Identification of a Novel Na�-independent Acidic Amino AcidTransporter with Structural Similarity to the Member of aHeterodimeric Amino Acid Transporter Family Associated withUnknown Heavy Chains*

Received for publication, January 2, 2002, and in revised form, February 26, 2002Published, JBC Papers in Press, March 20, 2002, DOI 10.1074/jbc.M200019200

Hirotaka Matsuo‡§, Yoshikatsu Kanai‡¶�, Ju Young Kim‡, Arthit Chairoungdua‡, Do Kyung Kim‡,Jun Inatomi‡, Yasuhiro Shigeta‡**, Hisako Ishimine§, Sophapun Chaekuntode‡,Kittipong Tachampa‡, Hye Won Choi‡, Ellappan Babu‡, Jun Fukuda§, 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, the **Department of Urology, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-Ku, Chiba260-8670, and ¶PRESTO, Japan Science and Technology Corporation, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan

We identified a novel Na�-independent acidic aminoacid transporter designated AGT1 (aspartate/glutamatetransporter 1). AGT1 exhibits the highest sequence sim-ilarity (48% identity) to the Na�-independent small neu-tral amino acid transporter Asc (asc-type amino acidtransporter)-2 a member of the heterodimeric aminoacid transporter family presumed to be associated withunknown heavy chains (Chairoungdua, A., Kanai, Y.,Matsuo, H., Inatomi, J., Kim, D. K., and Endou, H. (2001)J. Biol. Chem. 276, 49390–49399). The cysteine residueresponsible for the disulfide bond formation betweentransporters (light chains) and heavy chain subunits ofthe heterodimeric amino acid transporter family is con-served for AGT1. Because AGT1 solely expressed or co-expressed with already known heavy chain 4F2hc (4F2heavy chain) or rBAT (related to b0,�-amino acid trans-porter) did not induce functional activity, we generatedfusion proteins in which AGT1 was connected with4F2hc or rBAT. The fusion proteins were sorted to theplasma membrane and expressed the Na�-independenttransport activity for acidic amino acids. Distinct fromthe Na�-independent cystine/glutamate transporterxCT structurally related to AGT1, AGT1 did not acceptcystine, homocysteate, and L-�-aminoadipate and exhib-ited high affinity to aspartate as well as glutamate, sug-gesting that the negative charge recognition site in theside chain-binding site of AGT1 would be closer to the�-carbon binding site compared with that of xCT. TheAGT1 message was predominantly expressed in kidney.In mouse kidney, AGT1 protein was present in the baso-lateral membrane of the proximal straight tubules anddistal convoluted tubules. In the Western blot analysis,AGT1 was detected as a high molecular mass band in the

nonreducing condition, whereas the band shifted to a40-kDa band corresponding to the AGT1 monomer in thereducing condition, suggesting the association of AGT1with other protein via a disulfide bond. The finding ofAGT1 and Asc-2 has established a new subgroup of theheterodimeric amino acid transporter family whosemembers associate not with 4F2hc or rBAT but withother unknown heavy chains.

In the past, a large number of amino acid transport systemsin mammals have been distinguished based on differences insubstrate selectivity and ion dependence (1). For the last dec-ade, molecular cloning approaches have revealed the molecularnature of amino acid transport systems (2). The amino acidtransporters identified so far exhibit a variety of substrateselectivity and are composed of the members of several trans-porter families. Among them, the heterodimeric amino acidtransporter family, a subfamily of SLC7, is unique in twoaspects (3, 4). First, the members of this family are linked viaa disulfide bond with single membrane spanning type II mem-brane glycoproteins such as 4F2hc (4F2 heavy chain) and rBAT(related to b0,�-amino acid transporter) (3, 4). 4F2hc is theheavy chain of the cell surface antigen 4F2 (CD98) (5, 6). The4F2 antigen is a heterodimeric protein composed of two sub-units, an �80-kDa glycosylated heavy chain and a �40-kDanonglycosylated light chain (5, 6). The 4F2 light chain has beenrevealed to be an amino acid transporter. Six proteins have sofar been identified to be 4F2 light chains to form transporterssubserving systems L, y�L, x�

C, or asc (7–16). In addition, amember of the heterodimeric amino acid transporter family hasbeen identified that couples with the other type II membraneglycoprotein rBAT to form a system b0,� amino acid trans-porter (17–19). The conserved cysteine residue in the predictedextracellular loop between transmembrane domains 3 and 4 isresponsible for the disulfide bond formation between trans-porter proteins (light chains) and the heavy chains (20). Sec-ond, the heterodimeric amino acid transporter family is distinc-tive for its diversity in the substrate selectivity of its members.As already mentioned, they include transporters for neutralamino acids (systems L and asc), acidic amino acids as well ascystine (system x�

C), and both neutral and basic amino acids(systems y�L and b0,�) (4).

Recently, we identified a transporter designated Asc-2 (asc-type amino acid transporter 2) that exhibited relatively low but

* This work was supported in part by grants from the Ministry ofEducation, Culture, Sports, Science and Technology of Japan, the Ja-pan Society for the Promotion of Science, the Promotion and Mutual AidCorporation for Private Schools of Japan, the Japan Science and Tech-nology Corporation, the Japan Foundation for Applied Enzymology, andthe Japan Health Sciences Foundation. The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AB072352.

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

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 23, Issue of June 7, pp. 21017–21026, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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significant sequence similarity to the members of the het-erodimeric amino acid transporter family (21). Asc-2, however,does not associate with 4F2hc or rBAT and is presumed to linkto unknown heavy chains. Although Asc-2 itself is not sorted tothe plasma membrane when expressed in Xenopus oocytes, thefusion proteins in which Asc-2 is connected with rBAT or 4F2hcappeared on the plasma membrane and exhibits the functionalproperties corresponding to those of the transporter subservingNa�-independent small neutral amino acid transport systemasc (21). In the present study, we have identified a noveltransporter protein structurally related to Asc-2. The trans-porter is also proposed to associate with an additional protein,presumably through a conserved cysteine residue to form afunctional complex. We have generated fusion proteins inwhich the transporter protein is connected with rBAT or 4F2hcand shown that they appear on the plasma membrane andexhibit the Na�-independent transport activity with distinctselectivity for acidic amino acids.

EXPERIMENTAL PROCEDURES

cDNA Cloning of AGT1—The cDNA for a mouse expressed sequencetag (GenBankTM accession number AI314100) showing nucleotide se-quence similarity to rat BAT1 (17) was obtained from the Integratedand Molecular Analysis of Genomes and their Expression (IMAGE).The 1.8-kb XhoI fragment was excised from the cDNA (IMAGE cDNAclone number 1907807), labeled with [32P]dCTP (T7Quick prime; Amer-sham Biosciences), and used as a probe for screening a mouse kidneycDNA library (22). The oligo(dT)-primed cDNA library was preparedfrom mouse kidney poly(A)� RNA using the Superscript Choice System(Invitrogen) (23). The synthesized cDNA was ligated to �ZipLox EcoRIarms (Invitrogen). Screening of the library and the isolation of positiveplaques were performed as described elsewhere (22). The cDNAs inpositive �ZipLox phages were rescued into plasmid pZL1 by in vivoexcision in accordance with the manufacturer’s instructions (Invitro-gen). The cDNA insert was subcloned into pcDNA3.1(�) (Invitrogen) ata NotI site. The cDNA was sequenced in both directions by the dyeterminator cycle sequencing method (PerkinElmer Life Sciences andApplied Biosystems). Transmembrane regions of proteins were pre-dicted based on the SOSUI algorithm (24).

Construction of Fusion Proteins—Fusion proteins were constructedas described elsewhere with some modifications (21). To generate aAGT1-rBAT fusion protein, AGT1 cDNA fragment was amplified byPCR using a sense primer corresponding to nucleotides 4–23 of AGT1cDNA sequence extended at its 5� end by adding a HindIII restrictionsite and GCGCG (5�-GCGCGAAGCTTACCTATAGGCAGAAACATTC-3�) and a reverse primer corresponding to the end of the coding sequenceextended at its 5� end by adding a NotI restriction site and ATAT(5�-ATATGCGGCCGCACTTTCTTCATGTATGTGGT-3�). The PCRproduct was digested with HindIII and NotI and ligated to the HindIIIand NotI sites of a mammalian expression vector pcDNA3.1(�) (Invitro-gen). Mouse rBAT cDNA was amplified using a sense primer corre-sponding to the coding sequence starting just after the start codon(ATG) extended at its 5� end by adding NotI restriction site and ATAT(5�-ATATGCGGCCGCAGATGAGGACAAAGGCAAGAG-3�) and a re-verse primer corresponding to nucleotides 2228–2251 of mouse rBATcDNA sequence (GenBankTM accession number NM009205) (25) ex-tended at its 5� end by adding by XbaI restriction site and GCGCGC(5�-GCGCGCTCTAGAAATGCTTTAGTATTTGGCATAATC-3�). ThePCR product was digested with NotI and XbaI and then introduced intothe vector containing AGT1 (see above) precleaved with NotI and XbaI.The amino acid sequence around the junction of the resultant fusionprotein was reduced to EESAAADED. Three amino acids (AAA) wereinserted between the C terminus of AGT1 and the N terminus of rBATin which the first methionine residue was omitted.

For the AGT1–4F2hc fusion protein, mouse 4F2hc cDNA was ampli-fied using a sense primer corresponding to the coding sequence startingjust after the start codon (ATG) extended at its 5� end by adding a NotIrestriction site and ATAT (5�-ATATGCGGCCGCAAGCCAGGACAC-CGAAGTGGA-3�) and a reverse primer corresponding to nucleotides1820–1838 of mouse 4F2hc cDNA sequence (GenBankTM accessionnumber AB023408) (16) extended at its 5� end by adding an XbaIrestriction site and GCGC (5�-GCGCTCTAGACATGAGGCAGGGGT-GATGTTTT-3�). The PCR product was digested with NotI and XbaI andintroduced into the vector containing AGT1 (see above) predigested

with NotI and XbaI. The amino acid sequence around the junction of theAGT1–4F2hc fusion protein was reduced to EESAAASQD. Three aminoacids (AAA) were inserted between the C terminus of AGT1 and the Nterminus of 4F2hc, in which the first methionine residue was omitted.

Xenopus Oocyte Expression—cRNA for AGT1 was obtained by in vitrotranscription using T7 RNA polymerase for cDNA of AGT1 inpcDNA3.1(�) (Invitrogen) linearized with HindIII as described else-where (26). cRNAs for AGT1-rBAT fusion protein and AGT1–4F2hcfusion protein were obtained by in vitro transcription using T7 RNApolymerase for cDNAs of AGT1-rBAT fusion protein and AGT1–4F2hcfusion protein in pcDNA3.1(�) (Invitrogen) linearized with ApaI. TheXenopus oocyte expression studies and uptake measurements wereperformed as described previously (27). The uptake of 14C-labeledamino acids were measured 2 days after injection of cRNAs. Twenty-five nanograms of cRNAs were injected to each oocyte. For coexpressionof AGT1 and mouse 4F2hc or mouse rBAT, 12 ng of AGT1 cRNA and 13ng of mouse 4F2hc cRNA (16) or mouse rBAT cRNA (25) were injectedinto oocytes.

Amino Acid Uptake Measurements in Xenopus Oocytes—Groups ofsix to nine oocytes were incubated in 500 �l of standard uptake solution(100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and5 mM Tris, pH 7.4) or Na�-free uptake solution in which NaCl in thestandard uptake solution was replaced by choline chloride containing0.5–3.0 �Ci of the radiolabeled compounds (12). For Cl�-free uptakesolution, Cl� in the standard uptake solution was replaced by gluconateanion. Preliminary experiments to determine the time course of [14C]L-aspartic acid (300 �M) uptake into oocytes expressing the AGT1–4F2hcand AGT1-rBAT1 fusion proteins indicated that the uptake was lin-early dependent on incubation time up to 30 min (data not shown).Therefore, in all of the subsequent experiments, the uptake levelswere measured over 30 min, and the values were expressed aspmol/oocyte/min.

The Km and Vmax values of amino acid substrates were determinedusing an Eadie-Hofstee plot based on the amino acid uptakes mediatedby the AGT1–4F2hc and AGT1-rBAT fusion proteins measured at 1, 3,10, 30, 100, and 300 �M. The amino acid uptakes mediated by AGT1–4F2hc fusion protein or AGT1-rBAT fusion protein were calculated asdifferences between the means of the uptakes by the oocytes injectedwith cRNAs for the fusion proteins and those of the control oocytesinjected with water. For the uptake measurements in the present study,six to nine oocytes were used for each data point. Each data point in thefigures represents the mean � S.E. of uptake (n � 6–9). To confirm thereproducibility of the results, three separate experiments using differ-ent batches of oocytes and in vitro transcribed cRNA were performed foreach measurement. The results from the representative experimentsare shown in the figures.

Functional Expression in COS-7 Cells—cDNAs for AGT1 were sub-cloned in the mammalian expression vector pcDNA3.1(�) (Invitrogen)as described above. cDNAs for mouse 4F2hc and mouse rBAT were alsosubcloned into the pcDNA3.1(�) at EcoRI site and at EcoRI and ApaIsites, respectively. For functional expression, 1 �g of these plasmidswere solely transfected or cotransfected to COS-7 cells, which weregrown in Dulbecco’s modified Eagle’s medium containing 10% fetalbovine serum, by using LipofectAMINETM 2000 reagent (Invitrogen) asdescribed elsewhere (28). Amino acid uptake measurements were per-formed at 48 h after transfection as described elsewhere (17).

Anti-peptide Antibody—An oligopeptide (CIPDVSDDHIHEES) corre-sponding to amino acid residues 465–478 of AGT1 was synthesized. TheN-terminal cysteine residue was used for conjugation with keyholelimpet hemocyanin. An anti-peptide antibody was produced as de-scribed elsewhere (29).

Immunofluorescence and Confocal Laser Microscopy—The immuno-fluorescence detection of AGT1 and the epitope of 4F2hc in AGT1–4F2hc fusion protein expressed in Xenopus oocytes was performed asdescribed previously (21) with minor modifications. Briefly, 2 days afterinjection of cRNAs, Xenopus oocytes were fixed with 4% paraformalde-hyde in phosphate buffer at 4 °C overnight. The samples were dehy-drated in graded alcohol series, embedded in paraffin, and cut into 3-�mthick sections that were then deparaffinized and equilibrated with 0.05M Tris-buffered saline containing 0.1% Tween 20. The sections werethen treated with 5% goat serum as a blocking agent for 45 min at roomtemperature and were then washed and incubated overnight with anti-AGT1 antiserum (1:250) or affinity-purified anti-4F2hc antibody (21)(1:100) at 4 °C. Thereafter, they were treated with Alexa FluorTM 488-conjugated goat anti-rabbit IgG (Molecular Probes, Inc.; diluted 1:200)for 2 h at room temperature. The sections were then washed three timeswith 0.05 M Tris-buffered saline containing 0.1% Tween 20 andmounted with fluorescent mounting medium (DAKO, Carpinteria, CA).

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The images were acquired using an Olympus Fluoview (FV 500) laserscanning confocal microscope (Olympus Optical, Tokyo, Japan). AnArgon laser beam was used for excitation at 488 nm for Alexa FluorTM

488 visualization. Emission from Alexa FluorTM 488 was detected viaBA505IF filter (30). For absorption experiments, the sections weretreated with the primary antibodies in the presence of antigen peptides(200 �g/ml) (21).

Immunohistochemistry—Three-micrometer paraffin sections ofmouse kidney were processed for light microscopic immunohistochem-ical analysis as described previously (17). For immunostaining, thesections were incubated with anti-AGT1 antiserum (1:1,000) overnightat 4 °C. Thereafter, they were treated with Envision (�) rabbit perox-idase (DAKO) for 30 min. To detect immunoreactivity, the sections weretreated with diaminobenzidine (0.8 mM) (17). For absorption experi-ments, the tissue sections were treated with the primary antibodies inthe presence of antigen peptides (50 �g/ml) (21). The sections werecounterstained with hematoxylin.

Western Blot Analysis—The protein samples from mouse kidneywere prepared as described elsewhere (21), with minor modifications.Briefly, the mouse kidney was homogenized in 9 volumes of 50 mM

Tris-HCl (pH 7.5), 25 mM KCl, 1 mM MgCl2, 1 mM phenylmethylsulfonylfluoride, and 0.25 M sucrose, with 15 strokes of a Dounce homogenizer.The homogenate was centrifuged for 10 min at 8,000 � g, and thesupernatant was centrifuged further for 1 h at 100,000 � g. Aftercentrifugation 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 sampleswere heated at 100 °C for 5 min in the sample buffer either in thepresence or absence of 5% 2-mercaptoethanol and subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins were trans-ferred electrophoretically to a Hybond-P polyvinylidene difluoridetransfer membrane (Amersham Biosciences). The membrane wastreated with nonfat dried milk and diluted anti-AGT1 antiserum (1:10,000) and then with horseradish peroxidase-conjugated anti-rabbitIgG as a secondary antibody (Jackson ImmunoResearch Laboratories,Inc.). The signals were detected with an ECL plus system (AmershamBiosciences). To verify the specificity of immunoreactions by absorptionexperiments, the membranes were treated with primary antibodies inthe presence of antigen peptides (50 �g/ml) (17).

Northern Blot Analysis—RNA was prepared from the tissues of 4–5-week-old Jcl:ICR male mice and placenta of mice with late pregnancy bythe guanidinium isothiocyanate method using cesium trifluoroaceticacid (Amersham Biosciences) in accordance with the manufacturer’sinstructions. Poly(A)� RNA (3 �g/lane) selected by oligo(dT) cellulosechromatography (Amersham Biosciences) was separated on a 1% aga-rose gel in the presence of 2.2 M formaldehyde and was blotted onto anitrocellulose filter (Schleicher & Schuell) as described elsewhere (31).The polymerase chain reaction-amplified fragment of AGT1 cDNA,corresponding to 43–1836 base pairs, was labeled with 32P using theT7QuickPrime kit (Amersham Biosciences). Hybridization was per-formed for 20 h at 42 °C in 50% formamide. The final stringent wash ofthe filter was performed in 0.1� SSC, 0.1% SDS at 65 °C three times for20 min.

RESULTS

Structural Features of AGT1—A mouse cDNA clone with a2,141-bp insert was isolated from a mouse kidney cDNA li-brary. It contained an open reading frame from nucleotides 59to 1,495 encoding a putative 478-amino acid protein, desig-nated as AGT1 (aspartate/glutamate transporter 1). The startof the coding sequence was defined by the first ATG and thesurrounding sequences (CTCTCAATGG) corresponding to theKozak consensus translation initiation sequence (32). ThecDNA includes the poly(A) tail (16 As), which starts 23 nucle-otide downstream from a typical polyadenylation signalAATAAA at the nucleotide 2,103. The amino acid sequence ofAGT1 was identical to that of BCO14684, which was in aGenBankTM data base but not functionally characterized. TheAGT1 amino acid sequence exhibited remarkable sequenceidentity to that of mouse system asc transporter Asc-2 (48%identity), which is presumed to be associated with unknownheavy chains (21). AGT1 also exhibits sequence identity to ratsystem L transporters, LAT1 (35% identity) (7) and LAT2(37%) (12), the mouse system asc transporter, Asc-1 (37%) (16),

the y�L transporters, rat y�LAT1 (37%) (33) and humanKIAA0245/y�LAT2 (36%) (14, 34), the mouse system x�

C

transporter, xCT (37%) (15), and the rat system b0,� trans-porter, BAT1 (36%) (17), all of which are associated with either4F2hc or rBAT (Fig. 1). AGT1 also exhibited significant se-quence identity to the system y� transporters, CAT1–4 (30%)from mice and humans (35) and to the amino acid permeasesfrom bacteria and yeast (e.g. 30% identity to Saccharomycescerevisiae methionine permease MUP1 (36)).

As shown in Fig. 1, 12 transmembrane regions were pre-dicted on the AGT1 amino acid sequence. There is a conservedcysteine residue (AGT1 amino acid residue 129) in the putativeextracellular loop between predicted transmembrane domains3 and 4, through which LAT1, LAT2, Asc-1, y�LAT1, y�LAT2,xCT, and BAT1/b0,�AT are proposed to link to 4F2hc or rBATvia a disulfide bond (20). Protein kinase C-dependent phospho-rylation sites and a tyrosine phosphorylation site are predictedin the putative intracellular domains. A cAMP-dependentphosphorylation site is predicted in the putative intracellularloops that is conserved between AGT1 and Asc-2 (see legend forFig. 1).

Functional Expression of AGT1—The expression of AGT1 didnot induce functional activity in Xenopus oocytes (Fig. 2a) orCOS-7 cells (Fig. 2b). Therefore, AGT1 was coexpressed with4F2hc or rBAT, because AGT1 exhibited structural similarityto the members of the heterodimeric amino acid transporterfamily. The coexpression of AGT1 and 4F2hc, however, did notinduce amino acid transport activity in Xenopus oocytes (Fig.2a). This result was confirmed in COS-7 cells in which thecoexpression of AGT1 with 4F2hc or rBAT did not induce aminoacid transport activity (Fig. 2b). Then, following the functionalcharacterization of Asc-2 (21), we generated fusion proteins inwhich the C terminus of AGT1 was connected with the Nterminus of 4F2hc or rBAT. When expressed in Xenopus oo-cytes, AGT1–4F2hc and AGT1-rBAT fusion proteins exhibited[14C]L-aspartate uptake (Fig. 2a).

To examine whether the fusion proteins were expressed inthe oocyte plasma membrane, we performed confocal immuno-fluorescence microscopic analysis using specific antibodiesraised against C-terminal parts of AGT1 and 4F2hc. As shownin Fig. 3 (a and b), these antibodies did not exhibit specificstaining in the control oocytes injected with water instead ofcRNAs. When AGT1 was solely expressed, AGT1 protein didnot appear on the plasma membrane (Fig. 3d). In contrast,when the AGT1–4F2hc fusion protein was expressed in Xeno-pus oocytes, both anti-4F2hc antibody and anti-AGT1 antibodyrecognized the immunoreactivity on the plasma membrane(Fig. 3, e and f), indicating that the AGT1–4F2hc fusion proteinis expressed in the plasma membrane. In the absorption exper-iments in which the tissue sections were treated with theprimary antibodies in the presence of their antigen peptides,the immunostainings were not detected, confirming the speci-ficity of the immunoreactions (data not shown).

Transport Properties—When expressed in Xenopus oocytes,AGT1–4F2hc and AGT1-rBAT fusion proteins mediated theNa�- and Cl�-independent transport (Fig. 4). The uptake of[14C]L-aspartate mediated by the fusion proteins was saturableand followed Michaelis-Menten kinetics (Fig. 5). The Km valuesfor L-aspartate were calculated to be 25.5 � 5.9 �M in AGT1–4F2hc fusion protein and 20.1 � 6.1 �M in AGT1-rBAT fusionprotein.

Substrate Selectivity—The substrate selectivity of theAGT1–4F2hc fusion protein and the AGT1-rBAT fusion pro-tein was investigated by inhibition experiments in which theuptake of 20 �M [14C]L-aspartate was measured in the presenceof 2 mM of nonlabeled amino acids. As shown in Fig. 6, the

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[14C]L-aspartate uptake by the oocytes expressing AGT1–4F2hc fusion protein or AGT1-rBAT fusion protein was mark-edly inhibited by acidic amino acids such as L-aspartate andL-glutamate. L-Cysteine exhibited weaker but significant inhib-itory effect on the [14C]L-aspartate uptake. Other L-�-aminoacids including neutral amino acids and basic amino acids�-alanine and �-aminobutyric acid did not inhibit [14C]L-aspar-tate uptake mediated by AGT1–4F2hc fusion protein (Fig. 6).D-Amino acids such as D-aspartate, D-glutamate, D-asparagine,D-glutamine, D-alanine, D-serine, and D-valine did not exhibitsignificant inhibitory effects on the [14C]L-aspartate uptake.The [14C]L-aspartate uptake was not affected by the systemL-specific inhibitor 2-aminobicyclo-(2,2,1)-heptane-2-carboxylicacid or the system A-specific inhibitor �-(aminomethyl)isobu-tyric acid (Fig. 6).

Consistent with the results from the inhibition experiments,high uptake levels of 14C-labeled L-aspartate and L-glutamatewere observed for the AGT1–4F2hc fusion protein (Fig. 7). Theuptake of neutral amino acids including L-cysteine and L-cys-tine and basic amino acids was at the trace level. [14C]D-Aspar-tate and [14C]D-glutamate were not transported by the AGT1–4F2hc fusion protein, suggesting that AGT1 is stereoselectivefor aspartate and glutamate (Fig. 7). The AGT1-rBAT fusionprotein exhibited an amino acid uptake profile basically iden-tical to that of AGT1–4F2hc fusion protein (data not shown).The Km and Vmax values of [14C]L-glutamate uptake for AGT1–4F2hc fusion protein were 21.8 � 6.5 �M and 0.63 � 0.10

(normalized to that of [14C]L-aspartate determined on the samebatch of oocytes).

The effects of acidic amino acid analogues on AGT1-mediatedtransport were also investigated by inhibition experiments inwhich the uptake of 20 �M [14C]L-aspartate was measured inthe presence of 2 mM nonlabeled acidic amino acid analogues.As shown in Fig. 8, the [14C]L-aspartate uptake by the oocytesexpressing the AGT1–4F2hc fusion protein was markedly in-hibited by threo-�-hydroxyaspartate, L-serine-O-sulfate, L-cys-teine sulfinate, and L-cysteate as well as L-aspartate and L-glutamate, whereas it was not inhibited by L-�-aminoadipate,L-homocysteate, L-trans-pyrrolidine-2,4-dicarboxylate, anddihydrokainate.

Tissue Distribution of Expression—The expression of AGT1mRNA was analyzed by Northern blotting of poly(A)� RNAsfrom various mouse tissues. A strong 2.2-kb band was detectedonly in the kidney (Fig. 9).

Protein Characterization under Nonreducing and ReducingConditions—Western blot analyses were performed on themembrane fraction prepared from mouse kidney in the pres-ence or the absence of 2-mercaptoethanol (Fig. 10). The anti-body raised against AGT1 recognized the band of 250 kDa inthe absence of 2-mercaptoethanol (nonreducing condition). Inthe presence of 2-mercaptoethanol (reducing condition), the250-kDa band detected in the nonreducing condition disap-peared, and a 40-kDa band was detected, consistent with thepredicted molecular mass of AGT1 monomer (51 kDa). The

FIG. 1. Sequence alignment of AGT1 and the structurally related transporters. The deduced amino acid sequence of AGT1 (mouse) isshown aligned with those of system asc transporter Asc-2 (mouse) (21), system L transporter LAT1 (rat) (7), system y�L transporter y�LAT1 (rat)(33), system x�

C transporter xCT (mouse) (15), and system b0,� transporter BAT1 (rat) (17). Identical residues in at least two sequences are shaded.Predicted transmembrane regions of AGT1, numbered 1–12, are shown by bold lines above the sequences. The conserved cysteine residue (AGT1amino acid residue 129) in the predicted extracellular loop, through which LAT1, y�LAT1, xCT, and BAT1 are proposed to link to 4F2hc or rBATis indicated by an asterisk. Protein kinase C-dependent phosphorylation sites are predicted on the AGT1 sequence at residues 5, 36, 282, 312, and419, among which the ones at 5 and 312 are predicted intracellularly (labeled with �). A potential cAMP-dependent phosphorylation site is locatedat residue 237, which is predicted to be intracellular (labeled with #). A tyrosine phosphorylation site is predicted at residue 17 (labeled with &).Although a potential N-glycosylation site is located at residue 259, it is predicted to be in the membrane-spanning region. The residue numbersindicated above the aligned sequences are in reference to those in the amino acid sequence of AGT1.

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bands disappeared in the presence of antigen peptides in theabsorption experiment, confirming the specificity of immuno-reactions (data not shown).

Immunolocalization of AGT1 in the Mouse Kidney—Immu-nohistochemical analysis on mouse kidney revealed the strongimmunoreactivity for AGT1 in the proximal tubules in theouter medulla and the distal tubules in the cortex (Fig. 11a).AGT1 immunoreactivity appeared to be localized on the baso-lateral membrane of the proximal tubule S3 segments (Fig.11c) and the distal convoluted tubules (Fig. 11d). In the absorp-tion experiments in which the tissue sections were treated withthe primary antibodies in the presence of antigen peptides, theimmunostaining was not detected, confirming the specificity ofthe immunoreactions (Fig. 11b).

DISCUSSION

In the present study, we identified a novel transporter AGT1that exhibits structural similarity to Asc-2, a member of theheterodimeric amino acid transporter family presumed to beassociated with unknown heavy chains. By generating fusionproteins with 4F2hc or rBAT, we were able to express AGT1 onthe Xenopus oocyte plasma membrane. AGT1 transportedacidic amino acids at high affinity in a Na�-independentmanner.

In the family of heterodimeric amino acid transporters, thetransporter proteins are linked via a disulfide bond to thesingle membrane spanning heavy chains 4F2hc or rBAT (7–19). Among the members of this family, a cysteine residue isconserved in the extracellular loop between predicted trans-membrane domains 3 and 4 (Fig. 1). Through this cysteineresidue, the transporter proteins of this family are proposed toform a disulfide bond with heavy chains (20). The cysteineresidue is also conserved in AGT1 as well as Asc-2 (Fig. 1). It istherefore predicted that AGT1 is linked to the other protein(s)by forming a disulfide bond through the conserved cysteineresidue.

For the members of the heterodimeric amino acid trans-porter family, the association with the heavy chain type IImembrane glycoproteins is required for the light chain trans-

porter proteins to be sorted to the plasma membrane (8, 18, 37).In the previous investigation, we showed that the fusion pro-teins in which Asc-2 was connected with 4F2hc or rBAT weresorted to the plasma membrane and exhibited their functions,although Asc-2 was not functional when solely expressed orcoexpressed with 4F2hc or rBAT (21). This indicates that4F2hc and rBAT are capable of supporting the membranesorting of the light chain subunit transporter proteins whentheir fusion proteins are constructed even though not betweenthe right partners. AGT1 is also proposed to require additionalassociating proteins similar to 4F2hc or rBAT but not 4F2hcand rBAT themselves, because AGT1 was not functional whensolely expressed or coexpressed with 4F2hc or rBAT (Fig. 2)and also because AGT1 is not colocalized with 4F2hc or rBAT inkidney in vivo; AGT1 is expressed in the basolateral membraneof proximal straight tubules and distal convoluted tubules (Fig.11), whereas rBAT is present in the apical membrane of prox-imal tubules, and 4F2hc is most densely expressed in thebasolateral membrane of proximal convoluted tubules (17, 19,31, 38). We thus generated fusion proteins in which the Cterminus of AGT1 is connected with the N terminus of 4F2hc orrBAT to examine the functional properties of AGT1. The fusionproteins in fact appeared on the plasma membrane and exhib-ited the transport activity when expressed in Xenopus oocytes(Figs. 2 and 3, e and f).

The fusion proteins of heavy chain and light chain subunitsof heterodimeric amino acid transporters were first generatedby Pfeiffer et al. (19) for the characterization of system b0,�

transporter b0,�AT/BAT1 in Xenopus oocytes. They showedthat the fusion protein in which the C terminus of the lightchain b0,�AT/BAT1 was connected with the N terminus of itsassociating heavy chain rBAT was functional and exhibited theidentical properties to those obtained by the coexpression ofb0,�AT/BAT1 and rBAT (19). In addition, the mutant fusionprotein whose light chain portion was mutated was not func-tional, confirming that the detected transport activity was dueto the fusion protein itself and not to the associated oocyteendogenous light chain (19). For the characterization of Asc-2,

FIG. 2. Functional expression of the AGT1–4F2hc and AGT1-rBAT fusion proteins. a, the uptake of [14C]L-aspartate (20 �M) wasmeasured in the Na�-free uptake solution on Xenopus oocytes injected with water (water) or Xenopus oocytes injected with 4F2hc cRNA (4F2hc),AGT1 cRNA (AGT1), both AGT1 cRNA and 4F2hc cRNA (AGT1 � 4F2hc), AGT1–4F2hc fusion protein cRNA (AGT1–4F2hc), or AGT1-rBAT fusionprotein cRNA (AGT1-rBAT). b, the uptake of [14C]L-aspartate (20 �M) was measured on mock transfected COS-7 cells (mock) and COS-7 cellstransfected with 4F2hc (4F2hc), rBAT (rBAT), AGT1 (AGT1), both AGT1 and 4F2hc (AGT1 � 4F2hc), or both AGT1 and rBAT (AGT1 � rBAT).

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we generated fusion proteins in which the C terminus of Asc-2is connected with the N terminus of 4F2hc or rBAT (21). Weshowed that two Asc-2 fusion proteins connected with rBAT or4F2hc exhibited basically identical properties in their ion de-pendence, affinity, and substrate selectivity, suggesting thatthe rBAT or 4F2hc portion of the fusion proteins does not affectthe transport properties of light chain transporter subunits(21). Consistent with this, LAT1 fusion proteins connected with4F2hc or rBAT exhibited substrate selectivity and affinity ba-sically identical to those obtained by the coexpression of LAT1and its partner heavy chain 4F2hc (21). In the present study,the fusion proteins AGT1–4F2hc and AGT1-rBAT were alsoshown to exhibit basically identical transport properties.Therefore, it is suggested that to generate fusion proteins with4F2hc or rBAT is a useful strategy to examine the functionalproperties at least for the heterodimeric amino acid transport-ers and their related proteins.

The AGT1–4F2hc and AGT1-rBAT fusion proteins exhibitedNa� and Cl�-independent transport of acidic amino acids (Fig.4). Furthermore, the AGT1–4F2hc fusion protein showed highaffinity to L-aspartate and L-glutamate. For mammalian acidicamino acid transport systems, four transport systems have

been characterized so far: Na�-dependent X�A,G and X�

A andNa�-independent x�

G, and x�C (1). X�

A,G transports both glu-tamate and aspartate, whereas X�

A largely excludes glutamate

FIG. 3. Detection of the AGT1 and AGT1–4F2hc fusion proteinexpressed in Xenopus oocytes. Confocal immunofluorescence micro-scopic analyses were performed on the Xenopus oocytes injected withwater as a control (a and b), AGT1 cRNA (c and d), or the cRNA forAGT1–4F2hc fusion protein (e and f) using anti-4F2hc antibody (a, c,and e) or anti-AGT1 antibody (b, d, and f). When the fusion protein wasexpressed in Xenopus oocytes, it was detected in the oocyte plasmamembrane with anti-4F2hc antibody (arrows in e) and with anti-AGT1antibody (arrows in f). When AGT1 was solely expressed, it was notdetected in the oocyte plasma membrane (arrowheads in d).

FIG. 4. Ion dependence of the transport mediated by AGT1–4F2hc fusion protein. The [14C]L-aspartate uptake (20 �M) mediatedby the AGT1–4F2hc fusion protein measured in the standard uptakesolution (Control) was compared with that measured in the Na�-freeuptake solution (Na�-free) and that measured in the Cl�-free uptakesolution (Cl�-free).

FIG. 5. Concentration dependence of [14C]L-aspartate uptake.The [14C]L-aspartate uptake by the oocytes expressing the AGT1–4F2hc fusion protein (a) or the AGT1-rBAT fusion protein (b) wasmeasured at 1, 3, 10, 30, 100, and 300 �M of L-aspartate in a Na�-freeuptake solution and plotted against the L-aspartate concentration. TheL-aspartate uptake was saturable and fit to the Michaelis-Mentencurve. The insets show Eadie-Hofstee plots of L-aspartate uptake thatwere used to determine the kinetic parameters. The Km and Vmax valueswere 25.5 � 5.9 �M and 0.67 � 0.013 pmol/min/oocyte for the AGT1–4F2hc fusion protein; 20.1 � 6.1 �M and 0.55 � 0.052 pmol/min/oocytefor the AGT1-rBAT fusion protein.

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and longer analogues. x�G transports glutamate and its ana-

logues, largely excluding aspartate and short analogues. x�C is

similar to x�G except that it transports cystine as well as

glutamate (1). The functional properties of AGT1 is, however,not able to be assigned to any of these classically characterizedamino acid transport systems. The reason for this is probablyin its restricted localization in the basolateral membrane ofrenal tubules (Figs. 9 and 11). In addition, Na�-dependenttransport systems for acidic amino acids were present not onlyin the apical membrane but also in the basolateral membraneof the tubular epithelial cells (39), which may mask the contri-bution of Na�-independent transport system in the basolateralmembrane. The transporters subserving systems X�

A,G and

x�C have been identified so far by molecular cloning ap-

proaches (15, 40). Five isoforms of Na�-dependent high affinityglutamate transporters, which belong to SLC1 family, andNa�-independent cystine/glutamate transporter xCT, whichbelongs to SLC7 family, have been identified for systems X�

A,G

and x�C, respectively (15, 40, 41).

xCT is a heterodimeric amino acid transporter that is asso-ciated with 4F2hc (15). xCT accepts L-glutamate, L-homocyste-ate, and L-cystine. L-Aspartate is not transported at high rateby xCT (15). It is proposed that the substrate-binding site ofxCT possesses a negative charge recognition site in the sidechain-binding site so that xCT recognizes amino acids as an-ions. Thus, the length of the side chain of substrate amino

FIG. 6. Inhibition of the [14C]L-aspartate uptake by various amino acids. The [14C]L-aspartate uptake (20 �M) mediated by AGT1–4F2hcfusion protein (a) or AGT1-rBAT fusion protein (b) was measured in the presence of 2 mM nonradiolabeled indicated amino acids. The uptake wasmeasured in the Na�-free uptake solution, and the values are expressed as percentages of the control L-aspartate uptake in the absence ofinhibitors ((�)). The L-aspartate uptake was inhibited by L-aspartate, L-glutamate, and L-cysteine. The asterisks indicate statistical significance.**, p � 0.01, Student’s unpaired t test.

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acids, namely the distance between the �-carbon and the neg-ative charge on the side chain, would be an important deter-minant for the amino acids to be accepted by the substratebinding site of xCT (4). It is therefore understandable thatglutamate and homocysteate are well accepted by xCT,whereas aspartate, which has a shorter side chain, is not. Thedistance between two carboxyl groups of L-cystine is probablywell suited to meet the requirement, so that cystine is proposedto be recognized as an anionic amino acid to be accepted by thebinding site (4).

Although AGT1 is structurally related to xCT, AGT1 exhibitsa remarkable difference in the selectivity for acidic amino acidsubstrates. In contrast to xCT, AGT1 well accepts acidic aminoacids with shorter side chains such as L-aspartate, threo-�-

hydroxyaspartate, L-serine-O-sulfate, L-cysteine sulfinate, L-cysteate, and L-glutamate (Figs. 7 and 8). AGT1 does not acceptacidic amino acids with longer side chains such as L-homocyste-ate and L-�-aminoadipate (Fig. 8). L-Cystine is not transportedby AGT1. Again, the length of the side chain of acidic aminoacids is an important determinant for substrates of AGT1. It ispredicted that the negative charge recognition site in the side

FIG. 7. The uptake of radiolabeledamino acids mediated by AGT1–4F2hc fusion protein. The uptake ratesof 20 �M radiolabeled amino acids medi-ated by the AGT1–4F2hc fusion proteinwere measured in Na�-free uptake solu-tion. The high uptake rates were observedfor L-aspartate and L-glutamate. Cyst, L-cystine; MeAIB, �-(aminomethyl)isobu-tyric acid; AIB, �-aminoisobutyric acid;BCH, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid.

FIG. 8. The effects of acidic amino acid analogues on AGT1-mediated transport. The [14C]L-aspartate uptake (20 �M) mediatedby AGT1–4F2hc fusion protein was measured in the presence of 2 mM

nonradiolabeled indicated acidic amino acid analogues. The uptake wasmeasured in the Na�-free uptake solution, and the values are expressedas percentages of the control L-aspartate uptake in the absence ofinhibitors ((�)). The L-aspartate uptake was markedly inhibited bythreo-�-hydroxyaspartate (THA), L-serine-O-sulfate (SOS), L-cysteinesulfinate, and L-cysteate but was not inhibited by L-�-aminoadipate,L-homocysteate, L-trans-pyrrolidine-2,4-dicarboxylate (PDC), and dihy-drokainate (DHK). The asterisks indicate statistical significance. **,p � 0.01, Student’s unpaired t test.

FIG. 9. Tissue distribution of AGT1. High stringency Northernhybridization analysis using an AGT1 probe was performed againstpoly(A)� RNA (3 �g) from mouse tissues. A strong hybridization band at2.2 kb was detected only in the kidney.

FIG. 10. Western blot analyses under reducing and nonreduc-ing conditions. Western blot analyses were performed using an anti-AGT1 antibody on the membrane fractions prepared from the mousekidney in the presence or absence of 2-mercaptoethanol. The 250-kDaband detected in the absence of 2-mercaptoethanol (�) shifted to the40-kDa band in the presence of 2-mercaptoethanol (�).

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chain-binding site of AGT1 is closer to the �-carbon binding sitethan that of xCT. It is interesting that Asc-2 structurally re-lated to AGT1 also accepts amino acids with short side chains,although Asc-2 prefers neutral amino acids (21). It is proposedthat, although the substrate-binding site of AGT1 possessessimilar spatial configuration to that of Asc-2, AGT1 has ac-quired the additional mechanisms for negative charge recogni-tion in the course of evolution.

Na�-dependent high affinity glutamate transporters trans-port not only L-glutamate and L-aspartate but also D-aspartate(40), whereas AGT1 is quite stereoselective for both aspartateand glutamate (Figs. 6 and 7). Although five isoforms of Na�-dependent high affinity glutamate transporters exhibit re-markable differences in the inhibitor selectivity, L-trans-pyr-rolidine-2,4-dicarboxylate is accepted by all of the isoforms andin general is regarded as a selective inhibitor for Na�-depend-ent high affinity glutamate transporters (40, 41). Dihydrokain-ate and L-�-aminoadipate are selective for the isoforms GLT-1/EAAT2 and EAAT4, respectively (40, 42–44). AGT1-mediated L-aspartate uptake is not affected by these inhibitorsfor Na�-dependent high affinity glutamate transporters. AGT1is thus supposed to possess quite distinct mechanisms of sub-strate recognition compared with Na�-dependent high affinityglutamate transporters.

AGT1 is only expressed in kidney. In the immunohistochem-ical analysis, AGT1 immunoreactivity was detected in the ba-solateral membrane of the proximal straight tubules, particu-larly S3 segments in the outer medulla, and of the distaltubules in the cortex (Fig. 11, a, c, and d). In the S2 and S3segments of the proximal tubules, Na�-dependent high affinityglutamate transporter EAAC1 is present in the apical mem-brane (45). EAAC1 plays a critical role in the reabsorption ofacidic amino acids from the luminal fluid because EAAC1knockout mice exhibit acidic amino aciduria (46). Although thelevel of expression is less than that in the proximal tubules,

EAAC1 is also present in the apical membrane of the distalconvoluted tubules in agreement with previous physiologicalstudies showing significant glutamate reabsorption distal tothe proximal tubules, including the distal convoluted tubules(45, 47, 48). Although the functional role of AGT1 is not clear atthis moment, considering the distribution of AGT1 alongnephron segments apparently corresponding to that of EAAC1,it is speculated that AGT1 might function as an exit path forthe acidic amino acids at the basolateral membrane of tubularepithelial cells in the reabsorption of acidic amino acids fromthe luminal fluid. It is also possible that AGT1 might contrib-ute to provide tubular epithelial cells with metabolically im-portant acidic amino acids from basolateral side, althoughNa�-dependent glutamate transport systems with higher con-centrating capability have been reported to be present in thebasolateral membrane (39).

In the Western blot that we performed on mouse kidney, ahigh molecular mass band detected in the nonreducing condi-tion shifted to the lower molecular mass band, which seems tocorrespond to the AGT1 monomer in the reducing condition(Fig. 10). This observation is interesting because AGT1 is pro-posed to be linked to the other protein by a disulfide bondthrough the conserved cysteine residue, although it is stillunclear at this stage whether the high molecular mass band inthe nonreducing condition is because AGT1 forms a hetero-meric complex or because AGT1 oligomerizes with other cys-teine residues.

In summary, we identified and characterized a novel aminoacid transporter AGT1 with structural similarity to the mem-bers of heterodimeric amino acid transporter family particu-larly Asc-2 that is proposed to be associated with unknownheavy chains (21). AGT1 exhibits distinct Na�-independenttransport activity with substrate selectivity for acidic aminoacids. Similar to Asc-2, AGT1 appears to be associated withunknown protein(s) other than 4F2hc or rBAT. The finding ofAGT1 has established a subgroup of the heterodimeric aminoacid transporter family, which includes transporters such asAsc-2 and AGT1 associated not with 4F2hc or rBAT but withother unidentified proteins.

Acknowledgments—We are grateful to Dr. Tatsuo Sakai (JuntendoUniversity), Dr. Masayuki Masu (Tsukuba University), and Dr. KazukoKeino-Masu (National Defense Medical College) for helpful discussions.We also thank Michi Takahashi for technical assistance. Mouse rBATcDNA was kindly provided by Dr. Ken-ichi Miyamoto (Tokushima Uni-versity). Anti-AGT1 and anti-4F2hc antibodies were supplied by theKumamoto Immunochemical Laboratory, Transgenic Inc. (Kumamoto,Japan).

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Tachampa, Hye Won Choi, Ellappan Babu, Jun Fukuda and Hitoshi EndouKim, Jun Inatomi, Yasuhiro Shigeta, Hisako Ishimine, Sophapun Chaekuntode, Kittipong

Hirotaka Matsuo, Yoshikatsu Kanai, Ju Young Kim, Arthit Chairoungdua, Do KyungFamily Associated with Unknown Heavy Chains

Structural Similarity to the Member of a Heterodimeric Amino Acid Transporter -independent Acidic Amino Acid Transporter with+Identification of a Novel Na

doi: 10.1074/jbc.M200019200 originally published online March 20, 20022002, 277:21017-21026.J. Biol. Chem. 

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