three replicates were used for each treatment. GUS activity was measured at an excitation wavelength of 365 nm and emission wavelength of 455 nm on a spectrofluorometer (JASCO, Japan). Protein was determined by the method of Bradford (30Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar), and the specific GUS activity determined as the rate of increase in fluorescence of 4-methylumbelliferone (4-MU) divided by the protein concentration (pmol of 4-MU/h/μg protein). Quantitative Real-time PCR Assay—Expression was measured in different tissues of N. tabacum cv. SR1 using One Step SYBR RT-PCR (Takara, Japan), a method that relies on real-time monitoring of the release of a fluorescent reporter dye (SYBR-Green I) as the PCR product accumulates in the reaction mix (31Bustin S.A. J. Mol. Endocrinol. 2000; 25: 169-193Crossref PubMed Scopus (3076) Google Scholar). The cDNA was amplified using an ABI Prism 7500 (Applied Biosystems). Amplification of tubulin cDNA under identical conditions was performed as an internal control to normalize cDNA levels. Preparation of BY-2 and Yeast Vacuolar Membranes—Vacuolar membranes were prepared from BY-2 cells as described previously (32Maeshima M. Hara-Nishimura I. Takeuchi Y. Nishimura M. Plant Physiol. 1994; 106: 61-69Crossref PubMed Scopus (74) Google Scholar), except for a few modifications. All steps were performed at 4 °C. The cells were ground in a mortar with liquid nitrogen and grinding medium (0.25 mm sorbitol, 1 mm MgCl2, 2 mm EGTA, 0.5 mm phenylmethylsulfonyl fluoride, 1% (w/v) PVP-40, and 50 mm Tris acetate, pH 7.5). The frozen cell homogenates were thawed and centrifuged at 5,000 × g for 10 min, then the supernatant was centrifuged at 120,000 × g for 50 min, and the final precipitate suspended in 0.75 mm sucrose in 20 mm Tris acetate, pH 7.5, 2 mm dithiothreitol, 1 mm EGTA, and 1 mm MgCl2 (Tris-DEM) (microsomal membrane fraction). Further purification of vacuolar membranes was carried out by floating centrifugation. The microsomal membrane suspension was placed in a centrifuge tube and overlaid with the same volume of Tris-DEM buffer containing 0.25 mm sorbitol. After centrifugation at 120,000 × g for 30 min, the vacuolar membrane vesicles, which formed a band at the interface between the two solutions, were collected and suspended in 15 ml of Tris-DEM buffer containing 0.25 mm sorbitol. The suspension was centrifuged at 120,000 × g for 30 min and the pellet suspended in a small volume of the same buffer. Yeast vacuoles were prepared using the floating centrifugation method (33Yoshihisa T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1988; 263: 5158-5163Abstract Full Text PDF PubMed Google Scholar, 34Nakanishi Y. Yabe I. Maeshima M. J Biochem. (Tokyo). 2003; 134: 615-623Crossref PubMed Scopus (30) Google Scholar). For immunoblotting, NtTPK1-transformed S. cerevisae strain BJ5458 (Matα, ura3–52, trp1, lys2–801, leu2Δ1, his3Δ200, pep4::HIS3, prbΔ1.6R can1, GAL), lacking two major vacuolar proteases (17Nakanishi Y. Saijo T. Wada Y. Maeshima M. J. Biol. Chem. 2001; 276: 7654-7660Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), was used. Immunoblotting—An anti-NtTPK1 antibody was raised against a synthetic peptide containing the sequence NH2-CGRITLADLMESHHH-COOH from the C terminus of NtTPK1 (Operon Biotechnologies, Japan). Antibodies against YVC1, a marker for the S. cerevisiae tonoplast, were raised against the peptide NH2-CNLTAVITDLLEKLDIKDKKECOOH located at the C terminus of YVC1 (35Palmer C.P. Zhou X.L. Lin J. Loukin S.H. Kung C. Saimi Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7801-7805Crossref PubMed Scopus (173) Google Scholar). Polyclonal antibodies raised against v-PPase (34Nakanishi Y. Yabe I. Maeshima M. J Biochem. (Tokyo). 2003; 134: 615-623Crossref PubMed Scopus (30) Google Scholar), Sec61 alpha (36Yuasa K. Toyooka K. Fukuda H. Matsuoka K. Plant J. 2005; 41: 81-94Crossref PubMed Scopus (105) Google Scholar), or PAQ2 (plasma membrane aquaporin of Raphanus sativus) (37Suga S. Imagawa S. Maeshima M. Planta. 2001; 212: 294-304Crossref PubMed Scopus (74) Google Scholar) were used to identify the BY-2 cell tonoplast, plasma membrane, or endoplasmic reticulum (ER) membrane, respectively. Proteins were electrophoresed on SDS-10% polyacrylamide gels, then transferred to polyvinylidene fluoride membranes. The polyvinylidene difluoride membrane was blocked with 10% skim milk in PBS-T (140 mm NaCl, 16.0 mm Na2HPO4, 2.00 mm KH2PO4, 3.75 mm KCl, 0.1% Tween 20) (blocking buffer) for 1 h at room temperature, then incubated for 1 h at room temperature with primary antibody (1:4000 in blocking buffer), washed three times with PBS-T, incubated for 30 min at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (GE Healthcare) (1:5000 in blocking buffer), and subjected to chemiluminescence detection (ECL, Amersham Biosciences). Chemiluminescence signals on the polyvinylidene difluoride membrane were recorded using an LAS 3000 imaging system (Fujifilm, Japan). Generation of the YVC1 Knock-out Strain—Deletion of the YVC1 gene in the strain BJ5458 was achieved by homologous recombination of the integration cassette with the LEU2 marker, amplified from the plasmid pUG73 (38Gueldener U. Heinisch J. Koehler G.J. Voss D. Hegemann J.H. Nucleic Acids Res. 2002; 30: e23Crossref PubMed Scopus (758) Google Scholar). The linearized LEU2 PCR product was used for yeast transformation as described previously (39Gietz R.D. Schiestl R.H. Willems A.R. Woods R.A. Yeast. 1995; 11: 355-360Crossref PubMed Scopus (1712) Google Scholar). We named the YVC1-deleted strain SH1006. Preparation of Giant Yeast Cells and Isolation of Giant Yeast Vacuoles—Preparation of giant yeast from the yvc1 mutant. SH1006 was performed as described previously (34Nakanishi Y. Yabe I. Maeshima M. J Biochem. (Tokyo). 2003; 134: 615-623Crossref PubMed Scopus (30) Google Scholar, 40Yabe I. Horiuchi K. Nakahara K. Hiyama T. Yamanaka T. Wang P.C. Toda K. Hirata A. Ohsumi Y. Hirata R. Anraku Y. Kusaka I. J. Biol. Chem. 1999; 274: 34903-34910Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Briefly, at the early log phase (OD660 of 0.2), 1 ml of cells was incubated for 10 min at 30 °C in 0.1 m Tris-HCl, pH 9.4, 50 mm 2-mercaptoethanol and 0.1 m glucose, then for 15 min at 30 °C with 1 mg/ml of Zymolyase 20T in 1 m sorbitol, 2% (w/v) glucose, 50 mm Tris-HCl, pH 7.5, 0.17% yeast nitrogen base without amino acids and ammonium sulfate, and 0.25× dropout solution composed of all amino acids and adenines (17Nakanishi Y. Saijo T. Wada Y. Maeshima M. J. Biol. Chem. 2001; 276: 7654-7660Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The spheroplasts formed were obtained as described previously (34Nakanishi Y. Yabe I. Maeshima M. J Biochem. (Tokyo). 2003; 134: 615-623Crossref PubMed Scopus (30) Google Scholar). Giant vacuoles were prepared from giant spheroplasts by the osmotic shock method with the following modifications (34Nakanishi Y. Yabe I. Maeshima M. J Biochem. (Tokyo). 2003; 134: 615-623Crossref PubMed Scopus (30) Google Scholar, 40Yabe I. Horiuchi K. Nakahara K. Hiyama T. Yamanaka T. Wang P.C. Toda K. Hirata A. Ohsumi Y. Hirata R. Anraku Y. Kusaka I. J. Biol. Chem. 1999; 274: 34903-34910Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The spheroplasts were concentrated by floating centrifugation at 1,000 × g for 5 min, then diluted with 5 volumes of a hypotonic solution (0.15 m sorbitol, 0.1 m KCl, 1 mm MgCl2 20 mm Tris-MES, pH 7.5) in the recording chamber of the patch-clamp apparatus. Electrophysiological Experiments—For electrophysiological analysis, NtTPK1 was expressed from the pKT10 plasmid in the YVC1-lacking yeast strain SH1006. Currents were recorded in the whole vacuole mode, as described previously (40Yabe I. Horiuchi K. Nakahara K. Hiyama T. Yamanaka T. Wang P.C. Toda K. Hirata A. Ohsumi Y. Hirata R. Anraku Y. Kusaka I. J. Biol. Chem. 1999; 274: 34903-34910Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 41Kuroda T. Okuda N. Saitoh N. Hiyama T. Terasaki Y. Anazawa H. Hirata A. Mogi T. Kusaka I. Tsuchiya T. Yabe I. J. Biol. Chem. 1998; 273: 16897-16904Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). All experiments were carried out using the standard patch-clamp technique at 25 °C. Measurement of Polyamine Levels in Tobacco Plants—Different tissues (leaf, shoot, and root) collected from 3–4-week-old tobacco plants were ground in an Eppendorf tube and extracted for 2 h at 70°C with 5% (w/v) trichloroacetic acid. Levels of different polyamines were determined by HPLC as described previously (42Igarashi K. Kashiwagi K. Hamasaki H. Miura A. Kakegawa T. Hirose S. Matsuzaki S. J. Bacteriol. 1986; 166: 128-134Crossref PubMed Scopus (159) Google Scholar). Protein was determined using the Bradford assay (30Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Cloning of NtTPK1—We searched the EST data base of tobacco BY-2 cells for sequence homology with the known TPK family (43Czempinski K. Zimmermann S. Ehrhardt T. Muller-Rober B. EMBO J. 1997; 16: 2565-2575Crossref PubMed Scopus (138) Google Scholar) and identified a partial cDNA homologous sequence in the data base and isolated the upstream cDNA by 5′-RACE and PCR. The isolated tobacco homologous gene was named NtTPK1. The NtTPK1 cDNA contained an open reading frame of 1284 bp encoding a 428-amino acid protein (47.6 kDa) (GenBank™ accession number EU161633) (Fig. 1). In a further cloning approach, we isolated three more cDNAs from BY-2 cells and one from N. tabacum cv. SR1. The sequence of the cDNA from N. tabacum cv. SR1 was identical to the NtTPK1 sequence in the data base. The three sequences from BY-2 cells contained some amino acid differences compared with that of NtTPK1 and were named NtTPK1a, NtTPK1b, and NtTPK1c (Fig. 1). These different sequences were caused by an amphidiploid nature of tobacco plant. N. tabacum cv. SR1 is an amphidiploid species derived from ancestors related the Nicotiana sylvestris and Nicotiana tomentosiformis, which are both diploid species. Analysis of the predicted amino acid sequence for NtTPK1 showed 62, 60, and 60% identity with those of A. thaliana AtTPK2 (formerly KCO2), AtTPK3 (formerly KCO6), and AtTPK5 (formerly KCO5), respectively; in contrast, the percentage identity with AtTPK1 and AtTPK4 was 39 and 40%, respectively (supplemental Fig. 1. The phylogenetic tree is shown in supplemental Fig. 2.) The hydropathy profile of NtTPK1, generated using the Kyte and Doolittle algorithm (44Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17296) Google Scholar) predicted four potential transmembrane domains, designated M1–M4 (Fig. 1). The predicted protein sequence contained two pore (P) domains, P1, located between M1 and M2, and P2, located between M3 and M4. In the two P domains of NtTPK1, the K+ channel-selective motif (TXGYGD) (45Heginbotham L. Abramson T. MacKinnon R. Science. 1992; 258: 1152-1155Crossref PubMed Scopus (360) Google Scholar, 46Heginbotham L. Lu Z. Abramson T. MacKinnon R. Biophys. J. 1994; 66: 1061-1067Abstract Full Text PDF PubMed Scopus (690) Google Scholar) was conserved, whereas in NtTPK1, NtTPK1b, and NtTPK1c, several amino acid substitutions were found. Interestingly, NtTPK1b and NtTPK1c contained, respectively, VHG and GHG at the GYG site in P2. Analysis of the NtTPK1 sequence using the PROSITE pattern search data base showed a single EF-hand motif in its C-terminal region. The EF-hand, a structure common to several calcium-binding proteins, consists of two perpendicular 10–12-residue α helices separated by a 12-residue loop region, forming a single calcium-binding site (helix-loop-helix) (47Strynadka N.C. James M.N. Annu. Rev. Biochem. 1989; 58: 951-998Crossref PubMed Google Scholar). AtTPK1, AtTPK2, and AtTPK3 contain one (AtTPK2 and 3) or two (AtTPK1) EF-hands located in the C-terminal domain, while AtTPK4 has none (6Becker D. Geiger D. Dunkel M. Roller A. Bertl A. Latz A. Carpaneto A. Dietrich P. Roelfsema M.R. Voelker C. Schmidt D. Mueller-Roeber B. Czempinski K. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15621-15626Crossref PubMed Scopus (126) Google Scholar). Heterologous Expression of NtTPK1 in E. coli—We expressed NtTPK1 in X.laevis oocyte and the K+ uptake-deficient S. cerevisiae CY162, but did not observe any K+ transport activity (data not shown). This suggested that NtTPK1 might not be targeted to the plasma membrane, but to a subcellular membrane. Functional expression of plant K+ transporters in E. coli has been described previously (21Uozumi N. Nakamura T. Schroeder J.I. Muto S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9773-9778Crossref PubMed Scopus (82) Google Scholar). This expression system allows measurement of the K+ uptake activity of plant endosome membrane-associated K+ channels/transporters (23Uozumi N. Am. J. Physiol. Cell. Physiol. 2001; 281: C733-C739Crossref PubMed Google Scholar). NtTPK1, NtTPK1a, NtTPK1b, and NtTPK1c were expressed in E. coli strain LB2003, which is deficient in all three major K+ uptake systems (20Stumpe S. Bakker E.P. Arch. Microbiol. 1997; 167: 126-136Crossref PubMed Scopus (100) Google Scholar) and the growth of the cells was examined at limiting K+ concentrations. As shown in Fig. 2A, all four sets of cells grew as well as LB2003 cells expressing the Arabidopsis K+ channel KAT1 (pPAB404-KAT1), whereas cells transformed with empty vector (pPAB404) did not grow. These data suggested that NtTPK1, NtTPK1a, NtTPK1b, and NtTPK1c could take up K+ in the E. coli mutant. The ability of NtTPK1b and NtTPK1c to rescue the K+ uptake deficiency of E. coli suggested that the second signature sequence was not necessary for K+ uptake by two-pore K+ channels. To assess this hypothesis, we introduced substitutions in the GYG sequence in P1 and/or P2 of NtTPK1 (Fig. 1A); these were Y193H (P1-Y193H), G192A+Y193A+G194A (P1-AAA), and Y309H in P1 (P2-Y309H) and G308A+Y309A+G310A in P2 (P2-AAA), and the two double replacements, P1-Y193H/P2-Y309H and P1-AAA/P-2AAA (Fig. 2, B and C). Fig. 2B shows that P2-Y309H and P2-AAA allowed growth of LB2003, whereas P1-Y193H, P1-AAA, P1-Y193H/P2-Y309H, and P1-AAA/P-2AAA did not complement the K+ uptake deficiency. These results indicated that the first selectivity filter of the GYG sequence is absolutely required, but the second less important for K+ passage in NtTPK1 (Fig. 2C). Osmotic Stress-dependent Expression of NtTPK1 and Tissue Specificity of Expression—Three distinct NtTPK1 promoter sequences were isolated from N. tabacum cv. SR1 genomic DNA using the TAIL-PCR method. The length of the three sequences upstream of the NtTPK1 initiation codon was 1,101, 1,296, and 1,964 bp, and they were designated as promoter-A, promoter-B, and promoter-C, respectively. Promoter-A and promoter-B shared the same 327 bp sequence (–327 to –1) and all three promot
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