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. 1999 Dec;10(12):4135–4147. doi: 10.1091/mbc.10.12.4135

Luminal Heterodimeric Amino Acid Transporter Defective in Cystinuria

Rahel Pfeiffer *, Jan Loffing †, Grégoire Rossier ‡, Christian Bauch *, Christian Meier *, Thomas Eggermann §, Dominique Loffing-Cueni †, Lukas C Kühn ‡, François Verrey *,‖
Editor: Guido Guidotti
PMCID: PMC25748  PMID: 10588648

Abstract

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b0,+ type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b0,+AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b0,+ amino acid transporter complex. We demonstrate its b0,+-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b0,+AT protein is the product of the gene defective in non-type I cystinuria.

INTRODUCTION

The membrane glycoprotein rBAT (NBAT, D2) is related to the surface antigen 4F2hc/CD98. These proteins have a similar predicted membrane topology, with a single transmembrane domain and an intracellular NH2 terminus (type II membrane protein) (Bertran et al., 1992; Tate et al., 1992; Wells and Hediger, 1992). However, in the case of rBAT, an alternative four-transmembrane domain topology has also been proposed (Mosckovitz et al., 1994). When expressed alone in Xenopus laevis oocytes, rBAT produces a large amino acid transport, despite the fact that its structure does not resemble that of a transport protein. The induced transport system functions independent of Na+ and accepts cationic and zwitterionic dibasic amino acids (such as l-arginine, l-lysine, and l-cystine) with high affinity and, with a lower affinity, also zwitterionic (mono)amino acids (such as l-leucine, l-phenylalanine, and l-glutamine). These characteristics correspond to the b0,+ system first described in blastocysts, with the difference that rBAT also induces transport of l-cystine (Van Winkle, 1988). Extensive functional studies have shown that the b0,+ transport induced by rBAT expression in oocytes (also named b0,+-like transport) corresponds to an obligatory exchange of amino acids (Busch et al., 1994; Coady et al., 1994; Chillaron et al., 1996). The electrogenic uptake of cationic amino acids in exchange for neutral amino acids is stimulated by the membrane potential, and the uptake of l-cystine in exchange for monoamino acids is favored by the high concentration gradient maintained by the intracellular reduction of l-cystine to l-cysteine.

The glycoprotein rBAT has been shown to localize to the brush border membranes of kidney straight proximal tubule (S3 segment) (Kanai et al., 1992; Furriols et al., 1993) and small intestine, which are the sites of high-affinity l-cystine (re)absorption, a transport that can be competed by cationic amino acids (Foreman et al., 1980; Schafer and Watkins, 1984; Silbernagl, 1988; Riahi-Esfahani et al., 1995; Palacin et al., 1998). Mutations in the rBAT gene SLC3A1 have been shown to cause at least a large fraction of the cases of the human genetic disease cystinuria type I (OMIM 220100) (Calonge et al., 1994). This inherited malabsorption of l-cystine and dibasic amino acids across renal and intestinal epithelia leads to a severe kidney stone disease (Palacin et al., 1998, and references therein). Non-type I cystinuria (clinically divided in types II and III) differs from type I cystinuria in that heterozygotes have a larger amino aciduria than normals and type I heterozygotes (Rosenberg et al., 1966; Pras et al., 1998). Intestinal malabsorption is less severe in type III than in the other types. The locus of cystinuria type III has been mapped by linkage analysis to chromosome 19q13.1 (Bisceglia et al., 1997; Wartenfeld et al., 1997).

The apically restricted glycoprotein rBAT has been detected by SDS-PAGE analysis as a disulfide-linked oligomer of ∼130 kDa composed of an ∼85-kDa heavy chain (rBAT) and a second 40–50-kDa protein (light chain) in kidney cortex, jejunum, and rBAT-expressing Xenopus oocytes (Wang and Tate, 1995; Palacin et al., 1998). This is similar to the pattern obtained for the glycoprotein 4F2hc, which, in contrast to rBAT, is restricted to the basolateral membrane when expressed in epithelial cells (Quackenbush et al., 1986; Sordat, personal communication).

We and others have recently identified several amino acid permease-like light chains that need to associate with 4F2hc for surface expression (Kanai et al., 1998; Mannion et al., 1998; Mastroberardino et al., 1998; Torrents et al., 1998; Nakamura et al., 1999; Pfeiffer et al., 1999; Prasad et al., 1999; Rossier et al., 2000). These glycoprotein-associated amino acid transporters (gpaATs) function as amino acid exchangers and display L-type or y+L-type transport specificities with an epithelial, basolateral localization (y+LAT1 and LAT2) or a broad distribution (LAT1 [= E16/TA1, AmAT-L-lc] and y+LAT2) (Mastroberardino et al., 1998; Pfeiffer et al., 1999; Rossier et al., 2000; Sordat, personal communication). These highly lipophilic proteins share a topology prediction of 12 transmembrane domains with an intracellular NH2 terminus. The conserved cysteine residue that forms the disulfide bond with 4F2hc has been identified in the second putative extracellular loop (Pfeiffer et al., 1998) (see Figure 2E).

Figure 2.

Figure 2

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Selective coimmunoprecipitation of mb0,+AT with hrBAT (or hrBATFLAG) and of my+LAT1 with h4F2hc. Oocytes were injected with cRNA for a heavy chain (hc) (rBf = hrBATFLAG; rB = hrBAT; 4F2 = h4F2hc) and/or a light chain (lc) (b = mb0,+AT; y+L = my+LAT1) as indicated. (A) From oocytes expressing h4F2hc alone, anti-h4F2hc antibody precipitated a protein migrating as two bands of ∼66 and ∼80 kDa that correspond to the core- and terminally glycosylated forms of h4F2hc. As expected, coexpressed my+LAT1 was coprecipitated with h4F2hc and migrated as an additional band of ∼40 kDa when the sample was treated with the reducing agent β-mercaptoethanol (β-me) and as a heterodimeric complex of ∼130 kDa in its absence. In contrast, there was no coprecipitation of mb0,+AT with h4F2hc. (B) Anti-FLAG antibody precipitated a protein migrating as two bands of ∼80 and ∼90 kDa that presumably represent the core-glycosylated and the terminally glycosylated forms of hrBATFLAG. When the oocytes coexpressed mb0,+AT with hrBATFLAG, an additional band of ∼40 kDa was coprecipitated. In nondenaturing conditions, the proteins remained assembled mainly as heterodimers and migrated at a level of ∼135 kDa. my+LAT1 was not coprecipitated with rBATFLAG. (C) mb0,+AT was immunoprecipitated by anti-mb0,+AT antibody (∼40 kDa), and hrBAT and hrBATFLAG were coprecipitated and appear as additional bands of ∼80 and ∼90 kDa (compare with B). h4F2hc was not coprecipitated with mb0,+AT. (D) my+LAT1 was immunoprecipitated by anti-my+LAT1 antibody (∼40 kDa), and additional bands of ∼80 and ∼90 kDa corresponding to coprecipitated h4F2hc appeared when h4F2hc was coexpressed (compare with A). hrBAT was not coprecipitated with my+LAT1. (E) Scheme of the heterodimers formed by the heavy chain 4F2hc with LAT or y+LAT light chains and of the heterodimers formed by rBAT with b0,+AT. The single evident transmembrane domain of the heavy chains is labeled hc, and the 12 putative transmembrane domains of the lipophilic light chain are numbered 1–12. The dotted line between the COOH terminus of b0,+AT and the NH2 terminus of rBAT represents the linker sequence introduced to form a functional fusion protein. The localization of the conserved cysteine residues forming the intermolecular disulfide bond has been demonstrated for the heavy chain 4F2hc and the light chains Xenopus LAT1 (ASUR4) and Schistosoma mansoni SPRM1 (Pfeiffer et al., 1998). Potential N-glycosylation sites are indicated by forks.

We have now identified an additional member of the gpaAT family (mouse and human b0,+AT) that specifically associates with luminal rBAT, instead of basolateral 4F2hc, to form the b0,+ amino acid transporter complex. We show by in situ hybridization and immunofluorescence the localization of b0,+AT in the kidney and by expression studies in Xenopus oocytes the transport function of the transporter complex (b0,+ATc). Concurrently to our study, the members of the cystinuria consortium have identified in the gene encoding the b0,+AT transporter (SLC7A9) several mutations causing non-type I cystinuria (Palacin, personal communication). This latter study verifies at the genetic level that b0,+AT is involved in the amino acid transport that is defective in cystinuria, whereas our study confirms biochemically, functionally, and at the level of the localization that the SLC7A9 gene encodes the catalytic subunit of the transporter defective in cystinuria.

MATERIALS AND METHODS

Identification and Cloning of b0,+AT cDNAs from Mouse and Human

The cDNA for mb0,+AT was obtained by the ligation of two mouse expressed sequence tag (EST) cDNAs obtained from the I.M.A.G.E. consortium (accession numbers AA273998 and AA105180). Both plasmids were digested with BglII and NotI, and a 400-base pair (bp) fragment of the AA105180 cDNA corresponding to the 3′ end of the mRNA was isolated and cloned into the corresponding sites of AA273998. The mb0,+AT cDNA coding sequence was subcloned in the vector pSD5easy (Puoti et al., 1997). The cDNA representing an 850-bp 3′ fragment of hb0,+AT was obtained from the I.M.A.G.E. consortium (accession number R07056). The full-length clone of hb0,+AT was obtained using Marathon-Ready kidney cDNA (Clontech Laboratories, Palo Alto, CA) as template for 5′ rapid amplification of cDNA ends PCR. The reaction was set up according to the manufacturer’s protocol using adaptor primer 1, the gene-specific primer 5′-GGAACGATCCAAGAAGCAGGATAGAG-3′, and the Advantage-HF polymerase kit (Clontech Laboratories). The cycling parameters were as follows: 1 min at 94°C, 5 cycles of 30 s at 94°C/30 s at 68°C/3.5 min at 72°C, 5 cycles of 30 s at 94°C/30 s at 68°C/3.5 min at 70°C, and 25 cycles of 30 s at 94°C/4 min at 68°C. A 1017-bp fragment was recovered by gel extraction, cloned in a TA-type cloning vector (TOPO TA cloning kit, Invitrogen, Carlsbad, CA), and sequenced on both strands (Microsynth, Balgach, Switzerland). The 5′ fragment was ligated to the 3′ EST fragment in the cloning vector pT7T3pac using the restriction sites BamHI in the overlapping sequence and EcoRI in the polylinker.

Construction of the hrBAT-mb0,+AT Fusion Protein

The linker oligonucleotide encoding the amino acid sequence GAAPDGAPGC was introduced at the 3′ end of the mb0,+AT cDNA coding sequence starting just before the stop codon by PCR. An amplicon was generated using a sense primer corresponding to the SP6 promoter 5′ of the mb0,+AT cDNA and a reverse primer corresponding to the end of the coding sequence extended at its 5′ end by the linker sequence and an NsiI restriction site with an in-frame ATG codon (5′-TTGAATGCATCCAGGAGCACCATCAGGAGCA-GCACCCTCTGGGTCCTTTTCTGGTGGGAC-3′). The cDNA fragment encoding b0,+AT and the linker sequence was isolated after cutting its ends with XhoI and NsiI. An NsiI restriction site was introduced in hrBAT at the level of the ATG start codon by modifying the two preceding nucleotides (GA→TG) using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The primer sequences were 5′-GAAGACATAAGTCGGTGATGCATGGCTGAAGATAAAAGC-3′ and the corresponding antisense sequence. The b0,+AT-encoding fragment was introduced into the vector containing hrBAT previously linearized using SalI (compatible with XhoI) and NsiI.

FLAG Epitope at the COOH Terminus of hrBAT

A FLAG epitope (DYKDDDDK) was added to the COOH terminus of hrBAT (Bertran et al., 1992) using the QuickChange site-directed mutagenesis kit (Stratagene). Briefly, the entire plasmid DNA was amplified using primers each containing a 5′ extension coding for half of the added epitope (5′-GTCCTTGTAGTCACACGAGGTATACAGTATGTTCAGT-3′ and 5′-GACGATGACAAGTAGGCACCTTTATGAAGAGATGAAG-3′). The product was circularized by ligation and used for Escherichia coli transformation. The resulting construct was verified by sequence analysis.

cRNA Synthesis

Plasmids containing the cDNA of hrBAT, hrBAT-mb0,+AT fusion protein (both vector pSPORT), mb0,+AT, and my+LAT1 (both pSD5easy) were linearized using the restriction sites HindIII for the plasmid pSPORT and PvuII and BglII for pSD5easy containing mb0,+AT and my+LAT1, respectively. cRNA was synthesized with T7 (pSPORT) and SP6 (pSD5easy) RNA polymerase (Promega, Madison, WI), according to standard protocols.

Amino Acid Uptake in X. laevis Oocytes

The treatment of oocytes and uptake experiments were performed essentially as described previously (Pfeiffer et al., 1999). Oocytes were injected with 0.25–10 ng of cRNA dissolved in 33 nl of water and kept for 2–4 d at 16°C in ND96 buffer. Uptake experiments were performed for 1 min (the linearity of uptake was verified in preliminary time course experiments) in buffer supplemented with amino acid at the indicated concentrations and the corresponding 3H-labeled l-amino acid as tracer (except for [14C]l-cystine and [14C]l-isoleucine). Diamide (10 mM) was added to the buffer for experiments with l-cystine.

Amino Acid Uptake in Mouse M1 Cell Lines

The mouse M1 cell line was transfected with expression constructs for mb0,+AT, hrBAT, both (sequentially), or the fusion protein hrBAT-mb0,+AT and a selective marker. Stable cell lines were isolated by ring cloning or limit dilution (Bauch and Verrey, unpublished data). For uptake experiments, cells were seeded on 3.8-cm2 cell culture dishes. Before the uptake experiment, cells were washed three times and preincubated for 10 min with buffer containing 150 mM choline-Cl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, pH 7.4. Then, 80 μl of buffer supplemented with 2.5 μM l-cystine (containing [14C]l-cystine as tracer) and 10 mM diamide was given for 1 min. In control cells, an excess of 200 μM cold l-cystine was added. After removal of the uptake buffer, cells were washed three times and solubilized, and radioactivity was counted by liquid scintillation.

Immunoprecipitation

Oocyte labeling with [35S]methionine for 48 h and lysate preparation were performed as described by Mastroberardino et al. (1998). Polyclonal rabbit antibodies were raised against synthetic peptides corresponding to the NH2 termini of mb0,+AT and hrBAT (number 400, NH2-MEETSLRRRREDEKSTHC-COOH, and number 563, NH2-MAEDKSKRDSIEMSMKGC-COOH, respectively) coupled to keyhole limpet hemocyanin (Eurogentec, Seraing, Belgium) and were used, along with a polyclonal anti-FLAG antibody (Santa Cruz Biotechnology, Santa Cruz, CA), for immunoprecipitations. Antibodies were prebound to protein G plus protein A–agarose (Calbiochem, La Jolla, CA) for 2 h at room temperature. The beads were then added to lysate (each sample containing the same amount of incorporated radioactive methionine) that had been precleared by two incubations with uncoated beads. For precipitation with anti-FLAG antibody, the lysate was first treated with 3% SDS and 5 min of boiling and diluted 10-fold with buffer containing 1% NP40. The beads were rotated overnight at 4°C and then washed. The precipitate was eluted in SDS-PAGE sample buffer and heated to 65°C for 15 min. β-Mercaptoethanol was added where indicated, and SDS-PAGE analysis was performed. Gels were stained in Coomassie blue, fixed, incubated in Amplify (Amersham, Arlington Heights, IL), dried, and exposed to film.

Northern Blot

Total RNA from various tissues of two female and two male (for testes) B10D2 mice (kindly provided by Werner Held, Ludwig Institute for Cancer Research) and a B6D2 mouse (for placenta) (kindly provided by Friedrich Beerman, Swiss Institute for Experimental Cancer Research) was isolated using a cesium chloride method. Fifteen micrograms of total RNA was loaded on a 1.2% agarose/formaldehyde gel, transferred onto GeneScreen Plus membrane (New England Nuclear, Boston, MA), and UV cross-linked. mb0,+AT (bp 1–515) and mrBAT (EST cDNA AA388506, bp 21–363) cDNA fragments were labeled with [α-32P]dCTP by random priming. Hybridized and washed membranes were exposed on Kodak (Rochester, NY) Biomax film.

Tissue Fixation and Processing

Adult male Zur:ICR mice (Institut für Labortierkunde, Zürich, Switzerland) were anesthetized with an intraperitoneal injection of 50 μl of anesthetic solution, and tissues were then fixed and processed as described by Loffing et al. (1996).

In Situ Hybridization

A mouse rBAT cDNA fragment was generated by reverse transcriptase PCR using the following primers derived from a published sequence: 5′-AGCACCAGGAAGAGCTACACAGGGT-3′ and 5′-ACGTGCATCGTGCTACAGAAATATCTA-3′. This fragment was subcloned in pSPORT1. Digoxigenin-11-UTP–labeled riboprobes for mouse rBAT and b0,+AT were synthesized by in vitro transcription (DIG RNA labeling kit [Sp6/T7], Boehringer Mannheim [Indianapolis, IN]). Plasmids containing either mouse rBAT cDNA or b0,+AT cDNA were linearized using the appropriate restriction enzymes Antisense probes for mouse rBAT and b0,+AT were generated by T7 RNA polymerase and T3 RNA polymerase, respectively. For control, sense riboprobes for mouse rBAT and b0,+AT were transcribed by Sp6 RNA polymerase and T7 RNA polymerase, respectively. RNA probes were shortened by alkaline hydrolysis to fragments of an average length of ∼200 bp.

Cryostat sections (7 μm thick) were postfixed with 4% paraformaldehyde/PBS for 20 min at room temperature. The slides were then rinsed with diethylpyrocarbonate-treated water followed by two short washes in PBS. The cryosections were pretreated for 10 min with proteinase K (5 μg/ml; Sigma, St. Louis, MO) and 0.025% collagenase type 5 (Sigma) at room temperature followed by two short rinses in PBS. Acetylation (0.1 M triethanolamine and 0.25% acetic anhydrate) was performed for 20 min at room temperature. After two short rinses in PBS, sections were dehydrated in ethanol (70, 80, and 100%) and air dried for 20 min. Prehybridization was performed at 42°C for 2 h (50% formamide, 50 mM Tris-HCl, pH 7.6, 25 mM EDTA, pH 8, 20 mM NaCl, 0.2% SDS, 2.5× Denhardt’s solution, 0.25 mg/ml tRNA). Hybridization was performed at 42°C for 16–18 h (50% formamide, 20 mM Tris-HCl, pH 7.6, 1 mM EDTA, pH 8, 33 mM NaCl, 10 mM DTT, 0.5 mg/ml tRNA, 0.1 mg/ml DNA, 1× Denhardt’s solution, 12.5% dextran sulfate; optimal concentration of digoxigenin-labeled riboprobes was 8 ng/μl of hybridization mixture).

Slides were washed once with 2× SSC (SSC = 150 mM NaCl, 15 mM Na citrate) at room temperature for 15 min followed by three sequential washes at 49°C each for 1 h in 1× SSC/50% formamide followed by 0.5× SSC/50% formamide and 0.2× SSC/50% formamide. This treatment was followed by two short rinsing steps in 0.5× SSC and 0.2× SSC. Digoxigenin-labeled probes were detected with the DIG RNA detection kit (Boehringer Mannheim) according to the manufacturer’s instructions. The provided alkaline phosphatase–linked sheep anti-digoxigenin antibody was diluted 1:500. Nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate served as chromogenic substrates for the alkaline phosphatase–catalyzed color reaction. Endogenous activity of alkaline phosphatase was blocked by the addition of 5 mM levamisole (Sigma) to the substrate solution.

Immunohistochemistry

Serial sections (4 μm thick) were cut in a cryostat and placed on chrome-alum-gelatin–coated glass slides. After preincubation of the sections with 10% normal goat serum (DAKO, Glostrup, Denmark), the sections were incubated overnight at 4°C with a 1:500 dilution of antiserum 400, which was raised against a NH2-terminal peptide of b0,+AT (see above). After repeated washing with PBS, the binding sites of the primary antibody were revealed with a Cy3-conjugated donkey anti-rabbit immunoglobulin G (Jackson Immunoresearch, West Grove, PA) diluted 1:1000. Subsequently, sections were washed in PBS, mounted in DAKO-glycergel (DAKO) containing 2.5% 1,4-diazabicyclo [2,2,2]octane as fading retardant, and studied by epifluorescence with a Polyvar microscope (Reichert Jung, Vienna, Austria). For controls, consecutive cryosections were incubated with either preimmune serum or the anti-b0,+AT antiserum preabsorbed for 1 h with the corresponding antigenic peptide (20 μg/ml). Digitized images were acquired with a VISICAM charge-coupled device camera (Visitron, Puchheim, Germany) attached to the microscope and processed by Image-Pro Plus version 3.0 software (Media Cybernetics, Silver Spring, MD).

Fluorescent In Situ Hybridization

The cDNA hb0,+AT was labeled with Biotin-11-dUTP in a nick translation reaction (GIBCO-BRL, Gaithersburg, MD), and 1 μg was precipitated with 10 μg of salmon sperm DNA and 100 μg of cot1 DNA, dissolved in hybridization buffer (50% deionized formamide, 10% dextran sulfate, 2× SSC), and denatured at 75°C for 5 min. Metaphase spreads were prepared from human peripheral blood lymphocyte cultures by standard procedures, and the chromosomal DNA was denatured for 3 min at 75°C in buffer containing 70% formamide and 2× SSC, pH 7.0. After passing the denatured spreads through an ice-cold ethanol series (70%, 90%, and absolute for 3 min each, air drying), probe was dropped onto them, covered with an 18- × 18-mm coverslip, sealed with rubber cement, and incubated at 37°C for 12 h. High-stringency washes were performed three times for 5 min in 50% formamide/2× SSC at 45°C and in 0.1× SSC at 60°C, followed by a final wash of 5 min in 4× SSC/0.05% Tween 20 at room temperature. Hybridization signals were revealed using avidin-FITC (Vector Laboratories, Burlingame, CA), and chromosomes were counterstained with DAPI.

Data Bank Accession Numbers

Accession numbers are as follows: mb0,+AT, AJ249198; hb0,+AT, AJ249199.

RESULTS

Recently, LAT1 and y+LAT1 have been identified as light chains of the type II glycoprotein 4F2hc (Kanai et al., 1998; Mastroberardino et al., 1998; Torrents et al., 1998; Pfeiffer et al., 1999), a surface protein with a high level of structural similarity to rBAT (Bertran et al., 1992; Tate et al., 1992; Wells and Hediger, 1992). Performing database searches (BLAST programs, National Center for Biotechnology Information) with the sequences of hLAT1 and my+LAT1, we identified two overlapping mouse EST sequences that together form a cDNA encoding a full-length 487-amino acid protein (mb0,+AT) that is equally similar to human LAT1 and y+LAT1 (44% identity on 483–486 aligned amino acids; ToPLign Server, Sankt Augustin, Germany) (Figure 1). We further identified an ∼850-bp EST cDNA that corresponds to the 3′ end of the human orthologue mRNA. The missing part of the human sequence was obtained by 5′ rapid amplification of cDNA ends PCR. The deduced primary structures from mouse and human b0,+AT have the same number of amino acids and are 86.7% identical (Figure 1).

Figure 1.

Figure 1

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Alignment of mouse and human b0,+AT amino acid sequences with hLAT1 and hy+LAT1. Amino acids identical to those of mb0,+AT are shown in boldface type. Putative transmembrane domains (TMDs), the positions of which represent compromises based on the predictions obtained for the different sequences (TMpred server, Swiss Institute for Experimental Cancer Research), are numbered from 1 to 12. The conserved cysteine residue known to be involved in the disulfide bond of LAT1 (Pfeiffer et al., 1998) with h4F2hc is indicated by the letter C.

mb0,+AT Forms a Covalently Linked Heterodimer with hrBAT

We have previously shown by immunoprecipitation that the surface glycoprotein h4F2hc very efficiently covalently binds to at least three different members of the glycoprotein-associated amino acid transporter family, LAT1, LAT2 (Rossier et al., 2000), and y+LAT1, as demonstrated by coimmunoprecipitation of proteins coexpressed in Xenopus oocytes. Figure 2A shows that, in contrast, mb0,+AT did not bind to h4F2hc. Indeed, as expected, immunoprecipitation of h4F2hc yielded a band migrating at the level of ∼80 kDa on SDS-PAGE (core glycosylated form, ∼66 kDa), and coexpressed my+LAT1 was coprecipitated and migrated as an ∼40-kDa band when the samples were reduced (Mastroberardino et al., 1998; Pfeiffer et al., 1998, 1999). Coexpression of y+LAT strongly increased the proportion of the core-glycosylated form of 4F2hc that migrated at 66 kDa, as discussed previously (Pfeiffer et al., 1999). When reduction was omitted, the heterodimer formed by h4F2hc and my+LAT1 migrated as an ∼130-kDa complex (some complex was also visible after reduction). In contrast, no coprecipitated light chain was observed when h4F2hc was coexpressed with mb0,+AT, showing that y+LAT1 but not b0,+AT heterodimerizes with 4F2hc.

To test the hypothesis that b0,+AT heterodimerizes with rBAT, the 4F2hc-related apical protein known to be defective in many cases of cystinuria type I, coexpression experiments of mb0,+AT and hrBAT (extended at the COOH terminus by a FLAG epitope [hrBATFLAG]) were performed (Figure 2B). The anti-FLAG antibody was shown to be specific, because it immunoprecipitated hrBATFLAG (doublet of ∼80 and ∼90 kDa corresponding most probably to core-glycosylated and terminally glycosylated forms, respectively) but not hrBAT without FLAG, h4F2hc, or any of the light chains expressed alone. Coexpression of hrBATFLAG with mb0,+AT but not my+LAT1 led to the appearance of an additional band of ∼40 kDa under reducing conditions, corresponding to coprecipitated mb0,+AT. Under nonreducing conditions, the complex remained associated and migrated as a major heterodimeric complex of ∼135 kDa and as higher-molecular-mass bands corresponding possibly to higher-order complexes. The same pattern was obtained using anti-hrBAT antibody (data not shown). These results indicate that hrBAT and mb0,+AT are covalently linked by a disulfide bond, similar to the association of the other light chains with 4F2hc. A band at the same level (∼135 kDa) was also apparent when hrBATFLAG was expressed alone or in the presence of my+LAT1. This band is slightly weaker and corresponds most probably to the heterodimeric complex formed by hrBATFLAG and an endogenous oocyte light chain (Wang and Tate, 1995; Palacin et al., 1996). The fact that this putative endogenous light chain is hardly visible when the complex is dissociated by reduction indicates that it is only weakly labeled by the pulse-labeling procedure and thus that most of this endogenous protein must preexist the expression of exogenous rBAT. This situation is analogous to that of the Na,K-ATPase α subunit of Xenopus oocytes, which is present in large excess over the endogenous β subunit and can be activated by expressing exogenous β subunit (Geering et al., 1989).

Similar experiments were also performed with antibodies recognizing the light chains (Figure 2, C and D). Immunoprecipitation with anti-mb0,+AT antibody (Figure 2C) yielded the expected band of ∼40 kDa for mb0,+AT. Efficient coprecipitation of hrBAT and hrBATFLAG (80- and 90-kDa bands, respectively, after reduction) but not of h4F2hc was observed. In nonreducing conditions, an ∼135-kDa band that probably represents a heterodimer of mb0,+AT and hrBAT was visible only when hrBAT was coexpressed with mb0,+AT. In contrast, a high-molecular-mass aggregate, the nature of which is not known, also was visible in the absence of hrBAT. Figure 2D shows that anti-my+LAT1 antibody coprecipitates h4F2hc (∼66- and 80-kDa bands in reducing conditions and an ∼130-kDa band [heterodimer] in nonreducing conditions) with my+LAT but not hrBAT or hrBATFLAG (Pfeiffer et al., 1999). These results demonstrate that mb0,+AT heterodimerizes with hrBAT but not with h4F2hc, in contrast to my+LAT1, which associates with h4F2hc but not with hrBAT. Figure 2E shows a scheme of the heterodimers formed by 4F2hc with LAT or y+LAT light chains and of those formed by rBAT with b0,+AT. The cytosolic localization of the light chain COOH terminus is confirmed by the expression of a functional fusion protein when b0,+AT and rBAT are fused by a 10-amino acid linker sequence (dotted line; see below).

Transport Properties of mb0,+AT

To test whether b0,+AT functions as an amino acid transporter, we measured the uptake of radioactively labeled l-arginine in water-injected oocytes and oocytes expressing mb0,+AT (Figure 3A). No uptake of l-arginine over background levels was observed in oocytes expressing mb0,+AT alone. As expected, hrBAT induced an efficient uptake of l-arginine, and coexpression of mb0,+AT did not further increase the uptake of l-arginine. This is compatible with the hypothesis that hrBAT very efficiently associates with an endogenous oocyte transport protein light chain (Wang and Tate, 1995; Palacin et al., 1996) and that much of this light chain preexists hrBAT synthesis, as indicated by the result shown in Figure 2B and discussed above.

Figure 3.

Figure 3

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Both hrBAT and mb0,+AT are required for the expression of b0,+-type transport function. (A) No l-arginine uptake over background level was observed when oocytes expressed the b0,+AT light chain (b) alone. hrBAT (rB) alone, which associated with endogenous oocyte light chains, induced a high transport rate. Coexpression of the b0,+AT light chain with rBAT did not modify the transport rate significantly. The fusion protein hrBAT-mb0,+AT (brB) induced in oocytes an l-arginine uptake that was severalfold higher than that observed with fusion protein containing the mutant b0,+ATE244Q light chain moiety (bmrB) or that of water-injectedoocytes. (B) Oocytes were first injected with the light chain cRNA for mb0,+AT, mb0,+ATE244Q, or my+LAT. After 2 d of expression, hrBAT cRNA was injected and l-arginine uptake was measured 24 h later. The preexpression of the mutant light chain mb0,+ATE244Q nearly fully inhibited l-arginine uptake expression induced by rBAT, presumably by efficiently competing with functional endogenous chains for association with rBAT. Means of 12 oocytes ± SEM are shown. (C) Mouse M1 cell lines expressing rBAT, b0,+AT, both, or the fusion protein hrBAT-mb0,+AT (brB) were seeded on plastic dishes, and the uptake of l-cystine (2.5 μM) was measured. A single experiment with one cell line each is shown. Several cell lines were tested for single and double transfectants and gave comparable results. Uptakes significantly higher than by untransfected control cells were obtained only in double transfectant and fusion protein–expressing cell lines (Bauch and Verrey, unpublished data).

To prevent expressed hrBAT from heterodimerizing with this preexisting endogenous oocyte light chain, we constructed a cDNA encoding a fusion protein in which hrBAT was linked by its NH2 terminus to the COOH terminus of mb0,+AT. A linker sequence of 10 amino acids was introduced between the two protein moieties (see scheme in Figure 2E). This sequence was designed to contain, besides a negative charge, several alanine and proline residues (Mao et al., 1995). Although l-arginine uptake by oocytes expressing this fusion protein was lower than in hrBAT-expressing oocytes, there was a threefold to sixfold induction of amino acid transport over background levels. In contrast, when the conserved residue Glu-244 of the mb0,+AT moiety of the fusion protein was mutated to Gln (located in the putative intracellular loop between transmembrane domains 6 and 7), l-arginine uptake was completely abolished. This shows that amino acid transport is mediated by the fused b0,+AT light chain and not by an associated oocyte light chain. This point mutation in b0,+AT was chosen based on preliminary tests made with SPRM1, a Schistosoma mansoni member of the gpaAT family that heterodimerizes with 4F2hc (Mastroberardino et al., 1998). In this light chain, the equivalent mutation (E237Q) was shown to completely abolish transport function, whereas the cell surface expression was identical to that of wild-type SPRM1 (Mastroberardino et al., 1998) (data not shown).

To functionally confirm that in oocytes rBAT can associate with either endogenous light chain and/or with coexpressed b0,+AT light chain, the b0,+ transport function was measured after injection of different exogenous light chains potentially competing with the endogenous one for binding to rBAT (Figure 3B). The biosynthetic apparatus of oocytes was prefilled with various exogenous light chains by first injecting a large amount of their cRNAs (10 ng). These preinjected light chains were wild-type mb0,+AT, shown above to bind rBAT and which presumably is functional, mutant mb0,+AT, which binds to rBAT but is not functional, and my+LAT1, which does not bind to rBAT and thus is not expected to compete with endogenous light chains. cRNA of hrBAT (2.5 ng) was injected 2 d after the light chain cRNA, and l-arginine uptake experiments were performed 1 d later. In these conditions, my+LAT1 and mb0,+AT preexpression slightly decreased l-arginine uptake compared with control oocytes preinjected with water only, probably because of competition at the level of the translation machinery of the large amount of preinjected cRNA with the postinjected rBAT cRNA. In contrast, l-arginine uptake was completely inhibited by the preexpression of mutant mb0,+AT. Thus, in the presence of high amounts of exogenous mutant mb0,+AT light chain, hrBAT no longer efficiently binds to endogenous light chain. This strongly suggests that in the case of preexpression of wild-type mb0,+AT, hrBAT similarly associates mainly with this exogenous light chain and thus the measured transport is mediated by the complex hrBAT-mb0,+AT.

To verify in cells expressing no endogenous b0,+AT light chain that both rBAT and the light chain b0,+AT are required to form the functional high-affinity l-cystine transporter, mouse epithelial M1 cell lines expressing either one or both subunits were produced. Figure 3C shows an example of l-cystine (2.5 μM) uptake obtained with one set of cell lines. In none of the cell lines expressing a single subunit was the rate of [14C]l-cystine uptake (inhibited by cold l-cystine) significantly greater than the level measured in untransfected controls. However, a 6- to 40-fold higher transport rate was observed in the cell lines expressing both rBAT and b0,+AT. The fact that cell lines expressing the hrBAT-mb0,+AT fusion protein produced l-cystine uptake like those expressing the unlinked subunits supports our conclusion that the transports measured in Xenopus oocytes with the same fusion protein are representative of the function of the hetero-oligomeric b0,+ATc.

To test the specificity of the transport induced by the rBAT-b0,+AT complex in Xenopus oocytes, experiments were performed with the fusion protein hrBAT-mb0,+AT. Oocytes were injected with cRNA for the mutant fusion protein hrBAT-mb0,+ATE244Q or water for controls. The uptake of radioactively labeled amino acids (100 μM) was measured within the linear phase of transport and is given in picomoles per hour (Figure 4). The fusion protein transported, independent of the presence of Na+, the cationic dibasic amino acids l-arginine and l-lysine, the neutral dibasic l-cystine, and the neutral monoamino acids l-leucine and l-alanine, as expected for b0,+-type rBAT-associated amino acid transport. There was little transport of l-tyrosine. The β-branched neutral amino acids l-valine and l-isoleucine as well as l-glycine were not transported significantly in these conditions.

Figure 4.

Figure 4

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Specificity of amino acid uptake by oocytes expressing the mrBAT-b0,+AT fusion protein. The tested amino acids were given for 1 min at a concentration of 100 μM in the presence (A) or absence (B) of Na+. The black bars represent the test values for oocytes expressing the fusion protein mrBAT-b0,+AT. Oocytes injected with water (white bars) or the mutant fusion protein hrBAT-mb0,+ATE244Q (gray bars) represent controls. Means of 12 oocytes ± SEM pooled from two independent experiments are shown.

Figure 5 shows the plots of dose-response experiments performed for l-arginine, l-cystine, and l-leucine uptakes. Curves corresponding to Michaelis-Menten kinetics were fitted to the experimental points, and apparent affinity values (Km) were derived. The Km values (±SEM) for l-arginine and l-cystine were 72 ± 35 μM and 41 ± 20 μM, respectively. The Km for l-leucine was much higher (1.1 ± 0.7 mM) and was also higher than previously reported for oocytes injected with rBAT alone. The difference might represent a species variation between the mammalian b0,+AT light chain and the endogenous X. laevis light chain present in oocytes. The higher affinity of mb0,+AT toward l-arginine and l-cystine compared with l-leucine might reflect the function of the transporter, which has been shown to preferentially take up dibasic amino acids in exchange for neutral amino acids (Chillaron et al., 1996). Experiments with epithelial cells expressing both subunits of the b0,+ATc (see above) show apparent affinities for the uptake of amino acids similar to those measured here for Xenopus oocytes expressing the fusion protein hrBAT-mb0,+AT (Bauch and Verrey, unpublished observations).

Figure 5.

Figure 5

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Concentration dependence of l-cystine (○), l-arginine (▾), and l-leucine (●) uptake by oocytes expressing the hrBAT-mb0,+AT fusion protein. Uptake experiments were performed at five or six different amino acid concentrations, and background uptake by water-injected oocytes was subtracted. Sigmoidal curves corresponding to Michaelis-Menten kinetics were fitted to the experimental data using a nonlinear regression routine. For every concentration, data from means of 12 oocytes ± SEM pooled from two independent experiments are shown. The apparent Km derived from the curves for l-cystine, l-arginine, and l-leucine were 41 μM, 72 μM, and 1.1 mM, respectively.

Preliminary experiments have shown that l-arginine efflux from the oocytes takes place only when the fusion protein hrBAT-mb0,+AT containing the wild-type mb0,+AT moiety is expressed and that this efflux is strictly dependent on the presence of an extracellular amino acid (our unpublished results). This confirms results obtained with rBAT expressed alone and thus associated with endogenous oocyte light chain (Busch et al., 1994; Coady et al., 1994; Chillaron et al., 1996).

Common and Differential Localizations of b0,+AT and rBAT

Northern blot analysis of mouse tissues shows that a major b0,+AT transcript of ∼2 kilobase (kb) is expressed specifically in (small) intestine and kidney (Figure 6A). Weaker signals at a higher molecular weight (∼4.5 kb and/or ∼6 kb) can be seen in most other tested tissues. rBAT data (Figure 6B) are shown for comparison. In this case, the signal of the major transcript (∼2.5 kb) also is clearly visible only in kidney and intestine and is slightly visible in liver. A higher-molecular-weight transcript (∼4.5 kb) is expressed in brain, as reported previously in other species (Bertran et al., 1992, 1993; Yan et al., 1992).

Figure 6.

Figure 6

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Tissue distribution of rBAT and b0,+AT in the mouse. (A) Northern blot analysis of mouse tissue RNAs shows a major b0,+AT transcript of ∼2 kb specifically expressed in intestine and kidney. Weaker signals at a higher molecular weight (∼4.5 kb and/or ∼6 kb) can be seen in most other tested tissues. (B) The major rBAT transcript (∼2.5 kb) is also clearly visible only in kidney and intestine and is only slightly visible in liver on the original autoradiograph. A higher-molecular-weight transcript (∼4.5 kb) is visible only in brain.

The localization of rBAT and b0,+AT in the kidney was analyzed by in situ hybridization (rBAT and b0,+AT) and immunofluorescence (b0,+AT) (Figure 7). Panels b and g show that in mice, rBAT mRNA was localized not only to the straight part of the proximal tubules (S3 segments), as reported previously for the protein of rat (Furriols et al., 1993), but also, although at a lower level, to the preceding S1 and S2 segments, as reported previously for the mRNA of rat (Kanai et al., 1992). By contrast, b0,+AT mRNA was most highly expressed in the S1 segments (panels c and h) and was only barely detectable in S2 and S3 segments within the medullary rays and outer medulla (panel h). Immunofluorescence confirmed, at the protein level, that b0,+AT was highly abundant in S1 (panels d and i) and was present at lower levels in S2 and S3 segments. Staining intensity in S3 segments was consistently lower than that in S2 segments (panels i and k). The immunohistochemistry clearly demonstrated that, at the subcellular level, b0,+AT was present in the brush border along the lumen of the entire proximal tubule from the beginning of S1 (panel i) to the end of S3 (panel k). The same immunohistochemical staining pattern was found in rat kidneys (data not shown). No staining was observed in glomeruli, distal tubules, or cells in the renal interstitium. All technical control experiments, such as hybridization with corresponding sense riboprobes (panel a and f) and immunohistochemistry after preincubation of the antiserum with the antigenic peptide (panel e) or with the preimmune serum (data not shown), were negative.

Figure 7.

Figure 7

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Distribution of rBAT and b0,+AT in mouse kidney. Panels a–e show overviews of the cryosections (bar in a, 200 μm), and panels f–k show higher magnifications (bar in f, 50 μm). (a–c and f–h) In situ hybridizations. (b) Hybridization with a digoxigenin-labeled rBAT antisense riboprobe shows a strong signal in the outer stripe (OS) of the outer medulla, consistent with the previously reported preferential localization of rBAT mRNA in the S3 segments of proximal tubules. Weaker staining is also visible in proximal tubule profiles located in the renal cortex (Co), representing S1 and S2 segments. No staining is seen in the inner stripe (IS) of the outer medulla. (c) An inverse staining pattern is found in kidney sections hybridized with a b0,+AT antisense riboprobe. Proximal tubules in the renal cortex are heavily stained, whereas only very weak staining is detectable in the medullary rays and in the outer stripe. However, the hybridization signal at these locations is higher than in the adjacent inner stripe and is clearly above the level obtained from sections hybridized with a digoxigenin-labeled b0,+AT sense riboprobe (a). Higher magnifications of the renal cortex confirm the presence of rBAT mRNA (g) and b0,+AT mRNA (h) in the S1 segments of proximal tubules, starting from the first S1 cells at the urinary pole of the glomerulus (G), whereas other cell types within the glomerulus and distal tubules (D) are unstained. No signal is detected in the corresponding control section hybridized with a rBAT sense riboprobe (f). (d, e, i, and k) Immunofluorescence. (d) Immunohistochemical detection of b0,+AT at low magnification shows a bright immunofluorescent signal in proximal tubules within the renal cortex. Immunostaining, although at a lower intensity, is also visible in proximal tubules in the outer stripe. Preincubation of the antiserum with the antigenic peptide abolished the immunostaining completely (e). Higher magnifications of the cortex (i) and the outer medulla (k) show that b0,+AT is exclusively present in the brush border at the luminal pole of proximal tubular cell from S1 (i) to S3 (k). Arrows in panel k point to the abrupt transition from an S3 segment (with the stained brush border) to a thin descending limb (unstained). Note the decreasing staining intensities from S1 to S2 to S3. The immunofluorescent staining of the brush border of the proximal tubule at the urinary pole of the glomerulus (G) is slightly weaker than farther downstream in S1. Cells within the glomerulus, cortical distal tubules (D) (i), medullary thick ascending limbs (T) (k), and thin descending limbs (asterisk) (k) are not labeled with the antiserum.

Localization of the Human Gene Encoding hb0,+AT on Chromosome 19q

We performed fluorescent in situ hybridization of the hb0,+AT cDNA on human metaphase chromosomes (data not shown). Positive hybridization signals were found consistently in the chromosomal region 19q, as expected for a candidate gene of cystinuria type III, the locus of which has been mapped to 19q13.1 (Bisceglia et al., 1997; Wartenfeld et al., 1997).

DISCUSSION

Differential Polarity of gpaATs Associating with 4F2hc or rBAT in Epithelia

The amino acid transporter b0,+AT described in this study is a member of the recently characterized family of gpaATs. These transporters share high structural similarity, with 12 putative transmembrane domains and a conserved cysteine residue in the second putative extracellular loop that forms an intermolecular disulfide bridge with the so-called heavy chain (see model in Figure 2E) (Mastroberardino et al., 1998; Pfeiffer et al., 1998, 1999). The other light chains of the gpaAT family that have been described recently associate with 4F2hc, a type II glycoprotein required for their surface expression that displays a strict basolateral localization in renal and intestinal epithelia (Quackenbush et al., 1986; Sordat, personal communication). These light chains exhibit differential localizations and specificities of amino acid transport. For instance, LAT1 is quite ubiquitous and accepts only neutral amino acids in a Na+-independent manner. In contrast, y+LAT1 is expressed prominently in kidney proximal tubule and small intestine at the basolateral membrane (associated with 4F2hc) and transports cationic amino acids in a Na+-independent manner and neutral amino acids only in the presence of Na+ (Pfeiffer et al., 1999). Both LAT1 and y+LAT1 have been shown to function as amino acid exchangers.

We and others have proposed that the amino acid transport system composed of y+LAT1 and 4F2hc represents the basolateral exit pathway for cationic amino acids from kidney proximal tubule and small intestine cells (Torrents et al., 1998; Pfeiffer et al., 1999). Correspondingly, mutations in the gene encoding this member of the gpaAT family (SLC7A7) have been shown very recently to cause the genetic disease lysinuric protein intolerance (Borsani et al., 1999; Torrents et al., 1999). Interestingly, we have identified a second member of the gpaAT family that is also expressed in kidney proximal tubule and small intestine and is associated with 4F2hc at the basolateral membrane. Unlike the heterodimer formed by 4F2hc and y+LAT, this second basolateral heterodimeric amino acid transporter (LAT2) displays a specificity of the L type similar to LAT1 (Rossier et al., 2000).

In contrast to these gpaATs associated with 4F2hc and expressing their transport function at the basolateral membrane, we show here that b0,+AT associates covalently with rBAT and is expressed in the apical brush border membrane. Importantly, these differentially polarized heterodimers are expressed mainly in the same kidney and small intestine cells and thus can participate in the same transepithelial amino acid transports. The rBAT-b0,+AT complex is the high-affinity apical entry pathway for l-cystine and cationic amino acids (see also below), whereas 4F2hc-y+LAT1 is the basolateral exit pathway for cationic amino acids and 4F2hc-LAT2 might play a role for the basolateral exit of some neutral amino acids such as cysteine, the reduction product of l-cystine (Torrents et al., 1998, 1999; Borsani et al., 1999; Pfeiffer et al., 1999; Rossier et al., 2000). Interestingly, the primary structure of b0,+AT is equally similar to that of the LAT and y+LAT members of the mammalian gpaAT family (∼44% identity; see comparison with LAT1 and y+LAT1 in Figure 1). This extent of identity is not much lower than that of 4F2hc-associated transporters of LAT and y+LAT type (∼49% identity). It will be interesting to localize the region(s) involved in the selective interaction of the gpaAT family members with either rBAT or 4F2hc. Because the mutation of the cysteine residues involved in intermolecular disulfide bond formation, in two different light chains (Pfeiffer et al., 1998) and in 4F2hc (Estevez et al., 1998; Pfeiffer et al., 1998), has been shown to only slightly decrease the ability of the coexpressed heavy and light chains to form functional amino acid transporters at the surface of Xenopus oocytes, other regions of these proteins might play an important role in forming noncovalent interactions.

In conclusion, the interaction of the gpaAT family light chains with either 4F2hc or rBAT appears to determine their polarity of expression in epithelial cells. To date, it is not clear whether the heavy chains, besides determining the polarity of surface expression of the “catalytic” light chains, also participate in the transport and/or modulate its kinetics.

Function of b0,+ATc as an l-Cystine Transporter

Earlier functional studies have indicated that the major part of l-cystine reabsorption in the kidney proximal tubule takes place in the S1 and S2 segments via a low-affinity and high-capacity system and that the final clearing of the tubular fluid from l-cystine takes place in the S3 segments via a high-affinity, low-capacity system corresponding to b0,+-type transport (Palacin et al., 1998, and references therein). In the present study, we show that b0,+AT is expressed in the brush border membrane over the entire length of the proximal tubule, but at a much higher density in S1 and S2 than in S3. An earlier immunofluorescence study performed by others had shown that, in contrast to b0,+AT, rBAT is expressed exclusively in the S3 segment of the rat kidney proximal tubule. Here we confirm in mouse kidney at the mRNA level the predominant expression of rBAT at the level of S3. However, we show that rBAT is also expressed, although at a lower level, in the earlier segments S1 and S2. Thus, the localization of the high-affinity l-cystine transport in S3 corresponds to the site of high rBAT and low b0,+AT expression. The localization of the low-affinity, high-capacity l-cystine transport in S1 and S2 corresponds to that of high b0,+AT and low rBAT expression.

We propose that the high-affinity, b0,+-type l-cystine transport of the proximal kidney tubule S3 segment is mediated by the b0,+AT-rBAT complex, in accordance with the localization data mentioned above and with the oocyte and M1 cell expression data, which demonstrate that the b0,+AT-rBAT heterodimer functions as a high-affinity l-cystine transport system shared with cationic amino acids. To explain the discrepancy in rBAT and b0,+AT expression levels along the proximal tubule, we propose two hypotheses that are compatible with the phenotype of rBAT defect (type I cystinuria: no amino aciduria in heterozygotes) and b0,+AT defect (non-type I cystinuria: small amino aciduria in heterozygotes) (see references for cystinuria in INTRODUCTION). The first possibility is that rBAT is expressed in a large excess over b0,+AT in the S3 segment and that all functional b0,+ATs of the S1 and S2 segments are associated with rBAT as well. This complex could produce a low-affinity transport of l-cystine in the context of S1 and S2 cells, in contrast to the high-affinity transport observed in S3 cells and oocytes. Such differential transport kinetics could be due to the presence in S1 and S2, or, alternatively, in S3 (and Xenopus oocytes), of another noncovalently associated protein that would modulate the transport function. The second possibility is that b0,+AT is expressed in a large excess over rBAT in S1 and S2 and reaches the brush border membrane independent of association with rBAT. Because expression of functional surface transport requires the association of b0,+AT with rBAT in oocytes and in transfected M1 cells (and in the S3 segment), one could postulate that surface expression in S1 and S2 requires the interaction with another apical membrane protein that is not expressed in S3 or in the cells used for exogenous expression mentioned above. In this putative complex with another membrane protein, b0,+AT would express an amino acid transport with different kinetic properties, i.e., an l-cystine uptake with a lower apparent affinity, such as when it is associated with rBAT. However, this hypothesis remains quite speculative, in particular because database searches for rBAT-related proteins (candidates for association with b0,+AT in S1 and S2) were unsuccessful.

It is important to mention that, concurrent with our study, several mutations have been identified by others in the genes encoding the b0,+AT transporter (SLC7A9) of patients suffering from non-type I cystinuria (Palacin, personal communication). This confirms at the genetic level that b0,+AT is involved in intestinal and renal l-cystine transport. This genetic information and the results of our biochemical, functional, and localization study complement each other to demonstrate that b0,+AT, which is the product of the SLC7A9 gene, is the catalytic subunit of the hetero-oligomeric b0,+ATc defective in cystinuria.

ACKNOWLEDGMENTS

The authors thank Brigitte Kaissling, in whose laboratory J.L. and D.L.-C. are working, for her support, Christian Gasser for the artwork, and Alexandra Albers for performing some of the fluorescent in situ hybridization experiments. The laboratory of F.V. is supported by Swiss National Science Foundation grant 31-49727.96. The project is also supported by grant 279-1-1996 from the Swiss Cancer League to L.C.K.

REFERENCES

  1. Bertran J, Werner A, Chillaron J, Nunes V, Biber J, Testar X, Zorzano A, Estivill X, Murer H, Palacin M. Expression cloning of a human renal cDNA that induces high affinity transport of l-cystine shared with dibasic amino acids in Xenopus oocytes. J Biol Chem. 1993;268:14842–14849. [PubMed] [Google Scholar]
  2. Bertran J, Werner A, Moore ML, Stange G, Markovich D, Biber J, Testar X, Zorzano A, Palacin M, Murer H. Expression cloning of a cDNA from rabbit kidney cortex that induces a single transport system for cystine and dibasic and neutral amino acids. Proc Natl Acad Sci USA. 1992;89:5601–5605. doi: 10.1073/pnas.89.12.5601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bisceglia L, et al. Localization, by linkage analysis, of the cystinuria type III gene to chromosome 19q13.1. Am J Hum Genet. 1997;60:611–616. [PMC free article] [PubMed] [Google Scholar]
  4. Borsani G, et al. SLC7A7, encoding a putative permease-related protein, is mutated in patients with lysinuric protein intolerance. Nat Genet. 1999;21:297–301. doi: 10.1038/6815. [DOI] [PubMed] [Google Scholar]
  5. Busch AE, Herzer T, Waldegger S, Schmidt F, Palacin M, Biber J, Markovich D, Murer H, Lang F. Opposite directed currents induced by the transport of dibasic and neutral amino acids in Xenopus oocytes expressing the protein rBAT. J Biol Chem. 1994;269:25581–25586. [PubMed] [Google Scholar]
  6. Calonge MJ, et al. Cystinuria caused by mutations in rBAT, a gene involved in the transport of cystine. Nat Genet. 1994;6:420–425. doi: 10.1038/ng0494-420. [DOI] [PubMed] [Google Scholar]
  7. Chillaron J, et al. Obligatory amino acid exchange via systems b0,+-like and y+L-like: a tertiary active transport mechanism for renal reabsorption of cystine and dibasic amino acids. J Biol Chem. 1996;271:17761–17770. doi: 10.1074/jbc.271.30.17761. [DOI] [PubMed] [Google Scholar]
  8. Coady MJ, Jalal F, Chen X, Lemay G, Berteloot A, Lapointe JY. Electrogenic amino acid exchange via the rBAT transporter. FEBS Lett. 1994;356:174–178. doi: 10.1016/0014-5793(94)01262-8. [DOI] [PubMed] [Google Scholar]
  9. Estevez R, Camps M, Rojas A, Testar X, Deves R, Hediger M, Zorzano A, Palacin M. The amino acid transport system y+L/4F2hc is a heteromultimeric complex. FASEB J. 1998;12:1319–1329. doi: 10.1096/fasebj.12.13.1319. [DOI] [PubMed] [Google Scholar]
  10. Foreman JW, Hwang SM, Segal S. Transport interactions of cystine and dibasic amino acids in isolated rat renal tubules. Metab Clin Exp. 1980;29:53–61. doi: 10.1016/0026-0495(80)90098-0. [DOI] [PubMed] [Google Scholar]
  11. Furriols M, Chillaron J, Mora C, Castello A, Bertran J, Camps M, Testar X, Vilaro S, Zorzano A, Palacin M. rBat, related to l-cysteine transport, is localized to the microvilli of proximal straight tubules, and its expression is regulated in kidney by development. J Biol Chem. 1993;268:27060–27068. [PubMed] [Google Scholar]
  12. Geering K, Theulaz I, Verrey F, Häuptle MT, Rossier BC. A role for the β-subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes. Am J Physiol. 1989;257:C851–C858. doi: 10.1152/ajpcell.1989.257.5.C851. [DOI] [PubMed] [Google Scholar]
  13. Kanai Y, Segawa H, Miyamoto K, Uchino H, Takeda E, Endou H. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98) J Biol Chem. 1998;273:23629–23632. doi: 10.1074/jbc.273.37.23629. [DOI] [PubMed] [Google Scholar]
  14. Kanai Y, Stelzner MG, Lee WS, Wells RG, Brown D, Hediger MA. Expression of mRNA (D2) encoding a protein involved in amino acid transport in S3 proximal tubule. Am J Physiol. 1992;263:F1087–F1092. doi: 10.1152/ajprenal.1992.263.6.F1087. [DOI] [PubMed] [Google Scholar]
  15. Loffing J, Loffing-Cueni D, Hegyi I, Kaplan MR, Hebert SC, Le Hir M, Kaissling B. Thiazide treatment of rats provokes apoptosis in distal tubule cells. Kidney Int. 1996;50:1180–1190. doi: 10.1038/ki.1996.426. [DOI] [PubMed] [Google Scholar]
  16. Mannion BA, Kolesnikova TV, Lin SH, Wang S, Thompson NL, Hemler ME. The light chain of CD98 is identified as E16/TA1 protein. J Biol Chem. 1998;273:33127–33129. doi: 10.1074/jbc.273.50.33127. [DOI] [PubMed] [Google Scholar]
  17. Mao Q, Schunk T, Gerber B, Erni B. A string of enzymes, purification and characterization of a fusion protein comprising the four subunits of the glucose phosphotransferase system of Escherichia coli. J Biol Chem. 1995;270:18295–18300. doi: 10.1074/jbc.270.31.18295. [DOI] [PubMed] [Google Scholar]
  18. Mastroberardino L, Spindler B, Pfeiffer R, Skelly PJ, Loffing J, Shoemaker CB, Verrey F. Amino acid transport by heterodimers of 4F2hc/CD98 and members of a permease family. Nature. 1998;395:288–291. doi: 10.1038/26246. [DOI] [PubMed] [Google Scholar]
  19. Mosckovitz R, Udenfriend S, Felix A, Heimer E, Tate SS. Membrane topology of the rat kidney neutral and basic amino acid transporter. FASEB J. 1994;8:1069–1074. doi: 10.1096/fasebj.8.13.7926373. [DOI] [PubMed] [Google Scholar]
  20. Nakamura E, Sato M, Yang H, Miyagawa F, Harasaki M, Tomita K, Matsuoka S, Noma A, Iwai K, Minato N. 4F2 (CD98) heavy chain is associated covalently with an amino acid transporter and controls intracellular trafficking and membrane topology of 4F2 heterodimer. J Biol Chem. 1999;274:3009–3016. doi: 10.1074/jbc.274.5.3009. [DOI] [PubMed] [Google Scholar]
  21. Palacin M, Chillaron J, Mora C. Role of the b0,+-like amino acid-transport system in the renal reabsorption of cystine and dibasic amino acids. Biochem Soc Trans. 1996;24:856–863. doi: 10.1042/bst0240856. [DOI] [PubMed] [Google Scholar]
  22. Palacin M, Estevez R, Bertran J, Zorzano A. Molecular biology of mammalian plasma membrane amino acid transporters. Physiol Rev. 1998;78:969–1054. doi: 10.1152/physrev.1998.78.4.969. [DOI] [PubMed] [Google Scholar]
  23. Pfeiffer R, Rossier G, Spindler B, Meier C, Kühn LC, Verrey F. Amino acid transport of y+L-type by heterodimers of 4F2hc/CD98 and members of the glycoprotein-associated amino acid transporter family. EMBO J. 1999;18:49–57. doi: 10.1093/emboj/18.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pfeiffer R, Spindler B, Loffing J, Skelly P, Shoemaker C, Verrey F. Functional heterodimeric amino acid transporters lacking cysteine residues involved in disulfide bond. FEBS Lett. 1998;439:157–162. doi: 10.1016/s0014-5793(98)01359-3. [DOI] [PubMed] [Google Scholar]
  25. Pras E, Kochba I, Lubetzky A, Pras M, Sidi Y, Kastner D. Biochemical and clinical studies in Libyan Jewish cystinuria patients and their relatives. Am J Med Genet. 1998;80:173–176. [PubMed] [Google Scholar]
  26. Prasad PD, Wang H, Huang W, Kekuda R, Rajan DP, Leibach FH, Ganapathy V. Human LAT1, a subunit of system L amino acid transporter: molecular cloning and transport function. Biochem Biophys Res Commun. 1999;255:283–288. doi: 10.1006/bbrc.1999.0206. [DOI] [PubMed] [Google Scholar]
  27. Puoti A, May A, Rossier BC, Horisberger JD. Novel isoforms of the β and γ subunits of the Xenopus laevis Na channel provide information about the amiloride binding site and extracellular sodium sensing. Proc Natl Acad Sci USA. 1997;94:5949–5954. doi: 10.1073/pnas.94.11.5949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Quackenbush EJ, Gougos A, Baumal R, Letarte M. Differential localization within human kidney of five membrane proteins expressed on acute lymphoblastic leukemia cells. J Immunol. 1986;136:118–124. [PubMed] [Google Scholar]
  29. Riahi-Esfahani S, Jessen H, Roigaard H. Comparative study of the uptake of L-cysteine and L-cystine in the renal proximal tubule. Amino Acids. 1995;8:247–264. doi: 10.1007/BF00806822. [DOI] [PubMed] [Google Scholar]
  30. Rosenberg LE, Downing S, Durant JL, Segal S. Cystinuria: biochemical evidence for three genetically distinct diseases. J Clin Invest. 1966;45:365–371. doi: 10.1172/JCI105351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rossier, G., Meier, C., Bauch, C., Summa, V., Sordat, B., Verrey, F., and Kühn, L.C. (2000). LAT2: a new basolateral 4F2hc/CD98- associated amino acid transporter of kidney and intestine. J. Biol. Chem. (in press). [DOI] [PubMed]
  32. Schafer JA, Watkins ML. Transport of l-cystine in isolated perfused proximal straight tubules. Pfluegers Arch. 1984;401:143–151. doi: 10.1007/BF00583874. [DOI] [PubMed] [Google Scholar]
  33. Silbernagl S. The renal handling of amino acids and oligopeptides. Physiol Rev. 1988;68:911–1007. doi: 10.1152/physrev.1988.68.3.911. [DOI] [PubMed] [Google Scholar]
  34. Tate SS, Yan N, Udenfriend S. Expression cloning of a Na+-independent neutral amino acid transporter from rat kidney. Proc Natl Acad Sci USA. 1992;89:1–5. doi: 10.1073/pnas.89.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Torrents D, Estevez R, Pineda M, Fernandez E, Lloberas J, Shi Y, Zorzano A, Palacin M. Identification and characterization of a membrane protein (y+L amino acid transporter-1) that associates with 4F2hc to encode the amino acid transport activity y+L: a candidate gene for lysinuric protein intolerance. J Biol Chem. 1998;273:32437–32445. doi: 10.1074/jbc.273.49.32437. [DOI] [PubMed] [Google Scholar]
  36. Torrents D, et al. Identification of SLC7A7, encoding y+LAT-1, as the lysinuric protein intolerance gene. Nat Genet. 1999;21:293–296. doi: 10.1038/6809. [DOI] [PubMed] [Google Scholar]
  37. Van Winkle LJ. Amino acid transport in developing animal oocytes and early conceptuses. Biochim Biophys Acta. 1988;947:173–208. doi: 10.1016/0304-4157(88)90024-x. [DOI] [PubMed] [Google Scholar]
  38. Wang Y, Tate SS. Oligomeric structure of a renal cystine transporter: implications in cystinuria. FEBS Lett. 1995;368:389–392. doi: 10.1016/0014-5793(95)00685-3. [DOI] [PubMed] [Google Scholar]
  39. Wartenfeld R, Golomb E, Katz G, Bale SJ, Goldman B, Pras M, Kastner DL, Pras E. Molecular analysis of cystinuria in Libyan Jews: exclusion of the SLC3A1 gene and mapping of a new locus on 19q. Am J Hum Genet. 1997;60:617–624. [PMC free article] [PubMed] [Google Scholar]
  40. Wells RG, Hediger MA. Cloning of a rat kidney cDNA that stimulates dibasic and neutral amino acid transport and has sequence similarity to glucosidases. Proc Natl Acad Sci USA. 1992;89:5596–5600. doi: 10.1073/pnas.89.12.5596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yan N, Mosckovitz R, Udenfriend S, Tate SS. Distribution of mRNA of a Na+-independent neutral amino acid transporter cloned from rat kidney and its expression in mammalian tissues and Xenopus laevis oocytes. Proc Natl Acad Sci USA. 1992;89:9982–9985. doi: 10.1073/pnas.89.21.9982. [DOI] [PMC free article] [PubMed] [Google Scholar]

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