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J Biol Chem, Vol. 275, Issue 13, 9690-9698, March 31, 2000

Identification and Characterization of a Na⁺-independent Neutral Amino Acid Transporter That Associates with the 4F2 Heavy Chain and Exhibits Substrate Selectivity for Small Neutral D- and L-Amino Acids^*

Yoshiki Fukasawa, Hiroko Segawa^§¶, Ju Young Kim, Arthit Chairoungdua, Do Kyung Kim, Hirotaka Matsuo, Seok Ho Cha, Hitoshi Endou, and Yoshikatsu Kanai^**

From the  Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, the ^§ Department of Clinical Nutrition, School of Medicine, Tokushima University, Kuramoto-Cho 3, Tokushima 770-8503, the  First Department of Physiology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, and ^** PRESTO, Japan Science and Technology Corporation, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A cDNA was isolated from the mouse brain that encodes a novel Na⁺-independent neutral amino acid transporter. The encoded protein, designated as Asc-1 (asc-type amino acid transporter 1), was found to be structurally related to recently identified mammalian amino acid transporters for the transport systems L, y⁺L, x_C, and b^0,+, which are linked, via a disulfide bond, to the type II membrane glycoproteins, 4F2 heavy chain (4F2hc), or rBAT (related to b^0,+ amino acid transporter). Asc-1 required 4F2hc for its functional expression. In Western blot analysis in the nonreducing condition, a 118-kDa band, which seems to correspond to the heterodimeric complex of Asc-1 and 4F2hc, was detected in the mouse brain. The band shifted to 33 kDa in the reducing condition, confirming that Asc-1 and 4F2hc are linked via a disulfide bond. Asc-1-mediated transport was not dependent on the presence of Na⁺ or Cl. Although Asc-1 showed a high sequence homology (66% identity at the amino acid level) to the Na⁺-independent broad scope neutral amino acid transporter LAT2 (Segawa, H., Fukasawa, Y., Miyamoto, K., Takeda, E., Endou, H., and Kanai, Y. (1999) J. Biol. Chem. 274, 19745-19751), Asc-1 also exhibited distinctive substrate selectivity and transport properties. Asc-1 preferred small neutral amino acids such as Gly, L-Ala, L-Ser, L-Thr, and L-Cys, and -aminoisobutyric acid as substrates. Asc-1 also transported D-isomers of the small neutral amino acids, in particular D-Ser, a putative endogenous modulator of N-methyl-D-aspartate-type glutamate receptors, with high affinity. Asc-1 operated preferentially, although not exclusively, in an exchange mode. Asc-1 mRNA was detected in the brain, lung, small intestine, and placenta. The functional properties of Asc-1 seem to be consistent with those of a transporter subserving the Na⁺-independent small neutral amino acid transport system asc.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Amino acid transport across the plasma membrane is mediated by both Na⁺-dependent and Na⁺-independent transporters (1). Recent molecular cloning approaches have led to the identification of amino acid transporters corresponding to amino acid transport systems described in the past in culture cells or membrane vesicle preparations (2). Although the cDNAs for various Na⁺-dependent amino acid transporters have been successfully isolated, the molecular characteristics of Na⁺-independent amino acid transporters have not yet been determined, except for those of cationic amino acid transport systems (2). Recently, by means of expression cloning, we isolated a cDNA encoding the Na⁺-independent transporter LAT1, subserving the amino acid transport system L, which preferentially transports large neutral amino acids with branched or aromatic side chains (3). We and others have further demonstrated that LAT1 requires the presence of an additional protein, the heavy chain of the 4F2 antigen, to form a heterodimeric functional complex (3-6).

The 4F2 antigen (CD98) was originally identified as a cell surface antigen that is up-regulated upon lymphocyte activation (7, 8). It was later found to be involved in a variety of cellular activities such as cell proliferation, cell transformation, and cell adhesion (7-10). Structurally, the 4F2 antigen is a heterodimeric protein composed of two subunits, an 80-kDa glycosylated heavy chain and a 40-kDa nonglycosylated light chain (7, 8). The 4F2 heavy chain (4F2hc)¹ is an integral membrane protein with a single-membrane spanning domain, classified as a type II membrane glycoprotein (11). Now, the 4F2 light chain has been revealed to be an amino acid transporter. The transporter corresponding to the amino acid transport systems L, y⁺L, and x_C has been shown to be the 4F2 light chain, which requires 4F2hc for its functional expression (3-6, 12-16). Recently, a protein structurally related to the 4F2hc-associated transporters has been identified that couples with the cystinuria-related type II membrane glycoprotein rBAT (related to b^0,+ amino acid transporter) to form a system b^0,+ amino acid transporter, thereby establishing a family of amino acid transporters associated with type II membrane glycoproteins (LAT family) (17, 18).

Because most of the hitherto identified members of the LAT family have been shown to function as Na⁺-independent amino acid transporters (3-6, 12-17), it may be reasonable to assume that the as yet unidentified Na⁺-independent amino acid transporters would also belong to this family. Based on this assumption, we attempted to search for proteins structurally related to the 4F2hc-associated transporters and identified a novel transporter that exhibits functional properties apparently consistent with those of a transporter subserving system asc, a Na⁺-independent transport system for small neutral amino acids (1, 19).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cDNA Cloning-- The cDNA fragment (corresponding to nucleotides 35-416 of the nucleotide sequence with the GenBank^TM/EBI/DDBJ accession number N32639) that was formerly used to isolate a rat cDNA for LAT2 was labeled with [³²P]dCTP (T7Quick prime, Amersham Pharmacia Biotech) and used as a probe for screening a mouse brain cDNA library (13). The oligo(dT)-primed cDNA library was prepared from mouse brain poly(A)⁺ RNA using the Superscript Choice System (Life Technologies, Inc.) (20, 21). The synthesized cDNA was ligated to ZipLox EcoRI arms (Life Technologies, Inc.). Screening of the cDNA library and isolation of the positive plaques were performed as described elsewhere (20, 21). Hybridization was performed in 50% formamide at 37 °C. The filters were washed at 37 °C in 0.1× SSC (1× SSC = 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0)/0.1% SDS, and the cDNAs in positive ZipLox phages were rescued into plasmid pZL1 by in vivo excision in accordance with the manufacturer's instructions (Life Technologies, Inc.). The cDNA was subcloned into the EcoRI cleavage site of plasmid pBluescript II SK (Stratagene) and sequenced in both direction by dye terminator cycle sequencing method (Perkin-Elmer and Applied Biosystems). The transmembrane regions of the proteins were predicted from the amino acid sequence determined based on the SOSUI algorithm (13, 17, 22).

The mouse brain cDNA library was also screened using a cDNA probe corresponding to base pairs 211-656 of rat 4F2hc (GenBank^TM/EBI/DDBJ accession number AB015433), to isolate a mouse 4F2hc cDNA (GenBank^TM/EBI/DDBJ accession number AB023408).

Anti-peptide Antibody-- Oligopeptides (PSPLPITDKPLKTQC) corresponding to amino acid residues 517-530 of Asc-1 and (CEGLLLQFPFVA) amino acid residues 516-526 of mouse 4F2hc were synthesized. The C-terminal or N-terminal cysteine residues were introduced for conjugation with keyhole limpet hemocyanine. The anti-peptide antibodies were generated as described elsewhere (17, 23).

Western Blotting-- Brain membranes were prepared as described elsewhere (24), with minor modifications. Briefly, adult mouse brain was homogenized in 9 volumes of 0.3 M sucrose, 0.26 unit/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM tosyl-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 40 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 either in the presence or absence 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 nonfat dried milk and diluted anti-Asc-1 antiserum (1:10,000) or anti-4F2hc antiserum (1:10,000). 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) (17).

Xenopus Oocyte Expression-- cRNAs were obtained for Asc-1 by in vitro transcription using T7 RNA polymerase in the plasmid pBluescript II SK linearized with BamHI and for mouse 4F2hc in pZL1 linearized with HindIII as described elsewhere (25). The Xenopus oocyte expression studies and uptake measurements were performed as described previously (20, 26). The uptake of ¹⁴C-labeled amino acids was measured 3 days after injection of the cRNA. For the co-expression experiments, 12 ng of Asc-1 cRNA and 13 ng of 4F2hc cRNA in a molar ratio of 1:1 were mixed and injected into each oocyte. To solely express Asc-1 and 4F2hc in the Xenopus oocytes, 12 and 13 ng of the respective cRNAs were injected. Expression of rat LAT1 with rat 4F2hc in the Xenopus oocytes was performed as described previously (3).

Amino Acid Uptake Measurements-- Groups of 6-8 oocytes were incubated in 500 µl of standard uptake solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 5 mM Tris, pH 7.4) or Na⁺-free uptake solution in which NaCl in the standard uptake solution was replaced with choline-Cl, containing 0.5-3.0 µCi of the radiolabeled compounds (20). To prepare uptake solutions with varying pH levels for the 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 as the buffer systems (20). Preliminary experiments to determine the time-course of [¹⁴C]L-alanine (100 µM) uptake by oocytes expressing Asc-1 and 4F2hc indicated that the uptake was linearly dependent on the incubation time up to 40 min (data not shown). Therefore, in all the subsequent experiments, the uptake levels were measured over 30 min, and the values were expressed as pmol/oocyte/min.

The K_m and V_max values for the amino acid substrates were determined using Eadie-Hofstee equation based on the Asc-1-mediated amino acid uptake levels measured at 1, 3, 10, 30, 100, and 300 µM for glycine, L-alanine, L-serine, L-threonine L-cysteine, -aminoisobutyric acid (AIB), D-alanine, and D-serine, at 3, 10, 30, 100, 300, and 1000 µM for L-valine, L-methionine, and L-isoleucine, and at 10, 30, 100, 300, 1000, and 3000 µM for L-leucine, L-histidine, L-phenylalanine, and -alanine. The Asc-1-mediated amino acid uptake levels were calculated as the differences between the means of the uptake levels by oocytes injected with the cRNAs and those by the control oocytes injected with water.

Efflux Measurements-- 100 nl (~3 nCi) of [¹⁴C]amino acids (100 µM) was injected into oocytes via fine-tipped glass micropipettes. The individual oocytes were incubated for 5 min in ice-cold Na⁺-free uptake solution and then transferred to Na⁺-free uptake solution, with or without 100 µM nonradiolabeled amino acids, kept at room temperature (18 -22 °C). The radioactivity in the medium and the radioactivity remaining in the oocytes were measured. The values were expressed as percentages of radioactivity (radioactivity in medium or that in oocytes/(radioactivity in medium + radioactivity in oocytes) × 100%) (13).

The radiolabeled amino acids used were: ¹⁴C(U)-labeled L-leucine, L-alanine, L-serine, L-threonine, L-glutamine, L-isoleucine, L-valine, L-phenylalanine, L-tyrosine, L-histidine, L-aspartate, L-glutamate, L-lysine, L-arginine, L-proline, and L-cystine, [1-¹⁴C]glycine, L-[methyl-¹⁴C]methionine, L-[side chain-3-¹⁴C]tryptophan, [1-¹⁴C]AIB, and a-[1-¹⁴C](aminomethyl)isobutyric acid (MeAIB) from NEN Life Science Products, Inc., and L-[1-¹⁴C]cysteine, L-[³H(G)]asparagine, D-[1-¹⁴C]leucine, D-[1-¹⁴C]phenylalanine, D-[1-¹⁴C]serine, and -[1-¹⁴C]alanine from American Radiolabeled Chemicals, Inc. For each data point in the measurements of the uptake and efflux of radiolabeled amino acids in the present study, six to eight oocytes were used. Each data point in the figures represents the mean ± S.E. of the uptake and efflux values (n = 6-8). To confirm the reproducibility of the results, three separate experiments were performed for each measurement using different batches of oocytes and in vitro transcribed cRNA except for K_m and V_max determination (see Table I). Results from representative experiments are shown in the figures.

Northern Blot Analysis-- RNA was prepared from the tissues of 4-5-week-old male Jcl:ICR mice and the placenta of mice with late pregnancy by the guanidinium isothiocyanate method using cesium-trifluoroacetic acid (Amersham Pharmacia Biotech) in accordance with the manufacturer's instructions (27). Poly(A)⁺ RNA (3 µg/lane) selected by oligo(dT) cellulose chromatography (Amersham Pharmacia Biotech) was separated on a 1% agarose gel in the presence of 2.2 M formaldehyde and was blotted onto a nitrocellulose filter (Schleicher & Schuell) as described (20, 27). The Asc-1 cDNA fragment corresponding to base pairs 1-512 obtained by digestion of the plasmid with EcoRI and XhoI was labeled with ³²P using the T7QuickPrime kit (Amersham Pharmacia Biotech). Hybridization was performed for 20 h at 42 °C in 50% formamide. The final stringent wash of the filter was performed in 0.1× SSC/0.1% SDS at 65 °C three times for 20 min (5, 34). To obtain spleens with reticulocytes, mice were administered intraperitoneal injection of 60 mg/kg of 1-acetyl-2-phenylhydrazine on days 0, 1, and 3 to induce hemolytic anemia as described elsewhere (28). The spleens were removed on day 6, and poly(A)⁺ RNA was prepared as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Structural Features of Asc-1-- A cDNA clone with a 1,716-base pair insert was isolated from a mouse brain cDNA library. It contained an open reading frame from nucleotides 12 to 1,604 encoding a putative 530-amino acid protein, designated as Asc-1 (asc-type amino acid transporter 1). The Asc-1 amino acid sequence exhibited remarkable homology to the amino acid sequences of the rat system L transporters, LAT1 (45% identity) (3) and LAT2 (65% identity) (13), the human y⁺L transporters, y⁺LAT1 (45% identity) (14), and KIAA0245/y⁺LAT2 (45% identity) (14, 29), the mouse system x_C transporter, xCT (46% identity) (16) (Fig. 1), and the rat system b^0,+ transporter, BAT1 (44% identity) (17). Asc-1 also exhibited significant homology to the system y⁺ transporters, CAT1~4 (29~30%), from mice and humans (2) and to the amino acid permeases from bacteria and yeast (e.g. 31% identity to Saccharomyces cerevisiae methionine permease MUP1 (30)).

View larger version (107K): [in this window] [in a new window]   Fig. 1.   Sequence alignment of Asc-1 and the structurally related transporters associated with 4F2hc. The deduced amino acid sequence of Asc-1 (mouse) is shown aligned with those of rat system L transporters, LAT2 and LAT1, human system y+L transporters, y+LAT1 and KIAA0245/y+LAT2 (labeled as "y+LAT2"), and system xC transporter, xCT (mouse). Identical residues in at least two sequences are shaded. Predicted transmembrane regions of Asc-1, numbered 1-12, are shown by lines above the sequences. In Asc-1, a putative tyrosine kinase-dependent phosphorylation site is located at residue 115 (labeled with #). Protein kinase C-dependent phosphorylation sites are predicted on the Asc-1 sequence at residues 37, 67, 185, 343, and 523, among which those at residues 37, 343, and 523 are predicted to be located intracellularly (labeled with *). A potential cAMP-dependent phosphorylation site is located at residues 5 and 369, among which the one at residue 5 is predicted to be intracellular (labeled with +). The residue numbers indicated above the aligned sequences are with reference to those in the amino acid sequence of Asc-1.

As shown in Fig. 1, 12 transmembrane regions were predicted on the Asc-1 amino acid sequence. There is a conserved cysteine residue (Asc-1 amino acid residue 160) in the putative extracellular loop between predicted transmembrane domains 3 and 4, which is presumed to be linked to 4F2hc via a disulfide bond (31). A tyrosine kinase-dependent phosphorylation site was predicted at residue 115 in the putative intracellular loop between predicted transmembrane domains 2 and 3, which is conserved in the system L transporters LAT1 and LAT2 but not in the system y⁺L transporters y⁺LAT1 and KIAA0245/y⁺LAT2 or the system x_C transporter xCT (Fig. 1). Protein kinase C-dependent phosphorylation sites were predicted in the putative intracellular loops (see legend for Fig. 1), among which one at residue 343 is conserved in LAT1, LAT2, and KIAA0245/y⁺LAT2 but not in y⁺LAT1 and xCT.

The antibody raised against Asc-1 recognized bands of 118 and 33 kDa in the nonreducing condition in the mouse brain, whereas the 118-kDa band disappeared in the reducing condition (Fig. 2). The antibody against 4F2hc recognized a band of 118 kDa in the nonreducing condition, whereas the band shifted to 85 kDa in the reducing condition (Fig. 2).

View larger version (50K): [in this window] [in a new window]   Fig. 2.   Western blot analyses under reducing and nonreducing conditions. Western blot analyses were performed on the membrane fractions prepared from the mouse brain in the presence or absence of 2-mercaptoethanol. For Asc-1 (left), a 118-kDa band detected in the absence of 2-mercaptoethanol (labeled with ) shifted in the presence of 2-mercaptoethanol (labeled with as +) to 33 kDa. For 4F2hc (right), a 118-kDa band detected in the absence of 2-mercaptoethanol shifted in the presence of 2-mercaptoethanol to 85 kDa.

Transport Activity-- Asc-1 required 4F2hc for its functional expression in Xenopus oocytes. As shown in Fig. 3a, Asc-1 by itself did not induce [¹⁴C]L-alanine transport. 4F2hc also by itself induced L-alanine transport only slightly. The co-expression of Asc-1 and 4F2hc, however, resulted in significant leucine uptake, indicating that 4F2hc is indispensable for the functional expression of Asc-1.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Functional expression of Asc-1 in Xenopus oocytes. a, the effect of co-expression of Asc-1 and 4F2hc. The uptake of [14C]L-alanine (100 µM) was measured in Na+-free uptake solution in Xenopus oocytes injected with water, Asc-1 cRNA (asc-1), 4F2hc cRNA (4F2hc), and both Asc-1 cRNA and 4F2hc cRNA (asc-1 + 4F2hc) 3 days after the injection. The co-expression of Asc-1 and 4F2hc resulted in a significant uptake of [14C]L-alanine. b, ion dependence of [14C]L-alanine transport in the oocytes expressing Asc-1 and 4F2hc. The uptake of 100 µM [14C]L-alanine measured in the standard uptake solution (Na) was not altered in the Na+-free uptake solution in which Na+ was replaced with choline (Choline) or in the Cl-free uptake solution in which Cl was replaced with gluconate (gluconate). c, Concentration dependence of Asc-1-mediated [14C]L-alanine uptake. The Asc-1-mediated [14C]L-alanine uptake by oocytes co-expressing Asc-1 and 4F2hc was measured at 1, 3, 10, 30, 100, and 300 µM of the amino acids in a Na+-free uptake solution and plotted against the [14C]L-alanine concentration. The alanine uptake was saturable and fit to the Michaelis-Menten curve.

The functional properties of Asc-1 were examined by co-expressing it with 4F2hc in Xenopus oocytes. [¹⁴C]L-alanine uptake was not dependent on either Na⁺ or Cl (Fig. 3b). The uptake was saturable and followed Michaelis-Menten kinetics with a K_m value of 23.0 ± 5.1 µM (mean ± S.E. of four separate experiments) for the [¹⁴C]L-alanine uptake (Fig. 3c).

Substrate Selectivity-- The substrate selectivity of Asc-1 was investigated by inhibition experiments in which the uptake of 50 µM [¹⁴C]L-alanine was measured in the presence of 5 mM of nonlabeled amino acids. As shown in Fig. 4a, the L-alanine uptake was markedly inhibited by glycine, L-alanine, L-serine, L-threonine, and L-cysteine. Some other neutral L-amino acids exhibited weaker inhibitory effects on the Asc-1-mediated [¹⁴C]L-alanine uptake. Proline, cystine, acidic amino acids glutamate and aspartate, or the basic amino acids lysine and arginine did not inhibit Asc-1-mediated [¹⁴C]L-alanine uptake. The [¹⁴C]L-alanine uptake was affected to a less significant extent by system L-specific inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) (32) (Fig. 4a). Consistent with the results of the inhibition experiments, high uptake levels of ¹⁴C-labeled glycine, L-alanine, L-serine, L-threonine, and L-cysteine were observed, whereas lower levels of uptake were detected for L-methionine, L-leucine, L-isoleucine, L-valine, L-phenylalanine, and L-histidine (Fig. 4b). Asc-1 did not transport acidic amino acids and basic amino acids (Fig. 4b). D-Alanine, D-serine, D-threonine, D-cysteine, and some other D-amino acids exerted inhibitory effects on [¹⁴C]L-alanine uptake (Fig. 5a). Among the [¹⁴C]D-amino acids tested, [¹⁴C]D-alanine and [¹⁴C]D-serine were shown to be transported by Asc-1 (Fig. 5b).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Substrate selectivity for L-amino acids. a, inhibition of Asc-1-mediated [14C]L-alanine uptake by glycine and L-amino acids. The Asc-1-mediated [14C]L-alanine uptake (50 µM) was measured in the presence of 5 mM nonradiolabeled glycine, the indicated L-amino acids, and BCH. The uptake was measured in the Na+-free uptake solution, and the values are expressed as percentages of the control L-alanine uptake in the absence of inhibitors (()). The L-alanine uptake was markedly inhibited by small neutral amino acids such as glycine, L-alanine, L-serine, L-threonine, and L-cysteine. b, Asc-1-mediated [14C]amino acid uptakes. The uptake levels of 100 µM radiolabeled glycine and the indicated L-amino acids by Xenopus oocytes expressing Asc-1 and 4F2hc were measured in Na+-free uptake solution. The uptake levels of small neutral amino acids such as glycine, L-alanine, L-serine, L-threonine, and L-cysteine were significantly high. Lower levels of uptake were detected for L-methionine, L-leucine, L-valine, L-phenylalanine, and L-histidine. Proline, cystine, acidic amino acids, and basic amino acids were not transported by Asc-1. Cyst., L-cystine.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   The effects of D-amino acids on Asc-1-mediated transport. a, inhibition of Asc-1-mediated [14C]L-alanine uptake by D-amino acids. The Asc-1-mediated [14C]L-alanine uptake (50 µM) was measured in the presence of 5 mM of the indicated nonradiolabeled D-amino acids. The values are expressed as percentages of the control L-alanine uptake in the absence of inhibitors (()). The L-alanine uptake was markedly inhibited by small neutral D-amino acids such as D-alanine, D-serine, D-threonine, and D-cysteine. b, Asc-1-mediated amino acid uptake. The uptake levels of 100 µM radiolabeled D-amino acids by Xenopus oocytes expressing Asc-1 and 4F2hc were measured in Na+-free uptake solution. The uptake levels of D-alanine and D-serine were significantly high.

In the experiments in which [¹⁴C]L-alanine uptake (50 µM) was measured in the presence of compounds (5 mM) structurally related to alanine, it was shown that -alanine, alanine methylester, and AIB strongly inhibited Asc-1-mediated [¹⁴C]L-alanine uptake to an equivalent degree to that by L-alanine and D-alanine (Fig. 6a). N-Methylalanine, MeAIB, sarcosine, and -aminobutyric acid did not inhibit Asc-1-mediated [¹⁴C]L-alanine uptake. -Alanine and AIB were further confirmed to be transported by Asc-1 in the uptake measurements using ¹⁴C-labeled compounds (Fig. 6b). As shown in Table I, Asc-1 exhibited high affinity for glycine, L-alanine, L-serine, L-threonine, L-cysteine, and AIB (K_m = ~10-20 µM) and lower affinity for larger neutral amino acids and -alanine. It also exhibited high affinity for D-isomers of serine and alanine (Table I).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   The effects of alanine-related compounds on Asc-1-mediated transport. a, inhibition of Asc-1-mediated [14C]L-alanine uptake by the indicated compounds. The Asc-1-mediated [14C]L-alanine uptake (50 µM) was measured in the presence of 5 mM nonradiolabeled compounds. The values are expressed as percentages of the control L-alanine uptake in the absence of inhibitors (()). The L-alanine uptake was significantly inhibited by D-alanine, -alanine, alanine methylester, and MeAIB. b, Asc-1-mediated transport of alanine-related compounds. The uptake levels of 100 µM radiolabeled compounds by Xenopus oocytes expressing Asc-1 and 4F2hc were measured in Na+-free uptake solution. The uptake levels of -alanine and AIB, as well as L-alanine and D-alanine, were significantly high. Ala-Me, alanine methylester; N-MeAla, N-methylalanine.

Amino Acid Efflux Mediated via Asc-1-- The efflux of radioactivity from the oocytes preloaded with [¹⁴C]L-alanine was measured in the absence or presence of extracellular L-alanine. As shown in Fig. 7, L-alanine outside the oocytes induced the efflux of preloaded [¹⁴C]L-alanine from the oocytes expressing both Asc-1 and 4F2hc. Lower but significant efflux of radioactivity was detected in the absence of extracellular L-alanine from the oocytes expressing both Asc-1 and 4F2hc compared with that from control oocytes not expressing either Asc-1 or 4F2hc. No significant efflux was induced by L-alanine application from the control oocytes. The efflux was not dependent on the extracellular presence of Na⁺ (Fig. 7).

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Amino acid efflux via Asc-1. Oocytes expressing both Asc-1 and 4F2hc (black column) and control oocytes injected with water instead of cRNA (shaded column) were preloaded with [14C]L-alanine by microinjection. Efflux of the preloaded [14C]L-alanine was measured in the presence (L-Ala (+)) or absence (L-Ala ()) of 100 µM L-alanine in the medium. The efflux measurements were performed in the standard uptake solution (Na(+)) as well as the Na+-free uptake solution (Na()). The efflux values are expressed as percentages of the total radioactivity microinjected into the oocytes. Extracellularly applied L-alanine also induced the efflux of preloaded [14C]L-alanine.

To determine the extracellular substrate selectivity for induction of the efflux of preloaded [¹⁴C]L-alanine, nonlabeled amino acids (100 µM) were applied extracellularly (Fig. 8a). Glycine, L-alanine, L-serine, L-threonine, L-cysteine, L-methionine, and L-valine induced a high level of efflux of preloaded [¹⁴C]L-alanine compared with that observed in the absence of extracellular amino acid substrates (Fig. 8a). D-Isomers of alanine, serine, threonine and cysteine also induced a high level of efflux of preloaded [¹⁴C]L-alanine (Fig. 8b). Furthermore, as shown in Fig. 8c, whereas -alanine, alanine-methylester, and AIB induced the efflux of preloaded [¹⁴C]L-alanine, N-methylalanine, MeAIB, and BCH did not.

View larger version (27K): [in this window] [in a new window]   Fig. 8.   Extracellular substrate selectivity for the induction of Asc-1-mediated [14C] L-alanine efflux. a, the effects of L-amino acids. Oocytes expressing both Asc-1 and 4F2hc (closed column) and control oocytes injected with water instead of cRNA (open column) were preloaded with [14C]L-alanine by microinjection. Efflux of the preloaded [14C]L-alanine was measured in the presence of 100 µM of the indicated L-amino acids. Significant efflux of radioactivity was detected even in the absence of extracellular amino acids. Extracellularly applied amino acids, in particular small neutral amino acids, markedly increased the efflux. b and c, the effects of D-amino acids (b) and alanine-related compounds (c). Efflux of preloaded [14C]L-alanine was measured in the presence of 100 µM of the indicated D-amino acids (b) and alanine-related compounds (c). The values are expressed as percentages of the total radioactivity microinjected into the oocytes. For b and c, the difference between the efflux values from the oocytes expressed with both Asc-1 and 4F2hc and those of control oocytes injected with water instead of cRNA are plotted as ordinates. Ala-Me, alanine methylester; N-MeAla, N-methylalanine.

To determine which intracellular amino acids were effectively exchanged via Asc-1, the efflux of preloaded [¹⁴C]L-amino acids was measured in an uptake solution containing 100 µM L-alanine. A high level of efflux of intracellularly injected ¹⁴C-labeled small neutral amino acids such as glycine, L-alanine, L-serine, L-threonine, and L-cysteine was induced by extracellularly applied L-alanine (Fig. 9). A low level of efflux of ¹⁴C-labeled amino acids was detected for L-methionine, L-leucine, L-isoleucine, and L-valine (Fig. 9).

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Efflux of intracellularly injected [14C]amino acids via Asc-1. Oocytes expressing both Asc-1 and 4F2hc were preloaded with the indicated [14C]amino acids by microinjection (100 µM [14C]L-amino acids; 100 nl (~3 nCi/oocyte)). Efflux of the preloaded radioactivity was measured in the presence (solid column) or absence (shaded column) of 100 µM L-alanine in the uptake solution. The values are expressed as percentages of the total radioactivity microinjected into the oocytes. Glycine, L-alanine, L-serine, L-threonine, and L-cysteine were effectively released into the extracellular medium in exchange for L-alanine.

As shown in Fig. 10a, time-dependent efflux of radioactivity was detected from oocytes expressing Asc-1 even in the absence of L-alanine in the extracellular medium, compared with that from control oocytes not expressing either Asc-1 or 4F2hc. Addition of L-alanine to the extracellular medium further increased the [¹⁴C]L-alanine efflux mediated by Asc-1. In contrast, only a low level of efflux of preloaded [¹⁴C]L-leucine was detected from oocytes expressing LAT1 in the absence of extracellular L-leucine (Fig. 10b), although when L-leucine was applied extracellularly LAT1-mediated [¹⁴C]L-leucine efflux was greatly enhanced.

View larger version (14K): [in this window] [in a new window]   Fig. 10.   Comparison of amino acid efflux via Asc-1 and LAT1. The time-course of the levels of efflux of [14C]L-alanine via Asc-1 (a) was compared with that of the efflux of [14C]L-leucine via LAT1 (b). Oocytes expressing Asc-1 with 4F2hc or LAT1 with 4F2hc were preloaded with [14C]L-alanine or [14C]L-leucine, respectively, as described under "Experimental Procedures." The efflux of radioactivity was measured from the oocytes expressing Asc-1 with 4F2hc or LAT1 with 4F2hc in Na+-free uptake solution containing 0 µM (open squares) or 100 µM (closed squares) of nonlabeled L-alanine for Asc-1 or nonlabeled L-leucine for LAT1. The efflux measurements were also performed for control oocytes injected with water instead of the cRNAs in Na+-free uptake solution containing 0 µM (open circles) or 100 µM (closed circles) of nonlabeled L-alanine or L-leucine. Only the data at 60 min are included for the control oocytes in b. The values are expressed as percentages of the total radioactivity loaded into the oocytes (see "Experimental Procedures"). For Asc-1, the efflux value was higher in the absence of L-alanine compared with that from control oocytes. Extracellularly applied L-alanine greatly enhanced [14C]L-alanine efflux via Asc-1 (a). In contrast, [14C]L-leucine efflux was low in the absence of leucine and greatly augmented by the extracellularly applied leucine for LAT1 (b).

pH Dependence of Transport-- The effect of pH on Asc-1-mediated [¹⁴C]L-alanine transport was examined by varying the pH of the uptake solution. As shown in Fig. 11, the [¹⁴C]L-alanine uptake did not show any remarkable pH dependence within the pH range of 5.5-8.5.

View larger version (9K): [in this window] [in a new window]   Fig. 11.   The effects of pH on the Asc-1-mediated transport. The uptake of [14C]L-alanine (100 µM) was measured in Na+-free uptake solution of various pHs. The pH did not remarkably influence Asc-1-mediated transport.

Tissue Distribution of Expression-- The expression of Asc-1 mRNA was analyzed by Northern blotting of poly(A)⁺ RNAs from various mouse tissues. A strong 1.9-kilobase pair signal was observed in the brain and lung, and a weak one was observed in the placenta (Fig. 12). A 4.4-kilobase pair message was detected in the small intestine (Fig. 12). No hybridization signal was detected in anemic spleen (data not shown), indicating the low level of expression of Asc-1 mRNA in erythrocytes.

View larger version (33K): [in this window] [in a new window]   Fig. 12.   Tissue distribution of Asc-1. High stringency Northern hybridization analysis using an Asc-1 probe was performed against poly(A)+ RNA (3 µg) from mouse tissues. A strong hybridizing band at 1.9 kilobase pairs was detected in the brain and lung, and a weak one was detected in the placenta. A 4.4-kilobase pair band was detected in the small intestine.

	DISCUSSION

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In the present study, we have identified a novel protein Asc-1 structurally related to the formerly identified transporters for amino acid transport systems L, y⁺L, x_C, and b^0,+ (3, 13-17). When expressed with 4F2hc in Xenopus oocytes, Asc-1 mediates the transport of neutral amino acids, in particular, small amino acids without bulky or branched side chains, in a Na⁺-independent manner, reminiscent of the Na⁺-independent small neutral amino acid transport system asc (1, 19). Therefore, we named this transporter Asc-1 (asc-type amino acid transporter 1). Asc-1 is a member of the family of amino acid transporters associated with type II membrane glycoproteins (LAT family) (17).

It has been shown that 4F2hc and its associated transporters are linked via a disulfide bond to form heterodimeric complexes (4-6, 15). The results from the Western blot analyses using antibodies raised against Asc-1 and 4F2hc are consistent with the idea that Asc-1 and 4F2hc are linked to each other via a disulfide band (Fig. 2). The 118-kDa band detected in the nonreducing condition seems to correspond to the heterodimeric complex of Asc-1 and 4F2hc. In the transporters associated with 4F2hc, it is proposed that a conserved cysteine residue in the extracellular loop between putative transmembrane domains 3 and 4 is involved in the disulfide bond formation between 4F2hc and the transporters (31). The corresponding cysteine residue is also conserved in Asc-1 (Cys¹⁶⁰). In addition, it should be noted that although a 33-kDa band corresponding to the Asc-1 monomeric protein is detected in the nonreducing condition, no such band corresponding to the 4F2hc monomer is present, suggesting that the 33-kDa band is not due to the unexpected reduction of the protein but due to the presence of the Asc-1 protein not associated with 4F2hc (Fig. 2).

Among the members of LAT family, Asc-1 exhibits the highest structural similarity to LAT2. LAT2 is a Na⁺-independent neutral amino acid transporter with a broad substrate selectivity that accepts all neutral L--amino acids, including small amino acids without bulky side chains and large amino acids with branched or aromatic side chains (13), whereas Asc-1 preferentially transports only small amino acids. LAT1, the other neutral amino acid-specific transporter of the LAT family, preferentially transports only large neutral amino acids (3). The system L-selective inhibitor, BCH, while strongly inhibiting amino acid transport mediated by LAT1 and LAT2 (3, 13), is not an effective inhibitor of Asc-1. There seems to be an interesting transition in the spacial configuration of the substrate-binding sites among Asc-1, LAT2 and LAT1.

Asc-1 exhibits distinctive properties not only in its selectivity for small neutral L-amino acids but also in its acceptance of D-amino acids and amino acid-related compounds. Asc-1 transports D-isomers of serine and alanine (Fig. 5 and Table I). D-Threonine and D-cysteine are also shown to be transport substrates because they induce the efflux of preloaded radiolabeled amino acids via an exchange mechanism as discussed below (Fig. 8b). LAT2-mediated transport is also inhibited, although weakly, by D-isomers of some amino acids such as serine, cysteine, and asparagine, even though they are not transported by LAT2 (13). LAT1 accepts D-leucine, D-phenylalanine, and D-methionine (3), which are, in fact, low affinity substrates for the transporter.² Although the absence of marked stereoselectivity seems to be a common characteristic of the LAT family, that of Asc-1 seems to be quite distinctive in that it mediates high affinity transport of D-amino acids.

It is notable that the affinity of Asc-1 for D-serine is particularly high (Table I). It is shown that D-serine is present in the mammalian brain and is proposed to be an endogenous modulator of N-methyl-D-aspartate-type glutamate receptors (33). Although the kinetics of D-serine in the brain is not fully understood, it is proposed that the local concentration of D-serine around glutamatergic synapses is under the regulation of the transporters of D-serine (33). It was indicated that ASCT2, a Na⁺-dependent transporter of the neutral amino acid transport system ASC, accepts D-isomers of serine, threonine, and cysteine (20). Their affinity is, however, proposed to be relatively low. Therefore, it would be of interest to understand the role of Asc-1, which is strongly expressed in the brain (Fig. 12), in the mobilization of D-serine around the glutamatergic synapses and in the modulation of glutamatergic transmission.

AIB is a high affinity substrate of Asc-1, whereas its N-methyl-derivative MeAIB is not accepted by Asc-1 (Fig. 6 and Table I). Alanine-methylester appears to be a substrate of Asc-1, because it induces the efflux of preloaded [¹⁴C]L-alanine as discussed below (Fig. 8c). Therefore, it is proposed that the presence of -amino groups is essential in the substrates of Asc-1, whereas that of -carboxyl groups is less important because alanine-methylester, which dose not contain the latter, is accepted by Asc-1. In agreement with this, -alanine is a transport substrate, although the affinity of Asc-1 for it is lower than that for -amino acid substrates.

We previously reported that LAT1 mediates amino acid exchange, whereas LAT2 mediates the facilitated diffusion of substrate amino acids (3, 13). As shown in Figs. 7 and 10, extracellularly applied substrates markedly stimulate the efflux of preloaded [¹⁴C]L-alanine, whereas significant efflux is still detected in their absence. This indicates that Asc-1 mediates two modes of transport, "exchange transport" and "facilitated diffusion," with the exchange mode appearing to be the predominant mode. Such dual operation is often observed for transporters. For example, the organic anion transporter OAT1 mediates both the exchange and facilitated modes; glutamate transporters mediate K⁺-dependent "uptake transport" as well as K⁺-independent exchange transport (34-36).

Based on the exchange property, it is considered possible to determine whether the compounds that inhibit Asc-1-mediated transport are also its substrates or just inhibitors, even if radiolabeled compounds are not available (46). As shown in Fig. 8a, the profile of amino acid-induced efflux of preloaded [¹⁴C]L-alanine is basically identical to that of the uptake of radiolabeled amino acids, indicating that the efflux of radioactivity reflects the transport of extracellularly applied amino acids via the Asc-1-mediated exchange. D-Threonine, D-cysteine, and alanine-methylester are suggested to be transported by Asc-1, because they induce the efflux of preloaded [¹⁴C]L-alanine (Fig. 8, b and c).

By microinjecting radiolabeled compounds into oocytes expressing Asc-1, we assessed the intracellular substrate selectivity of Asc-1. As Fig. 9 indicates, intracellular substrate selectivity is basically identical to that of the extracellular substrate-binding site. The efflux of low affinity substrates, however, appears to be less than that expected, probably reflecting the competition between intracellularly loaded ¹⁴C-labeled compounds and high affinity substrates present endogenously in Xenopus oocytes.

System asc was initially characterized in horse erythrocytes and later demonstrated to be also present in other cell systems such as pigeon, Pacific hagfish and trout erythrocytes, exocrine pancreatic cells, and transformed kidney cells (37-42). The originally described system asc was a high affinity, stereospecific, and Na⁺-independent transport system for small neutral amino acids that operates preferentially but not exclusively in the exchange mode (37). System C, a low affinity transport system with a similar substrate selectivity to that of system asc, was described in pigeon erythrocytes (43, 44). Heterogeneity in substrate selectivity, transport mode, and pH dependence was described for system asc (37-42, 45). Asc-1 has high affinity for small neutral amino acids, is not pH-dependent, and operates preferentially in the exchange mode, which resembles the properties of system asc originally described in horse erythrocytes (37), except that Asc-1 is less stereospecific. Therefore, we propose that Asc-1 is the first isolated isoform of system asc transporters. As described under "Results," the mRNA for Asc-1 could not be detected in anemic spleen, indicating that Asc-1 is not an erythrocyte system asc transporter. It is suggested that transporters structurally related to Asc-1 and associated with 4F2hc are also present in erythrocytes, which exhibit the functional properties of classical erythrocyte system asc transporters.

We have identified a novel Na⁺-independent neutral amino acid transporter, Asc-1, that associates with 4F2hc and exhibits the functional properties that seem to be consistent with those of a transporter of system asc. Asc-1 belongs to the LAT family and is structurally related, in particular, to LAT2, a system L transporter. Therefore, it is expected that structure-function analyses based on a comparison between Asc-1 and LAT2 provide useful information to understand the structural features that are responsible for the apparently different substrate selectivities of the members of the LAT family.

	ACKNOWLEDGEMENTS

We are grateful to Hisako Ohba and Michi Takahashi for technical assistance. Anti-Asc-1 and anti-4F2hc antibodies were supplied by Kumamoto Immunochemical Laboratory Co., Ltd. (Kumamoto, Japan).

	FOOTNOTES

^* This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan (Grants-in-Aid for Scientific Research and High-Tech Research Center), the Scientific Research Promotion Fund of the Japan Private School Promotion Foundation, the Japan Science and Technology Corporation, the Kato Memorial Bioscience Foundation, the Research Fund of Mitsukoshi Health and Welfare Foundation (1998), the Uehara Memorial Foundation, and the Toyota Physical & Chemical Research Institute.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 GenBank^TM/EMBL Data Bank with accession number(s) AB026688.

^¶ Research fellow of the Japan Society for the Promotion of Science.

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.

² H. Segawa, H. Endou, and Y. Kanai, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: 4F2hc, 4F2 heavy chain; AIB, -aminoisobutyric acid; MeAIB, a-(aminomethyl)isobutyric acid; BCH, 2-Aminobicyclo-(2, 2, 1)-heptane-2-carboxylic acid; MES, 4-morpholineethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES