Expression Cloning of a Na+-independent
Aromatic Amino Acid Transporter with Structural Similarity to
H+/Monocarboxylate Transporters*
Do Kyung
Kim
,
Yoshikatsu
Kanai
§¶,
Arthit
Chairoungdua
,
Hirotaka
Matsuo
,
Seok Ho
Cha
, and
Hitoshi
Endou
From the
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
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ABSTRACT |
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.
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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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.
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RESULTS |
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.
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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.
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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).
-Aminoisobutyric acid,
-alanine, taurine, and
-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
-(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).
-Methyl-DOPA,
-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, -(aminomethyl)isobutyric acid;
-Me-Dopa, -methyl-DOPA;
-Me-Tyr, -methyl-tyrosine;
3-O-Me-Dopa, 3-O-methyl-DOPA; T3,
3,3',5-L-triiodothyronine; T4, thyroxine.
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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, -(aminomethyl)isobutyric acid;
L-DOPA,
L-3,4-dihydroxyphenylalanine; T3,
3,3',5-L-triiodothyronine, T4, thyroxine.
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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.
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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.
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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").
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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.
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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.
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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).
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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.
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DISCUSSION |
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
-carboxyl group, but not
-amino group, is essential for substrate
recognition by TAT1. Consistent with this, tyramine which lacks the
-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,
-methylation seems to interfere with the interactions
between substrates and the substrate-binding site of TAT1, because
-methyl-DOPA and
-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.
 |
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