Expression Cloning of a Na+-independent Aromatic Amino Acid Transporter with Structural Similarity to H+/Monocarboxylate Transporters*

Do Kyung KimDagger , Yoshikatsu KanaiDagger §, Arthit ChairoungduaDagger , Hirotaka MatsuoDagger ||, Seok Ho ChaDagger , and Hitoshi EndouDagger

From the Dagger  Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, the || First Department of Physiology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan, and § PRESTO, Japan Science and Technology Corporation (JST), 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan

Received for publication, October 17, 2000, and in revised form, January 17, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A cDNA was isolated from rat small intestine by expression cloning which encodes a novel Na+-independent transporter for aromatic amino acids. When expressed in Xenopus oocytes, the encoded protein designated as TAT1 (T-type amino acid transporter 1) exhibited Na+-independent and low-affinity transport of aromatic amino acids such as tryptophan, tyrosine, and phenylalanine (Km values: ~5 mM), consistent with the properties of classical amino acid transport system T. TAT1 accepted some variations of aromatic side chains because it interacted with amino acid-related compounds such as L-DOPA and 3-O-methyl-DOPA. Because TAT1 accepted N-methyl- and N-acetyl-derivatives of aromatic amino acids but did not accept their methylesters, it is proposed that TAT1 recognizes amino acid substrates as anions. Consistent with this, TAT1 exhibited sequence similarity (~30% identity at the amino acid level) to H+/monocarboxylate transporters. Distinct from H+/monocarboxylate transporters, however, TAT1 was not coupled with the H+ transport but it mediated an electroneutral facilitated diffusion. TAT1 mRNA was strongly expressed in intestine, placenta, and liver. In rat small intestine TAT1 immunoreactivity was detected in the basolateral membrane of the epithelial cells suggesting its role in the transepithelial transport of aromatic amino acids. The identification of the amino acid transporter with distinct structural and functional characteristics will not only facilitate the expansion of amino acid transporter families but also provide new insights into the mechanisms of substrate recognition of organic solute transporters.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transport of amino acids across the plasma membrane is mediated by Na+-dependent and Na+-independent transport systems (1). Molecular cloning approaches have revealed the molecular nature of most of the classically characterized amino acid transport systems (2-8). The exceptions have been the system B0 and system T. The transporters subserving these transport systems have not been identified in any of the already known amino acid transporter families: three Na+-dependent families (SLC1, SLC6, and the recently identified family for systems A and N) and one Na+-independent family (SLC7) (2-8).

System T was originally characterized in human erythrocytes (9, 10). It transports aromatic amino acids in a Na+-independent manner (9-12). Although it was once proposed that system T is a variant of system L which shows Na+-independent transport of neutral amino acids including aromatic amino acids, system T is distinct in that it accepts N-methyl amino acids whereas system L does not (11, 13, 14). Therefore, it is reasonable to assume that transporters subserving system T would belong to a different family with distinct mechanisms of substrate recognition. In order to identify the transporter for system T, we have performed expression cloning. We have revealed that the system T transporter, in fact, does not belong to any of the already identified amino acid transporter families, but exhibits structural similarity to monocarboxylate transporters, which could explain the unique characteristics of system T in its substrate recognition.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of Rat Small Intestine Poly(A)+ RNA-- Xenopus laevis oocyte expression studies and uptake measurements were performed as described elsewhere (15, 16). Defolliculated oocytes were injected with poly(A)+ RNA (50 ng) obtained from small intestine epithelial scrape of adult male Harlan Sprague-Dawley rats. Three days after injection, the uptake of [14C]L-tryptophan was measured for 30 min in the regular uptake solution (NaCl 100 mM, KCl 2 mM, CaCl2 1 mM, MgCl2 1 mM, HEPES 10 mM, Tris 5 mM, pH 7.4) containing 100 µM [14C]L-tryptophan (0.5 µCi/ml) and 5 mM L-lysine.

Expression Cloning-- Expression cloning using a Xenopus oocyte expression system was performed as described (17-21). Four-hundred µg of poly(A)+ RNA obtained from small intestine epithelial scrape was size-fractionated by preparative gel electrophoresis (19, 20). RNA from each fraction (50 ng) was expressed in Xenopus oocytes. Positive fractions showing peak stimulation of [14C]L-tryptophan (100 µM) uptake in the presence of 5 mM L-lysine were used to construct a directional cDNA library (19, 20). cRNA synthesized in vitro from pools of ~500 clones was injected into Xenopus oocytes (19, 20). A positive pool was sequentially subdivided and analyzed until single clone (TAT1)1 was identified. The cDNA was sequenced in both directions by the dye terminator cycle sequencing method (PerkinElmer Life Sciences).

Functional Characterization-- Xenopus oocytes were injected with 25 ng of TAT1 cRNA synthesized in vitro from the TAT1 cDNA in plasmid pZL1 (Life Technologies, Inc.) linearized with NotI. Three days after injection the uptake of 14C-labeled amino acids was measured as described above in the regular uptake solution or Na+-free uptake solution in which NaCl in the regular uptake solution was replaced by choline chloride, containing 0.5-2.0 µCi/ml radiolabeled compounds. For Cl--free uptake solution, Cl- in the Na+ uptake solution was replaced by gluconate anion. To prepare uptake solution with varied pH for pH dependence experiments, MES-NaOH (pH 5.5 and 6.0), PIPES-NaOH (pH 6.5 and 7.0), HEPES-NaOH (pH 7.5 and 8.0), and Tris-HCl (pH 8.5) were used for buffer systems (22). Preliminary experiments to determine the time course of [14C]L-tryptophan (1 mM) uptake into oocytes expressing TAT1 indicated that the uptake was linearly dependent on incubation time up to 10 min (data not shown), so for all the following experiments uptakes were measured for 10 min and the values were expressed as picomole/oocyte/min.

Km and Vmax of amino acid substrates were determined using Eadie-Hofstee equation based on the TAT1-mediated amino acid uptakes measured at 0.03, 0.1, 0.3, 1, 3, 6, and 10 mM. To measure the Ki values, TAT1-mediated transport of [14C]L-tryptophan was measured in the Na+-free uptake solution with varied concentration of [14C]L-tryptophan (0.03, 0.1, 0.3, 1, 3, 6, and 10 mM) with or without addition of inhibitors. The Ki values were determined by double reciprocal plot analysis (23). TAT1-mediated amino acid uptakes were calculated as differences between the means of uptakes of the oocytes injected with TAT1 cRNA and those of the control oocytes injected with water.

For the efflux measurement, 100 nl (2 nCi) of [14C]L-tryptophan (~400 µM) was injected into oocytes with fine-tipped glass micropipette as described elsewhere (24, 25). The individual oocytes were incubated for 5 min in the ice-cold Na+-free uptake solution, and then transferred to the Na+-free uptake solution with or without 1 mM non-radiolabeled L-tryptophan kept at room temperature (18-22 °C). The radioactivity in the medium and the remaining radioactivity in the oocytes were measured. The values were expressed as % radioactivity (radioactivity of medium or oocytes/(radioactivity of medium + radioactivity of oocytes) × 100%) (24, 25).

For the uptake and efflux measurements in the present study, six to nine oocytes were used for each data point. Each data point in the figures represents the mean ± S.E. of uptake (n = 6-9). To confirm the reproducibility of the results, three separate experiments using different batches of oocytes and in vitro transcribed cRNA were performed for each measurement. Results from the representative experiments are shown in figures.

For electrophysiological measurements, Xenopus oocytes were clamped at -60 mV by two electrode voltage camping using GeneClamp 500 (Axon Instruments) (15). The electric current induced by the bath-applied 1 mM L-tryptophan was monitored.

Northern Analysis-- Poly(A)+ RNA (3 µg/lane) isolated from rat tissues was separated on 1% agarose gel in the presence of 2.2 M formaldehyde and blotted onto a nitrocellulose filter (Schleicher & Schuell) (16). The PstI fragment of TAT1 cDNA corresponding to 1274-2030 base pairs was labeled with 32P using T7 QuickPrime kit (Amersham Pharmacia Biotech). Hybridization was for 20 h at 42 °C in 50% formamide. The final stringent wash of the filter was in 0.1 × SSC, 0.1% SDS at 65 °C for 3 × 20 min (16).

Anti-peptide Antibody-- Oligopeptides (SSSSGIFKKESDSC) corresponding to amino acid residues 500-512 of TAT1 was synthesized. The C-terminal cysteine residue was introduced for conjugation with keyhole limpet hemocyanine. The anti-peptide antibodies were generated and affinity purified as described elsewhere (26, 27).

Western Blot Analysis-- Small intestine epithelium membranes of male Harlan Sprague-Dawley rats were prepared as described elsewhere (24), with minor modifications. Briefly, rat small intestine scrape was homogenized in 9 volumes of 0.3 M sucrose, 0.26 units/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM tosyl-L-lysine chloromethyl ketone, and 0.1 mM tosyl-arginine chloromethyl ketone, with 10 strokes of a motor-driven Teflon-potter homogenizer. The homogenate was centrifuged for 10 min at 8,000 rpm, and the supernatant was centrifuged further for 20 min at 8,000 rpm. After filtration, the cytosol was centrifuged for 60 min at 45,000 rpm, and the membrane pellet was resuspended in 0.25 M sucrose, 100 mM KCl, 5 mM MgCl2, and 50 mM Tris (pH 7.4). The protein samples were heated at 100 °C for 5 min in sample buffer in the presence of 5% 2-mercaptoethanol and subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred electrophoretically to a Hybond-P polyvinylidene difluoride transfer membrane (Amersham Pharmacia Biotech), and the membrane was treated with non-fat dried milk and diluted affinity purified anti-TAT1 antidody (1:500). The membrane was then treated with horseradish peroxidase-conjugated anti-rabbit IgG as the secondary antibody (Jackson ImmunoResearch Laboratories, Inc). The signals were detected with an ECL plus system (Amersham Pharmacia Biotech) (24, 27). To verify the specificity of immunoreactions by absorption experiments, the membranes were treated with primary antibodies in the presence of antigen peptides (200 µg/ml) (27).

Immunohistochemistry-- Three-micrometer paraffin sections of rat small intestine were processed for light microscopic immunohistochemical analysis as described previously (28). For immunostaining, sections were incubated with affinity-purified anti-TAT1 antibody (1:1000) overnight at 4 °C. Thereafter, they were treated with Envision + rabbit peroxidase (DAKO) for 30 min. To detect immunoreactivity, the sections were treated with diaminobenzidine (0.8 mM) (27). For absorption experiments, the tissue sections were treated with the primary antibodies in the presence of antigen peptides (200 µg/ml) (28). The sections were counterstained with hematoxylin.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

When poly(A)+ RNA from rat small intestine was expressed in X. laevis oocytes, small but significant augmentation of [14C]L-tryptophan uptake was detected in the presence of excess of L-lysine. L-Lysine (5 mM) was added to the uptake solution to inhibit the transport activity of oocyte-endogenous transporters activated by rBAT and 4F2hc derived from small intestine poly(A)+ RNA (Fig. 1). The size fractionation of the small intestine poly(A)+ RNA revealed that the fraction of 2.2-2.6 kilobases exhibited the peak activity of [14C]L-tryptophan uptake (Fig. 1). From this fraction, a cDNA library was constructed and screened for [14C]L-tryptophan uptake by expression in Xenopus oocytes. A 2.5-kilobase cDNA was isolated which encodes a protein designated as TAT1 (T-type amino acid transporter 1).


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Fig. 1.   Functional expression of rat small intestine poly(A)+ RNA in Xenopus oocytes. Uptake of [14C]L-tryptophan (100 µM) was measured on the Xenopus oocytes injected with water, rat small intestine poly(A)+ RNA, and its size fractions in the standard uptake solution containing 5 mM L-lysine.

When expressed in Xenopus oocytes, TAT1 induced [14C]L-tryptophan transport which was not dependent on Na+ or Cl- in the medium (Fig. 2, a and b). The uptake of [14C]L-tryptophan was saturable and followed Michaelis-Menten kinetics (Fig. 2c) with a Km values of 3.72 ± 0.62 mM (mean ± S.E. of three separate experiments) for the [14C]L-tryptophan uptake.


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Fig. 2.   Functional expression of TAT1 in Xenopus oocytes. a, uptake of [14C]L-tryptophan (100 µM) was measured on the Xenopus oocytes injected with water or TAT1 cRNA in the Na+-free uptake solution. b, ion dependence of TAT1-mediated transport. TAT1-mediated [14C]L-tryptophan uptakes (100 µM) measured in the standard uptake solution (Na) was compared with that measured in the Na+-free uptake solution (Choline) and that measured in the Cl--free uptake solution (Gluconate). c, concentration dependence of TAT1-mediated [14C]L-tryptophan uptake. The TAT1-mediated [14C]L-tryptophan uptake by oocytes expressing TAT1 was measured at 0.03, 0.1, 0.3, 1, 3, 6, and 10 mM L-tryptophan in a Na+-free uptake solution, and plotted against the [14C]L-tryptophan concentration. The L-tryptophan uptake was saturable and fit to the Michaelis-Menten curve. Inset, Eadie-Hofstee plot of the L-tryptophan uptake on which kinetic parameters were determined.

The substrate selectivity of TAT1 was investigated by inhibition experiments in which 10 µM [14C]L-tryptophan uptake was measured in the presence of 10 mM amino acids. The L-tryptophan uptake was highly inhibited by L-isomers of tyrosine and phenylalanine, whereas other L-amino acids did not inhibit TAT1-mediated [14C]L-tryptophan uptake (Fig. 3a). alpha -Aminoisobutyric acid, beta -alanine, taurine, and gamma -aminobutyric acid had no inhibitory effect on the TAT1-mediated uptake (data not shown). D-Isomers of tryptophan and phenylalanine exhibited small but significant inhibition on the TAT1-mediated uptake, whereas D-tyrosine did not inhibit TAT1-mediated uptake at 10 mM (Fig. 3a). Classical inhibitors for amino acid transport systems, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (for system L) and alpha -(aminomethyl)isobutyric acid (for system A), and substrates for H+/monocarboxylate transporters, lactate and pyruvate, did not inhibit TAT1-mediated uptake (Fig. 3b). Among amino acid-related compounds tested, L-DOPA (L-3,4-dihydroxyphenylalanine) and 3-O-methyl-DOPA inhibited [14C]L-tryptophan uptake as strongly as L-phenylalanine (Fig. 3c). Droxydopa exhibited a weaker inhibitory effect (Fig. 3c). alpha -Methyl-DOPA, alpha -methyltyrosine, tyramine, and thyroid hormones, 3,3',5-L-triiodothyronine and thyroxine, did not exhibit significant effects on TAT1-mediated [14C]L-tryptophan uptake (Fig. 3c).


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Fig. 3.   Inhibition of TAT1-mediated [14C]L-tryptophan uptake by amino acids and related compounds. The TAT1-mediated [14C]L-tryptophan uptake (10 µM) was measured in the presence of 10 mM non-radiolabeled indicated L-amino acids and D-amino acids (D-isomers) (a), model substrates for system L (BCH), system A (MeAIB), and monocarboxylate transporters (lactate and pyruvate) (b), and amino acid-related compounds (c). The uptake was measured in the Na+-free uptake solution; the values are expressed as percent of the control L-tryptophan uptake in the absence of inhibitors (-). BCH, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid; MeAIB, alpha -(aminomethyl)isobutyric acid; alpha -Me-Dopa, alpha -methyl-DOPA; alpha -Me-Tyr, alpha -methyl-tyrosine; 3-O-Me-Dopa, 3-O-methyl-DOPA; T3, 3,3',5-L-triiodothyronine; T4, thyroxine.

Consistent with the results from the inhibition experiments, 14C-labeled L-tryptophan, L-tyrosine, and L-phenylalanine were transported by TAT1 (Fig. 4a). Among D-amino acids, D-phenylalanine, for which a 14C-labeled compound was available, was confirmed to be transported by TAT1 (Fig. 4a). [14C]Lactate and [14C]pyruvate were not transported by TAT1 (Fig. 4a). [14C]L-DOPA was shown to be transported by TAT1 whereas thyroid hormones, 3,3',5-L-triiodothyronine and thyroxine, were not substrates of TAT1 (Fig. 4b). Kinetic parameters of the amino acid substrates are listed in Table I.


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Fig. 4.   TAT1-mediated amino acid uptake. a, the uptake of 14C-labeled compounds measured in the Na+-free uptake solution at the concentration of 1 mM. b, TAT1-mediated uptakes of [14C]L-tryptophan, [14C]L-DOPA and 125I-labeled thyroid hormones (T3 and T4) measured in the Na+-free uptake solution at the concentration of 100 µM. MeAIB, alpha -(aminomethyl)isobutyric acid; L-DOPA, L-3,4-dihydroxyphenylalanine; T3, 3,3',5-L-triiodothyronine, T4, thyroxine.

                              
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Table I
Kinetic parameters of amino acid substrates

As shown in Fig. 5a, N-methyl- and N-acetyl-derivatives of aromatic amino acid substrates exhibited inhibitory effects on the TAT1-mediated uptake, whereas the methylesters did not inhibit TAT1-mediated [14C]L-tryptophan uptake. The inhibition of [14C]L-tryptophan uptake by N-methyl-L-tryptophan was shown to be competitive in a double reciprocal plot analysis with a Ki value of 3.1 mM while that for L-tryptophan measured in the same procedure was 1.9 mM (Fig. 5b). N-Acetyl-L-tryptophan also competitively inhibited L-tryptophan uptake with a Ki value of 1.5 mM (data not shown).


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Fig. 5.   The effects of N-methyl- and N-acetyl-derivatives and methylesters of aromatic amino acids on TAT1-mediated [14C]L-tryptophan uptake. a, inhibition of TAT1-mediated [14C]L-tryptophan uptake by the N-methyl- ("N-Me-") and N-acetyl- ("N-Ac-") amino acids, and amino acid-methylesters ("-Me"). The TAT1-mediated [14C]L-tryptophan uptake (10 µM) was measured in the Na+-free uptake solution in the presence of 10 mM non-radiolabeled compounds and expressed as percent of the control L-tryptophan uptake in the absence of inhibitors ((-)). b, inhibitory effect of N-methyl-L-tryptophan on TAT1-mediated [14C]L-tryptophan uptake. TAT1-mediated uptake of [14C]L-tryptophan (0.03, 0.1, 0.3, 1, 3, 6, and 10 mM) was measured in the Na+-free uptake solution in the presence (3 mM L-tryptophan (filled triangle) or 3 mM N-methyl-L-tryptophan (filled square)) or absence (filled circle) of inhibitors. Lineweaver-Burk plot analyses were performed.

The effect of pH on TAT1-mediated [14C]L-tryptophan transport was examined by varying the pH of the uptake solution. As shown in Fig. 6, the [14C]L-tryptophan uptake did not show any remarkable pH dependence within the pH range of 5.5 to 8.5. 


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Fig. 6.   pH dependence of TAT1-mediated transport. TAT1-mediated [14C]L-tryptophan uptake (1 mM) was measured in Na+-free uptake solution of various pH values.

In order to determine whether TAT1 is an exchanger or a facilitated transporter, the efflux of radioactivity from the oocytes preloaded with [14C]L-tryptophan was measured in the absence or presence of extracellular L-tryptophan. As shown in Fig. 7, time-dependent efflux of radioactivity was detected from the oocytes expressing TAT1, which was not affected by the presence or absence of L-tryptophan (1 mM) in the extracellular medium. In contrast, only a low level of efflux of preloaded [14C]L-tryptophan was detected from the control oocytes injected with water instead of TAT1 cRNA (Fig. 7).


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Fig. 7.   Efflux of [14C]L-tryptophan via TAT1. The time course of the levels of efflux of [14C]L-tryptophan via TAT1 was determined on the oocytes expressing TAT1 preloaded with [14C]L-tryptophan. The efflux of radioactivity was measured from oocytes expressing TAT1 in Na+-free uptake solution containing 0 mM (open square) or 1 mM (closed square) nonlabeled L-tryptophan. The efflux measurements were also performed for control oocytes injected with water instead of TAT1 cRNA in Na+-free uptake solution containing 0 mM (open circle) or 1 mM (closed circle) nonlabeled L-tryptophan. The values are expressed as a percentage of the total radioactivity loaded into the oocytes (see "Experimental Procedures").

The transport mediated by TAT1 was electroneutral. The application of 1 mM L-tryptophan to the Xenopus oocytes clamped at -60 mV did not induce significant current, whereas 13.7 ± 0.9 pmol/min (mean ± S.E., n = 8) of [14C]L-tryptophan uptake was detected by 1 mM L-tryptophan in the same batch of oocytes expressing TAT1.

The TAT1 cDNA (2,540 base pairs) contains a single open reading frame encoding a putative 514-amino acid protein (Fig. 8). The start of the coding sequence was defined by the first ATG downstream of the in-frame stop codon and the surrounding sequences (CGGGCTATGG) corresponding to the Kozak consensus translation initiation sequence (29). The cDNA includes a poly(A) tail (59 As) which starts 20 nucleotides downstream from a typical polyadenylation signal AATAAA at nucleotide 2,462. As shown in Fig. 8, 12 transmembrane regions were predicted on the TAT1 amino acid sequence. A leucine-zipper motif was found at residues 363-384 on the predicted 9th transmembrane domain (Fig. 8).


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Fig. 8.   Sequence alignment of TAT1 and the structurally related proteins. The deduced amino acid sequence of TAT1 (rat) is shown aligned with those of mouse XPCT protein with unknown function (32) and rat monocarboxylate transporters MCT1 (46) and MCT2 (47). Identical residues in at least two sequences are shadowed. Predicted transmembrane regions of TAT1, numbered 1-12, are shown by lines above the sequences. In TAT1, potential cAMP-dependent phosphorylation sites are located at residues 276 and 510, both of which are predicted to be intracellular (labeled with #). Protein kinase C-dependent phosphorylation sites are predicted on the TAT1 sequence at residues 232, 249, 256, 271, and 473, among which those at residues 256, 271, and 473 are predicted to be located intracellularly (labeled with *). A leucine-zipper motif at residues 363-384 of TAT1 is indicated by filled circles above four leucine residues. The residue numbers indicated above the aligned sequences are with reference to those in the amino acid sequence of TAT1.

The search of protein data bases (August 2000) revealed that TAT1 sequence is novel and exhibits relatively low but significant similarity to those of mammalian H+/monocarboxylate transporters MCT1-4 (31-33% identity) (30) (Fig. 8). It was further revealed that TAT1 is homologous to the XPCT (X-linked PEST-containing transporter) protein whose function is not identified (49% identity) (31, 32) (Fig. 8).

The Northern blot indicated that the 2.6-kilobase TAT1 message was expressed at high level in jejunum, ileum, colon, and placenta, and at lower level in liver (Fig. 9). On longer exposure, faint messages were also detected in lung, heart, and spleen. In jejunum, ileum, colon, and liver, an additional 7.8-kilobase message was also detected (Fig. 9).


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Fig. 9.   Tissue distribution of TAT1. High stringency Northern blot analysis of poly(A)+ RNA (3 µg) from rat tissues probed with 32P-labeled TAT1 cDNA. kb, kilobase.

In Western blot analysis, the antibody raised against TAT1 recognized a single band of 70 kDa in the membrane fraction prepared from rat small intestine epithelium scrape, consistent with the predicted molecular mass of TAT1 protein (56 kDa) (Fig. 10). The band disappeared in the presence of antigen peptides in the absorption experiment, confirming the specificity of immunoreactions (Fig. 10).


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Fig. 10.   Western blot analysis of TAT1 in rat small intestine. A single band at 70 kDa was seen for the membrane fractions prepared from rat small intestine epithelium scrape (lane 1). The absorption test was performed by preincubation of the antibody with TAT1 peptides (200 µg/ml) (lane 2).

Immunohistochemical analysis on rat small intestine revealed that the TAT1 protein is present in the epithelial cells (Fig. 11a). The transporter protein was abundant at the tip of the villus and the amount decreased from the tip of the villus to its base (Fig. 11a). In the epithelial cells, TAT1 immunoreactivity was detected in the basolateral membrane as well as in the cytoplasm and not in the apical membrane (Fig. 11b). In the absorption experiments in which the tissue sections were treated with the primary antibodies in the presence of antigen peptides, the immunostaining was not detected, confirming the specificity of the immunoreactions (data not shown).


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Fig. 11.   Localization of TAT1 protein in rat small intestine. Results of immunohistochemical analysis of rat jejunum sections showing the localization of TAT1 protein. a, low magnification view showing that TAT1 immunoreactivity was located in the epithelial cells. The immunoreactivity was strong at the tip of the villus (arrowheads) and decreased from the tip of the villus to its base (arrows). Scale bar = 100 µm. b, high magnification view showing TAT1 immunoreactivity in the basolateral membrane (arrows) as well as in the cytoplasm and not in the apical membrane (arrowheads). Scale bar, 30 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TAT1 exhibits Na+- and Cl--independent transport of aromatic amino acids such as tryptophan, tyrosine, and phenylalanine. The substrate selectivity of TAT1 corresponds well to that of the classically characterized system T (9-12). Consistent with the Km values (2-5 mM) reported for system T in human erythrocytes and mouse preimplantation conceptuses (9, 11), TAT1 exhibits low affinity to aromatic L-amino acid substrates (Km = 2-7 mM). TAT1-mediated transport is inhibited by D-isomers of tryptophan and phenylalanine and, in fact, D-phenylalanine is transported by TAT1, consistent with the properties of system T (9, 11, 12, 33).

In human erythrocytes, it was shown that system T accepts N-methyl- and N-acetyl-derivatives of tryptophan (11, 14). In this study, we have shown that the tryptophan uptake mediated by TAT1 is, in fact, inhibited by N-methyl- and N-acetyl-derivatives of aromatic amino acids, whereas their methylesters have no effect on TAT1-mediated transport (Fig. 5). The inhibition by N-methyl- and N-acetyltryptophan is in a competitive manner with Ki values close to that for L-tryptophan. These observations indicate that the alpha -carboxyl group, but not alpha -amino group, is essential for substrate recognition by TAT1. Consistent with this, tyramine which lacks the alpha -carboxyl group is not accepted by TAT1 (Fig. 3c). It is, thus, proposed that TAT1 recognizes amino acid substrates as anions.

Besides regular amino acids, TAT1-mediated transport is inhibited by amino acid-related compounds such as L-DOPA and 3-O-methyl-DOPA, indicating that the binding site of TAT1 can accept some variations of aromatic side chains (Fig. 3c). Triiodothyronine and thyroxine are, however, not accepted by TAT1 (Fig. 3c and 4), in contrast with the system L transporter (34). Interestingly, alpha -methylation seems to interfere with the interactions between substrates and the substrate-binding site of TAT1, because alpha -methyl-DOPA and alpha -methyltyrosine do not inhibit TAT1-mediated transport (Fig. 3c).

Recently it has been shown that many amino acid transporters mediate the exchange of substrates amino acids (2, 3, 20, 24, 25). As shown in Fig. 7, however, the efflux of preloaded [14C]L-tryptophan was detected even in the absence of extracellular L-tryptophan, which is totally different from the properties of typical amino acid exchangers such as LAT1 (L-type amino acid transporter 1) and y+LAT1 (y+L-type amino acid transporter 1) (20, 25). Additionally, extracellularly applied L-tryptophan did not enhance the TAT1-mediated efflux (Fig. 7). This suggests that TAT1-mediated transport is due to the facilitated diffusion rather than the obligatory exchange of substrate amino acids.

Consistent with the proposed mechanisms of substrate recognition, the deduced amino acid sequence of TAT1 shows low but significant similarity to H+/monocarboxylate transporters which mediate H+-coupled transport of monocarboxylates such as lactate and pyruvate (30). In contrast to H+/monocarboxylate transporters, however, TAT1 dose not transport lactate and pyruvate. In addition, the transport mediated by TAT1 is an electroneutral facilitated diffusion and not driven by the pH gradient across the plasma membrane, suggesting that TAT1-mediated transport is not coupled with H+-transport. These properties of TAT1 are consistent with those of system T, whereas they are distinct from those of H+/monocarboxylate transporters (10, 12, 14, 35-39). It would be interesting to know what structural traits are responsible for the diversity in the substrate selectivity and the ion coupling observed among such structurally related transporters.

A leucine-zipper motif was found on the predicted 9th transmembrane domain (Fig. 8), suggesting that TAT1 might form homo- or heteromeric complexes. The association with additional proteins has been well characterized in heterodimeric amino acid transporters (LAT family) composed of 12-membrane spanning transporters and single membrane spanning proteins (2, 4). It has recently been shown that some other 12-membrane spanning transporter proteins are also associated with single membrane spanning proteins (30, 40). For example, a single membrane spanning protein, CD147, has been shown to be tightly associated with monocarboxylate transporters MCT1 and MCT4 which are structurally related to TAT1. It facilitates the cell surface expression of MCT1 and MCT4. It is interesting that the leucine-zipper motif is conserved between TAT1 and XPCT (Fig. 8), which may indicate the common mechanisms of protein-protein interaction for these homologous proteins.

Halestrap and Price (30) reported nine structurally related mammalian sequences for MCT family. Although MCT1-4 were functionally identified as monocarboxylate transporters, the function of the other members of the family (MCT5-9) remained to be clarified (30). TAT1 exhibits relatively high similarity (49% identity at the amino acid level) to the XPCT protein (also named as MCT8 in Ref. 30) which is encoded by the XPCT/Xpct gene identified in the X-chromosome inactivation center (32). XPCT possesses a long N terminus intracellular region which contains a characteristic PEST domain enriched for proline (P), glutamate (E), serine (S), threonine (T), and aspartate (32) (Fig. 8). TAT1 also shares part of the PEST domain. The sequence similarity between TAT1 and members of the MCT family will provide a clue to the functional identification of members of the MCT family with unknown function, which will facilitate the establishment of a new family of amino acid transporters.

Since system T was first characterized in human erythrocytes, several investigators have reported the functional characterization of system T in other cells and tissues such as placenta, liver, and preimplantation conceptuses (11, 12, 33). Functional roles of system T have, however, remained to be clarified. In mouse preimplantation conceptuses, it has been proposed that system T is one of the redundant aromatic amino acid transport systems which would ensure maintaining tryptophan homeostasis presumably important during preimplantation development (11). System T has also been proposed to participate in the massive transport of aromatic amino acids in liver, quantitatively the most important site of aromatic amino acid metabolism in the body (12). In placenta, system T has been proposed to reside on the basal membrane, while system L activity is detected in the brush-border membrane (33). TAT1 expressed in liver and placenta (Fig. 9) may be responsible for the system T activity detected in these organs.

In intestine where TAT1 messages are strongly expressed, it was once proposed that the presence of distinct T-system could explain the transport differences between Hartnup's disease and blue diaper syndrome (9). In order to understand the role of TAT1 in the intestine, we have performed immunohistochemical analyses on rat small intestine and revealed that the TAT1 protein is present in the basolateral membrane of the epithelial cells (Fig. 11, a and b). TAT1 protein is abundant at the tip of the villus and the amount decreased from the tip of the villus to its base (Fig. 11a), consistent with the distribution of oligopeptide transporter PepT1 which plays a major role in the absorption of protein digestion products from the intestinal lumen (41-43). It is, thus, reasonable to assume that, in concert with the system L transporter LAT2 which is present in the basolateral membrane of the epithelial cells (44), the facilitative transporter TAT1 works as an exit path of aromatic amino acids in their transepithelial transport. Because system L transporter LAT2 exhibits broad substrate selectivity covering all the neutral amino acids (22, 44, 45), it would be beneficial to have an additional aromatic amino acid-selective transporter for the efficient trans-epithelial transport of aromatic amino acids so as not to be competed out by the other neutral amino acids. Therefore, we predict that TAT1 plays an essential role in the absorption of aromatic amino acids from the intestinal lumen and its defect may be involved in the pathogenesis of disorders caused by the disruption of aromatic amino acid absorption such as blue diaper syndrome (9).

    ACKNOWLEDGEMENTS

We are grateful to Michi Takahashi for technical assistance. We thank Kumamoto Immunochemical Laboratory, Transgenic Inc., Kumamoto, Japan, for supplying the anti-TAT1 antibody. We also thank Sumitomo Pharmaceuticals Co. Ltd. for providing droxydopa.

    FOOTNOTES

* This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan, the Japan Society for the Promotion of Science, the Promotion and Mutual Aid Corporation for Private Schools of Japan, the Japan Science and Technology Corporation, the Toyota Physical & Chemical Research Institute, Japan Foundation for Applied Enzymology, and the Japan Health Sciences Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB047324.

To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan. Tel.: 81-422-47-5511 (ext. 3453); Fax: 81-422-79-1321; E-mail: ykanai@kyorin-u.ac.jp.

Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M009462200

    ABBREVIATIONS

The abbreviations used are: TAT1, T-type amino acid transporter 1; MCT, monocarboxylate transporter; MES, 4-morpholineethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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