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• W2057487010 abstract "The tnaT gene ofSymbiobacterium thermophilum encodes a protein homologous to sodium-dependent neurotransmitter transporters. Expression of the tnaT gene product inEscherichia coli conferred the ability to accumulate tryptophan from the medium and the ability to grow on tryptophan as a sole source of carbon. Transport was Na+-dependent and highly selective. TheKm for tryptophan was ∼145 nm, and tryptophan transport was unchanged in the presence of 100 μm concentrations of other amino acids. Tryptamine and serotonin were weak inhibitors with KI values of 200 and 440 μm, respectively. By using a T7 promoter-based system, TnaT with an N-terminal His6 tag was expressed at high levels in the membrane and was purified to near-homogeneity in high yield. The tnaT gene ofSymbiobacterium thermophilum encodes a protein homologous to sodium-dependent neurotransmitter transporters. Expression of the tnaT gene product inEscherichia coli conferred the ability to accumulate tryptophan from the medium and the ability to grow on tryptophan as a sole source of carbon. Transport was Na+-dependent and highly selective. TheKm for tryptophan was ∼145 nm, and tryptophan transport was unchanged in the presence of 100 μm concentrations of other amino acids. Tryptamine and serotonin were weak inhibitors with KI values of 200 and 440 μm, respectively. By using a T7 promoter-based system, TnaT with an N-terminal His6 tag was expressed at high levels in the membrane and was purified to near-homogeneity in high yield. γ-aminobutyric acid 5-hydroxytryptamine neurotransmitter:sodium symporter 2,4-dinitrophenol N,N′-dicyclohexylcarbodiimide isopropyl-1-thio-β-d-galactopyranoside serotonin transporter transmembrane dopamine transporter norepinephrine transporter Transporters responsible for reuptake of neurotransmitters across the plasma membrane of neurons and glia fall into two gene families (1Amara S. Nature. 1992; 360: 420-421Crossref PubMed Scopus (48) Google Scholar). The majority of small neurotransmitters, including glycine, γ-aminobutyric acid (GABA),1 dopamine, norepinephrine, and 5-hydroxytryptamine (5-HT, serotonin), are transported by proteins belonging to the family designated the neurotransmitter:sodium symporter (NSS) family 2.A.22 by Saier (2Saier M.H. J. Cell. Biochem. 1999; 75 Suppl. 32: 84-94Crossref Google Scholar). Glutamate, however, is transported by a family of mono- and dicarboxylic amino acid transporters, the dicarboxylate/amino acid:cation symporters family (2Saier M.H. J. Cell. Biochem. 1999; 75 Suppl. 32: 84-94Crossref Google Scholar). Proteins in both families play important roles in brain function as indicated by the profound behavioral effects of drugs that influence their activity, such as cocaine and amphetamines, which interact with amine transporters in the NSS family (3Ritz M.C. Lamb R.J. Goldberg S.R. Kuhar M.J. Science. 1987; 237: 1219-1223Crossref PubMed Scopus (2018) Google Scholar, 4Wall S.C. Innis R.B. Rudnick G. Mol. Pharmacol. 1993; 43: 264-270PubMed Google Scholar, 5Giros B. Jaber M. Jones S.R. Wightman R.M. Caron M.G. Nature. 1996; 379: 606-612Crossref PubMed Scopus (2033) Google Scholar, 6Rocha B.A. Fumagalli F. Gainetdinov R.R. Jones S.R. Ator R. Giros B. Miller G.W. Caron M.G. Nat. Neurosci. 1998; 1: 132-137Crossref PubMed Google Scholar, 7Wall S.C. Gu H.H. Rudnick G. Mol. Pharmacol. 1995; 47: 544-550PubMed Google Scholar, 8Sulzer D. Chen T.K. Lau Y.Y. Kristensen H. Rayport S. Ewing A. J. Neurosci. 1995; 15: 4102-4108Crossref PubMed Google Scholar, 9Sitte H.H. Huck S. Reither H. Boehm S. Singer E.A. Pifl C. J. Neurochem. 1998; 71: 1289-1297Crossref PubMed Scopus (176) Google Scholar, 10Jones S.R. Gainetdinov R.R. Wightman R.M. Caron M.G. J. Neurosci. 1998; 18: 1979-1986Crossref PubMed Google Scholar, 11Pifl C. Agneter E. Drobny H. Sitte H.H. Singer E.A. Neuropharmacology. 1999; 38: 157-165Crossref PubMed Scopus (33) Google Scholar, 12Pifl C. Sitte H.H. Reither H. Singer E.A. Pure Appl. Chem. 2000; 72: 1045-1050Crossref Scopus (4) Google Scholar), and many antidepressant drugs that inhibit serotonin and norepinephrine transporters (13Hrdina P.D. Foy B. Hepner A. Summers R.J. J. Pharmacol. Exp. Ther. 1990; 252: 410-418PubMed Google Scholar, 14Asberg M. Martensson B. Clin. Neuropharmacol. 1993; 16: S32-S44PubMed Google Scholar, 15Briley M. Moret C. Clin. Neuropharmacol. 1993; 16: 387-400Crossref PubMed Scopus (192) Google Scholar, 16Tatsumi M. Groshan K. Blakely R.D. Richelson E. Eur. J. Pharmacol. 1997; 340: 249-258Crossref PubMed Scopus (755) Google Scholar, 17Charney D.S. J. Clin. Psychiatry. 1998; 59: 11-14PubMed Google Scholar).Among the sequences found to be homologous to the NSS family of transporters are a number of “orphan” transporters, for which no function is known. These orphans include v7-3 (18Uhl G. Kitayama S. Gregor P. Nanthakumar E. Persico A. Shimada S. Mol. Brain Res. 1992; 16: 353-359Crossref PubMed Scopus (50) Google Scholar), NTT4 (19Liu Q. Mandiyan S. Lopez-Corcuera B. Nelson H. Nelson N. FEBS Lett. 1993; 315: 114-118Crossref PubMed Scopus (53) Google Scholar, 20el Mestikawy S. Giros B. Pohl M. Hamon M. Kingsmore S. Seldin M. Caron M. J. Neurochem. 1994; 62: 445-455Crossref PubMed Scopus (38) Google Scholar),inebriated (21Soehnge H. Huang X. Becker M. Whitley P. Conover D. Stern M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13262-13267Crossref PubMed Scopus (33) Google Scholar), blot (22Johnson K. Knust E. Skaer H. Dev. Biol. 1999; 212: 440-454Crossref PubMed Scopus (18) Google Scholar), and NTT5 (23Farmer M.K. Robbins M.J. Medhurst A.D. Campbell D.A. Ellington K. Duckworth M. Brown A.M. Middlemiss D.N. Price G.W. Pangalos M.N. Genomics. 2000; 70: 241-252Crossref PubMed Scopus (36) Google Scholar), among others. The largest number of orphan sequences in this family is found in prokaryotic organisms. Although these orphan sequences are highly similar to those encoding functional transporters, it is possible that these proteins fulfill other functions. For example, within the ATP-binding cassette family of transporters are the sulfonylurea receptor (24Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P.T. Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1278) Google Scholar) and the cystic fibrosis transmembrane regulator chloride channel (25Riordan J.R. Chang X.-B. Biochim. Biophys. Acta. 1992; 1101: 221-222Crossref PubMed Google Scholar). In the dicarboxylate/amino acid:cation symporters neurotransmitter transporter family is EAAT4, a ligand-gated ion channel (26Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. Nature. 1995; 375: 599-603Crossref PubMed Scopus (1006) Google Scholar); SGLT3, a member of the sodium:solute symporter (SSS) sugar transporter family, also is not a transporter but rather a glucose-gated ion channel. 2A. Diez-Sampedro, B. A. Hirayama, E. M. Wright, and H. Koepsell, personal communication. 2A. Diez-Sampedro, B. A. Hirayama, E. M. Wright, and H. Koepsell, personal communication.Moreover, some proteins, such as adenylate cyclase (28Barzu O. Danchin A. Prog. Nucleic Acids Res. Mol. Biol. 1994; 49: 241-283Crossref PubMed Scopus (59) Google Scholar) and patched (29Hooper J.E. Scott M.P. Cell. 1989; 59: 751-765Abstract Full Text PDF PubMed Scopus (335) Google Scholar) also have 12 transmembrane segments but no known transport function. For the orphan transporters in the NSS family, it is important to know if any of the newly discovered prokaryotic sequences actually encode functional transporters.Symbiobacterium thermophilum is a symbiotic thermophile, the growth of which is dependent on co-culture with an associatedBacillus strain (30Ohno M. Shiratori H. Park M.J. Saitoh Y. Kumon Y. Yamashita N. Hirata A. Nishida H. Ueda K. Beppu T. Int. J. Syst. Evol. Microbiol. 2000; 50: 1829-1832Crossref PubMed Scopus (69) Google Scholar, 31Suzuki S. Horinouchi S. Beppu T. J. Gen. Microbiol. 1988; 134: 2353-2362Google Scholar). The 16 S rDNA-based taxonomy showed that S. thermophilum occupies a novel phylogenetic branch in the Gram-positive group without clustering with any other genus (30Ohno M. Shiratori H. Park M.J. Saitoh Y. Kumon Y. Yamashita N. Hirata A. Nishida H. Ueda K. Beppu T. Int. J. Syst. Evol. Microbiol. 2000; 50: 1829-1832Crossref PubMed Scopus (69) Google Scholar). This bacterium produces a thermostable tryptophanase directed by the tna1 gene which, when cloned (32Hirahara T. Horinouchi S. Beppu T. Appl. Microbiol. Biotechnol. 1993; 39: 341-346Crossref PubMed Scopus (22) Google Scholar), was found to be part of a tna operon with an unusual gene organization. The operon differs from the conserved structure among enterobacteriaceae in that it consists of three open reading frames (33Martin K. Morlin G. Smith A. Nordyke A. Eisenstark A. Golomb M. J. Bacteriol. 1998; 180: 107-118Crossref PubMed Google Scholar). Furthermore, in the region downstream from the tryptophanase gene, this unique bacterium appears to encode a transporter, TnaT, that belongs, based on sequence homology, to the NSS family. 3K. Ueda and T. Beppu, GenBankTM accession number BAA24689.2.3K. Ueda and T. Beppu, GenBankTM accession number BAA24689.2. In this communication, we demonstrate that the tnaT gene encodes a Na+-dependent tryptophan transporter and that this transporter can be expressed in the cell membrane and purified in high yield.RESULTSS. thermophilum is a symbiotic thermophile (30Ohno M. Shiratori H. Park M.J. Saitoh Y. Kumon Y. Yamashita N. Hirata A. Nishida H. Ueda K. Beppu T. Int. J. Syst. Evol. Microbiol. 2000; 50: 1829-1832Crossref PubMed Scopus (69) Google Scholar) that produces thermostable tryptophanase and β-tyrosinase enzymes (32Hirahara T. Horinouchi S. Beppu T. Appl. Microbiol. Biotechnol. 1993; 39: 341-346Crossref PubMed Scopus (22) Google Scholar,36Hirahara T. Suzuki S. Horinouchi S. Beppu T. Appl. Environ. Microbiol. 1992; 58: 2633-2642Crossref PubMed Google Scholar). In cloning the tryptophanase gene from this organism, we discovered a downstream sequence with high homology to mammalian neurotransmitter transporters. The sequence was deposited as AB010832and identified as a putative tryptophan transporter by virtue of its location in the tryptophanase operon and its similarity to other transporters in the sodium-dependent neurotransmitter transporter family. Fig. 1 shows an alignment of the sequence with the full consensus sequence of the NSS family (pfam00209) from the Conserved Domain data base (37Marchler-Bauer A. Panchenko A.R. Shoemaker B.A. Thiessen P.A. Geer L.Y. Bryant S.H. Nucleic Acids Res. 2002; 30: 281-283Crossref PubMed Scopus (525) Google Scholar) and with the sequence of rat serotonin transporter (SERT), whose substrate, 5-HT, is closest to tryptophan among the known neurotransmitter transporters.Wild type E. coli cells express endogenous transporters capable of catalyzing tryptophan influx. Fig.2 shows a time course of tryptophan accumulation by E. coli K12 (open circles). To analyze tryptophan transport resulting from expression of the S. thermophilum tnaT gene, we used E. coli strain CY15212, obtained from the Yale E. coli Stock Center (CGSC 7672). This strain is inactivated in the three genes, mtr,tnaB, and aroP, that encode tryptophan transporters (38Yanofsky C. Horn V. Gollnick P. J. Bacteriol. 1991; 173: 6009-6017Crossref PubMed Google Scholar). As shown in Fig. 2 (squares), this strain is incapable of accumulating [3H]tryptophan. However, transformation of CY15212 with an expression plasmid encoding theS. thermophilum tnaT gene led to robust [3H]tryptophan uptake, as shown in Fig. 2 (filled circles).Figure 2Time course of tryptophan uptake.l-[5-3H]Tryptophan transport at room temperature was measured at the indicated times as described under “Materials and Methods.” The results are presented as the amount ofl-[5-3H]tryptophan taken up by the cells per mg of cell protein. Open circles, wild type E. coli K12 cells; squares, CY15212 cells; filled circles, CY15212 cells transformed with a plasmid encoding thetnaT gene. Data are means ± S.D. from six measurements in three separate experiments.View Large Image Figure ViewerDownload (PPT)A consequence of tryptophan uptake by cells expressing TnaT is that the transporter facilitated growth of cells on tryptophan-containing minimal media. Fig. 3 shows the dramatic increase in growth by BL21 cells transformed with pET26-TnaT over the first 12 h of incubation (filled circles) in contrast to the relatively slow growth in Trp-free medium or by control cells lacking the tnaT insert. The increase in cell growth indicates that tryptophan taken up by cells is accumulated within the cell where it can be metabolized and is not merely bound to the cell surface.Figure 3Stimulation of growth on tryptophan by TnaT. After an overnight aerobic culture of E. coliBL21(DE3) harboring pET-TnaT in LB medium, the cells were harvested by centrifugation followed by one wash with distilled water. The cells were then inoculated into Trp minimal medium containing (per liter); tryptophan, 5 g; K2HPO4, 11.2 g; KH2PO4, 4.8 g; (NH4)2SO4, 2 g; MgSO4·7H2O, 0.25 g; NaCl 0.5 g; FeSO4·7H2O, 0.5 mg; yeast extract (Difco) 0.05 g; and 1 mm IPTG at ∼5 × 105cells/ml and cultured aerobically at 37 °C for 2 days. Growth was measured by direct cell counting. Control BL21(DE3) cells harboring pET26b were used as a control.View Large Image Figure ViewerDownload (PPT)Trp transport by TnaT was saturable, as shown in Fig.4. Under the conditions used, theVmax for transport was 242 ± 9 pmol per min per mg of cell protein, and the Km was 145 ± 14 nm. The inset of Fig. 4 shows an Eadie-Hofstee (39Hofstee B.H.J. Science. 1952; 116: 329-331Crossref PubMed Scopus (490) Google Scholar) transformation of the transport rate data. The rate shows simple saturation with tryptophan concentration.Figure 4Saturation of tryptophan transport rate.CY15212 cells expressing TnaT were incubated with the indicated concentrations of l-[5-3H]tryptophan for 20 s at room temperature. Accumulation of tryptophan was determined as described under “Materials and Methods.” A nonlinear least squares fit of the data yielded a Km of 145 ± 14 nm and a Vmax of 242 ± 9 pmol per min per mg of cell protein. Inset, linear transformation of the data by the method of Hofstee (39Hofstee B.H.J. Science. 1952; 116: 329-331Crossref PubMed Scopus (490) Google Scholar). Data are means ± S.D. from six measurements in three separate experiments.View Large Image Figure ViewerDownload (PPT)To test the specificity of tnaT-encoded tryptophan transport, we measured the initial rate of transport in the presence of 100 μm concentrations of the 20 naturally occurring amino acids and also cystine, trans-proline, tryptamine, and serotonin. The results are shown in Fig. 5. Aside from tryptophan, none of the amino acids tested significantly inhibited tryptophan influx. A small inhibition was observed with tryptamine and serotonin, and the concentrations of these amines that inhibited influx by 50% were found to be 200 ± 18 and 440 ± 16 μm, respectively (not shown). With one exception, inhibitors of mammalian biogenic amine transporters also failed to block TnaT-mediated tryptophan transport. The following compounds failed to inhibit at 100 μm (not shown): imipramine, desipramine, fluoxetine, citalopram, nomifensine, mazindol, GBR-12909, GBR-12935, amphetamine, and 3,4-methylenedioxymethamphetamine. Cocaine (1 mm) also did not inhibit (not shown). Sertraline, a serotonin reuptake inhibitor with nm affinity, inhibited tryptophan uptake with aKI of 98 ± 3 μm (not shown).Figure 5Substrate specificity of TnaT. CY15212 cells expressing TnaT were incubated withl-[5-3H]tryptophan in the absence (control) or presence of 100 μm of the indicated reagents. (The single letters correspond to individual amino acids according to the standard single letter code.) The incubation time was 20 s at room temperature, as described under “Materials and Methods.” Data are presented as the percentage of the control transport rate. Data are means ± S.D. from four measurements in two separate experiments.View Large Image Figure ViewerDownload (PPT)In TM1 of all amino acid transporters in the NSS family, there is a glycine residue that is replaced by aspartate in the biogenic amine transporters. In TnaT, the corresponding residue is a glycine at position 24. Mutation of this residue to an aspartate led to a transporter that was unable to transport tryptophan (Fig.6A), serotonin, or 1-methyl-4-phenylpyridinium (not shown). This was not due to a block in expression because the His6-tagged G24D mutant was expressed in the membrane of CY15212 cells at 69 ± 15% (n = 3) of the level of wild type His6-tagged TnaT (Fig. 6B).Figure 6Tryptophan uptake and immunoblotting of wild type and G24D His6-TnaT expressed in the membrane of CY15212. His6-tagged wild type (WT) and G24D TnaT were expressed in CY15212 cells using the pQE82–6xHis-TnaT construct as described under “Materials and Methods.” A,l-[5-3H]tryptophan accumulation into CY15212 cells expressing His6-tagged wild type and G24D TnaT performed in parallel with samples used for the immunoblot inB. Total uptake in the wild type was ∼10 pmol per min per mg of total cell protein. B, membranes were prepared as described under “Materials and Methods,” and after solubilization the supernatant fluid was concentrated using nickel-Sepharose, and a representative immunoblot of the eluted material is shown in duplicate.View Large Image Figure ViewerDownload (PPT)A defining characteristic of the sodium-dependent neurotransmitter transporter family is the requirement for sodium ions. In almost all of the family members studied, sodium is required, and the transmembrane sodium gradient provides a driving force for solute accumulation. In many of the transporters in this family, chloride ion is also required, and the Cl− gradient also provides part of the driving force. The data in Fig. 7show that TnaT absolutely requires Na+ but not Cl− for transport. Transport was minimal below 0.1 mm Na+ and was half-maximal at ∼1 mm Na+. The inset shows that influx was essentially the same in NaCl and sodium isethionate medium but was not detectable in medium in whichN-methyl-d-glucamine-Cl replaced NaCl. Thus, tryptophan transport catalyzed by the tnaT gene product was Na+-dependent but not Cl−-dependent.Figure 7Sodium and chloride dependence of TnaT-mediated tryptophan uptake. Main graph,l-[5-3H]tryptophan influx into CY15212 cells expressing TnaT was measured as described under “Materials and Methods” using the indicated concentrations of Na+. To adjust sodium concentration, NaCl was replaced byN-methyl-d-glucamine-Cl. Nonlinear least squares fits of the data revealed that influx was half-maximal at 1.06 ± 0.07 mm Na+. Inset,l-[5-3H]tryptophan influx was measured in NaCl-containing buffer (NaCl), and in buffers where all Na+ was replaced byN-methyl-d-glucamine-Cl (−Na), or where all Cl− was replaced by isethionate (−Cl). Transport activities are shown as a percentage of the control sample (NaCl). In both graphs, data are means ± S.D. from four measurements in two separate experiments.View Large Image Figure ViewerDownload (PPT)Neurotransmitter transport utilizes transmembrane ion gradients. Within the NSS family, the coupled inward movement, or symport, of Na+ and neurotransmitter molecule is an almost universal feature. However, at least one member, the K+-coupled amino acid transporter, catalyzes substrate symport with K+ as well as Na+ (40Castagna M. Shayakul C. Trotti D. Sacchi V.F. Harvey W.R. Hediger M.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5395-5400Crossref PubMed Scopus (102) Google Scholar). Most of the known transporters in the NSS family also require Cl−, which is symported with substrate in the cases that have been examined (41Rudnick G. J. Bioenerg. Biomembr. 1998; 30: 173-185Crossref PubMed Scopus (68) Google Scholar). Among bacterial transport systems, some are energized directly by ATP, whereas others are coupled to transmembrane ion gradients.As an approach to determine the driving force used by TnaT to accumulate tryptophan, we used 2,4-dinitrophenol (DNP), a proton ionophore, to dissipate the transmembrane electrochemical potential for H+ (Δμ̃H+). As representatives of transporters coupled to ATP or to ion gradients, we used the histidine transporter, which is in the ATP-binding cassette family and known to be driven by ATP hydrolysis (42Ames G.F. Mimura C.S. Shyamala V. FEMS Microbiol. Rev. 1990; 75: 429-446Crossref Google Scholar), and the proline transporter, a Na+-coupled symporter (43Ramos S. Kaback H.R. Biochemistry. 1977; 16: 854-859Crossref PubMed Scopus (133) Google Scholar, 44Cairney J. Higgins C.F. Booth I.R. J. Bacteriol. 1984; 160: 22-27Crossref PubMed Google Scholar). Because dissipation of Δμ̃H+ can deplete ATP supplies by allowing futile H+ pumping through the F0F1-ATPase (45Hugenholtz J. Hong J.S. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3446-3449Crossref PubMed Scopus (14) Google Scholar), we also includedN,N′-dicyclohexylcarbodiimide (DCCD) to inhibit the ATPase. The results in Table Idemonstrate that, in both the presence and the absence of DCCD, DNP strongly inhibited both proline and tryptophan accumulation while having virtually no effect on histidine transport. These results strongly suggest that TnaT-mediated tryptophan transport, like the endogenous proline transport of E. coli, is coupled to the transmembrane electrochemical Na+ potential, which depends on Δμ̃H+.Table IThe effect of 2,4-dinitrophenol on transport of histidine, proline, and tryptophanl-[2,5-3H]Histidinel-[U-14C]Prolinel-[5-3H]TryptophanControl100 ± 5100 ± 7100 ± 10DCCD103 ± 10103 ± 893 ± 7DNP102 ± 107.0 ± 0.115 ± 5DCCD → dNP91 ± 27.5 ± 0.615 ± 10CY15212 cells expressing TnaT were treated with DCCD (1 mm, 20 min), DNP (1 mm, 5 min), or DCCD followed by DNP, and accumulation of l-[2,5-3H]histidine,l-[U-14C]proline, andl-[5-3H]tryptophan was measured as described under “Materials and Methods.” Transport levels are shown as a percentage of the untreated control sample. Data are means ± S.D. from six measurements in three separate experiments. Open table in a new tab For purification of the TnaT protein, the gene was tagged at the N terminus with 6 histidine residues and expressed in BL21(DE3)/pLysE using a T7 promoter-mediated expression system as described under “Materials and Methods.” After induction by IPTG, the cells were disrupted; membranes were isolated, and the membrane proteins were extracted with dodecyl maltoside. The His-tagged TnaT protein was purified using nickel chromatography and analyzed by SDS-PAGE. The purified protein migrated as a single band of ∼45 kDa relative to the predicted molecular size of the tagged construct of 57,258 kDa (Fig.8). In prokaryotic proteins the N-terminal Met is often cleaved, which would give a predicted mass of 57,127 kDa. Preliminary matrix-assisted laser desorption ionization mass spectrometry analysis of the purified TnaT gave a molecular mass of 57,012 ± 44 kDa (mean ± S.D., n = 2) or 99.8% of the predicted mass, suggesting that the protein is full-length and unmodified. From an initial culture of 1 liter, we obtained ∼0.5 mg of highly purified protein.Figure 8Coomassie-stained SDS-PAGE of nickel chromatography purification of TnaT. TnaT was prepared as described under “Materials and Methods.” Lane 1 is the flow-through that did not bind to the nickel column. Lanes 2and 3 are sequential fractions from the 20 mmimidazole elution. Lanes 4 and 5 are fractions eluted with 40 and 250 mm imidazole, respectively. The molecular masses of standards in kDa are shown on theright by arrows.View Large Image Figure ViewerDownload (PPT)DISCUSSIONThe tnaT gene is typical of an increasing number of prokaryotic sequences with striking homology to the NSS family of Na+-coupled neurotransmitter and amino acid transporters. To illustrate this observation, a BLAST search (46Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59138) Google Scholar) of GenBankTM was performed using a highly conserved portion of the consensus sequence (37Marchler-Bauer A. Panchenko A.R. Shoemaker B.A. Thiessen P.A. Geer L.Y. Bryant S.H. Nucleic Acids Res. 2002; 30: 281-283Crossref PubMed Scopus (525) Google Scholar) for the NSS family. At the time the search was performed, it yielded, in addition to tnaT, more than 40 other sequences from bacteria and Archaea, although no significant matches were found in sequences from yeast, fungi, or plants. The sequence similarities between the predicted prokaryotic proteins, including TnaT, and mammalian members of the NSS transporter family are extensive (Fig. 1). For the 30 prokaryotic sequences with the greatest sequence similarity to the NSS family, the MEMSAT2 transmembrane topology prediction method (47Jones D.T. Taylor W.R. Thornton J.M. Biochemistry. 1994; 33: 3038-3049Crossref PubMed Scopus (705) Google Scholar) found 12 well aligned transmembrane domains (TM) in 12 of these sequences, including TnaT, 11 TMs in 16 other sequences, and 10 TMs in 2 sequences. The sequences containing fewer than 12 TMs lacked the last one or two TMs and were homologous with the 12 TM sequences through the first 10 or 11 TMs.The apparent variability in the lengths of these transporter-like sequences highlights the uncertainty regarding their function. Without an unequivocal demonstration that these sequences encode functional transporters, we cannot rule out the possibility that they are responsible for other membrane functions. The characterization of TnaT as a functional Na+-coupled tryptophan transporter opens up this large family of orphan transporters to experimental study. We hope that these studies will shed light also on the structure and function of eukaryotic NSS neurotransmitter transporters. For example, if we find that those proteins with 10 or 11 TMs are functional as transporters, it will help to define which TMs are required for substrate binding, ion coupling, and other functions.Like almost all of the transporters in the NSS family, TnaT required Na+ for its function, although unlike many other NSS transporters, we found that Cl− was not required (Fig. 7). Moreover, TnaT-mediated Trp influx saturates at relatively low concentrations (Fig. 4) and is highly selective, similar to other NSS transporters. This high degree of functional and sequence similarity between TnaT and the mammalian members of the NSS family is even more remarkable in light of the complete absence of homologous sequences in yeast, fungi, or higher plants. This situation was found also in the sodium:solute symporter family (2Saier M.H. J. Cell. Biochem. 1999; 75 Suppl. 32: 84-94Crossref Google Scholar), which also has many members in prokaryotes and animals but almost none in yeast, fungi, or higher plants (48Turk E. Wright E.M. J. Membr. Biol. 1997; 159: 1-20Crossref PubMed Scopus (192) Google Scholar).One possible reason for the restricted distribution of this family to some prokaryotes and animals lies in the Na+ dependence of the NSS family. Prokaryotic and animal cells are known to maintain transmembrane Na+ gradients that are used for driving metabolite accumulation. Yeast and plants, however, extrude Na+ but rarely use solute-Na+ symport for metabolite accumulation (49Blumwald E. Aharon G.S. Apse M.P. Biochim. Biophys. Acta. 2000; 1465: 140-151Crossref PubMed Scopus (760) Google Scholar, 50Versaw W.K. Metzenberg R.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3884-3887Crossref PubMed Scopus (70) Google Scholar). If the coupling of substrate transport to Na+ is an integral property of NSS transporters, and they exclusively function, therefore, only as Na+ symporters, the restriction of their distribution to organisms that utilize a transmembrane Na+ gradient for metabolite transport would be understandable. This may also explain the distribution among prokaryotes. For example, among bacilli,Bacillus halodulans, an alkaliphilic species that uses the Na+ gradient as a driving force, has an NSS homologue (51Takami H. Nakasone K. Takaki Y. Maeno G. Sasaki R. Masui N. Fuji F. Hirama C. Nakamura Y. Ogasawara N. Kuhara S. Horikoshi K. Nucleic Acids Res. 2000; 28: 4317-4331Crossref PubMed Scopus (445) Google Scholar), whereas Bacillus subtilis, a neutralophile, does not (52Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessieres P. Bolotin A. Borchert S. Borriss R. Boursier L. Brans A. Braun M. Brignell S.C. Bron S. Brouillet S. Bruschi C.V. Caldwell B. Capuano V. Carter N.M. Choi S.K. Codani J.J. Connerton I.F. Danchin A. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3102) Google Scholar). A consequence of this explanation, however, is that all bacterial NSS proteins should catalyze Na+ symport, a prediction that we are currently testing.There is a high degree of sequence identity between TnaT and mammalian transporters for serotonin (21%), and dopamine (24%), and the consensus NSS sequence from the Conserved Domains data base (37Marchler-Bauer A. Panchenko A.R. Shoemaker B.A. Thiessen P.A. Geer L.Y. Bryant S.H. Nucleic Acids R" @default.
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• W2057487010 cites W1506737211 @default.
• W2057487010 cites W1517440098 @default.
• W2057487010 cites W1534623935 @default.
• W2057487010 cites W1558462642 @default.
• W2057487010 cites W1895466054 @default.
• W2057487010 cites W1895819679 @default.
• W2057487010 cites W1957153965 @default.
• W2057487010 cites W1966183574 @default.
• W2057487010 cites W1969174524 @default.
• W2057487010 cites W1970614608 @default.
• W2057487010 cites W1974260392 @default.
• W2057487010 cites W1976336543 @default.
• W2057487010 cites W1981596100 @default.
• W2057487010 cites W1985442468 @default.
• W2057487010 cites W1990944246 @default.
• W2057487010 cites W1993490647 @default.
• W2057487010 cites W2001669389 @default.
• W2057487010 cites W2006492629 @default.
• W2057487010 cites W2013883286 @default.
• W2057487010 cites W2014793858 @default.
• W2057487010 cites W2015379298 @default.
• W2057487010 cites W2020727025 @default.
• W2057487010 cites W2022310648 @default.
• W2057487010 cites W2031723027 @default.
• W2057487010 cites W2034403739 @default.
• W2057487010 cites W2035769503 @default.
• W2057487010 cites W2050630707 @default.
• W2057487010 cites W2053146824 @default.
• W2057487010 cites W2055169159 @default.
• W2057487010 cites W2056497347 @default.
• W2057487010 cites W2062589931 @default.
• W2057487010 cites W2064039245 @default.
• W2057487010 cites W2065123532 @default.
• W2057487010 cites W2070260945 @default.
• W2057487010 cites W2075109419 @default.
• W2057487010 cites W2081753415 @default.
• W2057487010 cites W2084460997 @default.
• W2057487010 cites W2089606003 @default.
• W2057487010 cites W2089934327 @default.
• W2057487010 cites W2091254626 @default.
• W2057487010 cites W2093048098 @default.
• W2057487010 cites W2093926288 @default.
• W2057487010 cites W2094673798 @default.
• W2057487010 cites W2095054691 @default.
• W2057487010 cites W2095217951 @default.
• W2057487010 cites W2095253451 @default.
• W2057487010 cites W2100882223 @default.
• W2057487010 cites W2106882534 @default.
• W2057487010 cites W2118007656 @default.
• W2057487010 cites W2122565898 @default.
• W2057487010 cites W2126084933 @default.
• W2057487010 cites W2131257296 @default.
• W2057487010 cites W2138563618 @default.
• W2057487010 cites W2139876538 @default.
• W2057487010 cites W2148959338 @default.
• W2057487010 cites W2149928224 @default.
• W2057487010 cites W2152517415 @default.
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• W2057487010 cites W2158714788 @default.
• W2057487010 cites W2165174899 @default.
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