Cloning and Expression of a b0,+-like Amino Acid Transporter Functioning as a Heterodimer with 4F2hc Instead of rBAT

We have cloned a transporter protein from rabbit small intestine, which, when coexpressed with the 4F2 heavy chain (4F2hc) in mammalian cells, induces a b0,+-like amino acid transport activity. This protein (4F2-lc6 for the sixth member of the 4F2 light chain family) consists of 487 amino acids and has 12 putative transmembrane domains. At the level of amino acid sequence, 4F2-lc6 shows significant homology (44% identity) to the other five known members of the 4F2 light chain family, namely LAT1 (4F2-lc1), y+LAT1 (4F2-lc2), y+LAT2 (4F2-lc3), xCT (4F2-lc4), and LAT2 (4F2-lc5). The 4F2hc/4F2-lc6 complex-mediated transport process is Na+-independent and exhibits high affinity for neutral and cationic amino acids and cystine. These characteristics are similar to those of the b0,+-like amino acid transport activity previously shown to be associated with rBAT (protein related to b0,+ amino acid transport system). However, the newly cloned 4F2-lc6 does not interact with rBAT. This is the first report of the existence of a b0,+-like amino acid transport process that is independent of rBAT. 4F2-lc6 is expressed predominantly in the small intestine and kidney. Based on the characteristics of the transport process mediated by the 4F2hc/4F2-lc6 complex and the expression pattern of 4F2-lc6 in mammalian tissues, we suggest that 4F2-lc6 is a new candidate gene for cystinuria.

Cystinuria is a genetic disorder in the transport of neutral and cationic amino acids and cystine in the intestine and kidney (1)(2)(3)(4)(5). Three groups of investigators have independently cloned a protein associated with an amino acid transport function with specificity toward neutral and cationic amino acids and cystine (6 -8). This protein, referred to as NBAT, D2, or rBAT 1 (the term rBAT is used in this paper to refer to this protein), is expressed in the intestinal and renal tubular cells (9). When expressed heterologously in Xenopus laevis oocytes, rBAT induces an amino acid transport activity similar to system b 0,ϩ (7,8), originally described in mouse blastocysts as a Na ϩ -independent high affinity system for neutral and cationic amino acids (10). Subsequent studies have revealed that the gene coding for rBAT is defective in some patients with cystinuria (3,5,11,12). Interestingly rBAT is not very hydrophobic, a characteristic rather unusual for a transport protein. The prevailing hypothesis is that rBAT does not constitute the transport system per se, but may be an essential subunit associated with the transport system (12). The molecular identity of the other subunits(s) that interact(s) with rBAT to form the transport system remains unknown. The fact that defects in the rBAT gene account for some but not all cystinuria cases indicates that other genes, hitherto unidentified, are also involved in this disease. Biochemical evidence suggests that cystinuria is comprised of three genetically distinct diseases (13). Mutational analyses have shown that defects in the rBAT gene are exclusively associated with type I cystinuria. However, there are cases of type I cystinuria with no detectable defects in the rBAT gene, and the identity of the gene responsible for these cases is not known. Similarly, the genes associated with type II and type III cystinuria have also not yet been identified.
rBAT exhibits significant homology to 4F2hc, the heavy chain of the cell surface antigen 4F2 (7,8). Expression of 4F2hc in X. laevis oocytes also induces an amino acid transport activity that is distinct from the activity induced by rBAT (14,15). The characteristics of 4F2hc-related transport activity resemble those of y ϩ L (16), a system which mediates Na ϩ -independent transport of cationic amino acids and Na ϩ -dependent transport of neutral amino acids (17). Recent studies have shown that 4F2hc is a subunit common to at least three different amino acid transport systems, namely L, y ϩ L, and x c Ϫ (18 -25). The corresponding light chains that associate with 4F2hc to form these three transport systems have been cloned and functionally characterized. These light chains are referred to as LAT1 and LAT2 (specific for system L) (18 -22), y ϩ LAT1 and y ϩ LAT2 (specific for system y ϩ L) (23,24), and xCT (specific for system x c Ϫ ) (25). Defects in the y ϩ LAT1 gene have been recently shown to be responsible for the genetic disorder lysinuric protein intolerance (26,27). None of these genes (4F2hc), LAT1, LAT2, y ϩ LAT1, y ϩ LAT2, or xCT) has been shown to be associated with cystinuria.
Here we report the cloning of a protein from the rabbit small intestine, which, when coexpressed with 4F2hc in mammalian cells, induces a Na ϩ -independent amino acid transport activity specific for neutral and cationic amino acids and cystine. Even though this transport activity is similar to the activity associated with rBAT, the newly cloned protein does not interact with rBAT. This protein is the sixth member of the 4F2 light chain family to be identified that interacts with the 4F2 heavy chain to constitute an amino acid transport system. Accordingly, we have named this protein 4F2-lc6. The other five are 4F2-lc1 (LAT1), 4F2-lc2 (y ϩ LAT1), 4F2-lc3 (y ϩ LAT2), 4F2-lc4 (xCT), and 4F2-lc5 (LAT2). Based on the functional characteristics of the transport system induced by the 4F2hc/4F2-lc6 complex, we hypothesize that 4F2-lc6 may represent a new candidate gene for cystinuria. These studies provide the first evidence for the existence of a b 0,ϩ -like amino acid transport activity independent of rBAT.

EXPERIMENTAL PROCEDURES
Materials-Radiolabeled amino acids and nucleotides were purchased from NEN Life Science Products, American Radiolabeled Chemicals, or Amersham Pharmacia Biotech. Unlabeled amino acids were from Sigma. Nitropure nitrocellulose transfer membranes used in the library screening were purchased from Micron Separations, Inc. (Westboro, MA). The Ready-to-go oligolabeling kit was purchased from Amersham Pharmacia Biotech. Lipofectin, TRIzol reagent, and oligo(dT)cellulose were purchased from Life Technologies, Inc. Restriction enzymes were from New England Biolabs. The human retinal pigment epithelial (HRPE) cell line 165, used in expression studies, has been described earlier from our laboratory (28,29). The human rBAT cDNA and rat 4F2hc cDNA were kindly provided by Matthias A. Hediger (Department of Medicine, Harvard University, Boston, MA). The human 4F2hc cDNA was isolated by screening a JAR human placental trophoblast cell line cDNA library (30) using the rat 4F2hc cDNA as a probe.
cDNA Library Screening and DNA Sequencing-The cDNA probe used for the screening was a 1.7-kilobase pair fragment of human LAT1 cDNA (20) obtained by digestion of the full-length cDNA with KpnI/ PvuII. The probe was labeled with [␣-32 P]dCTP by random priming using the Ready-to-go oligolabeling beads. The screening of the rabbit intestinal cDNA library (31) was done under low stringency conditions using this probe as described previously (31,32). Both sense and antisense strands of the cDNA were sequenced by primer walking using Taq DyeDeoxy terminator cycle sequencing in an automated Perkin-Elmer Applied Biosystems 377 Prism DNA sequencer.
Functional Expression of the cDNA in HRPE Cells-The vaccinia virus expression system was used to functionally characterize the cloned cDNA as described previously (20, 30 -32). 4F2-lc6, rat and human 4F2hc, and human rBAT were all cloned into pSPORT such that the sense transcription is under the control of T7 promoter. The cDNAs were transfected into HRPE cells grown in 24-well tissue culture plates using Lipofectin, and the functional expression of the cDNA was analyzed 12 h later by measuring radiolabeled amino acid uptake. One microgram of the plasmid carrying the specific cDNA (4F2-lc6, 4F21hc, or rBAT) was used per well. Sister wells transfected identically with vector alone served as control. The transport buffer was composed of 25 mM Hepes/Tris (pH 7.5), supplemented with 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgSO 4 , and 5 mM glucose. When the effect of Na ϩ on amino acid uptake was studied, the NaCl in the buffer was replaced with N-methyl-D-glucamine chloride. The incubation time for the transport measurements was 15 min in most cases, following which the uptake medium containing the radioactive substrate was aspirated off and the cells were washed with 2 ϫ 2 ml of ice-cold transport buffer. The cells were then solubilized in 0.5% SDS in 0.2 N NaOH, transferred to vials, and radioactivity associated with the cells quantitated by liquid scintillation spectrometry. The experiments were repeated two to three times with independent transfections, each done in duplicate or triplicate. Data are presented as means Ϯ S.E. of these replicate measurements.
Functional Expression in X. laevis Oocytes-Isolation of mature oocytes from X. laevis, cRNA synthesis, and microinjection of cRNA into oocytes have been described previously (16,30,31). Uptake of radiolabeled amino acids into oocytes was measured for 60 min in the uptake buffer, composed of 100 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , and 1 mM CaCl 2 , buffered with 3 mM Hepes/Tris, pH 7.5.
Northern Blot Analysis-Poly(A) ϩ RNA, isolated from different rabbit tissues (small intestine, kidney, heart, lung, liver, and skeletal muscle), was used for Northern blot analysis. The membrane filter containing the size-fractionated RNA (5 g/lane) was probed sequentially, first with the 4F2-lc6 cDNA and then, following stripping, with cyclophilin cDNA.
RT-PCR-Poly(A) ϩ RNA samples prepared from rabbit tissues were used for RT-PCR. The upstream primer was 5Ј-CCGCCTACCTCT-TCTCCT-3Ј, and the downstream primer was 5Ј-CGCTGGGTTAGT-GATGAC-3Ј. These primers correspond to nucleotide positions 405-422 and 1367-1384 of the rabbit intestinal 4F2-lc6 cDNA. The RT-PCR products were subcloned into pGEM-T vector and the inserts were analyzed for restriction pattern and nucleotide sequence.

Structural Features of 4F2-lc6 -
The rabbit intestinal 4F2-lc6 cDNA is 1858 bp long, with an open reading frame consisting of 1464 bp (including the termination codon). The cDNA codes for a protein of 487 amino acids (Fig. 1A). The open reading frame is flanked by a 82-bp 5Ј-noncoding sequence and a 312-bp 3Ј-noncoding sequence (GenBank accession no. AF155119). The predicted molecular mass of the protein is 53.6 kDa. Hydrophobicity analysis of the amino acid sequence suggests the presence of 12 putative transmembrane domains. When modeled similar to most of the transporter proteins with both the N terminus and C terminus facing the cytoplasmic surface of the lipid bilayer, there is one potential site for Nlinked glycosylation (Asn-271) in the extracellular loop between transmembrane domains 7 and 8 (Fig. 1B). The protein contains three potential sites for protein kinase C-dependent phosphorylation (Ser-169, Ser-345, and Thr-399) and one potential site for cAMP-and cGMP-dependent protein kinase phosphorylation (Thr-350) in putative intracellular domains. At the level of amino acid sequence, the rabbit 4F2-lc6 exhibits significant homology to the other known members of the 4F2 light chain family, namely 4F2-lc1 (LAT1), 4F2-lc2 (y ϩ LAT1), 4F2-1c3 (y ϩ LAT2), 4F2-lc4 (xCT), and 4F2-lc5 (LAT2). The amino acid sequence identity is 43-44%, and similarity is 63-65%.
Functional Characteristics of 4F2-lc6 -The structural similarity of 4F2-lc6 to the known light chains associated with 4F2hc suggested that 4F2-lc6 may be a protein which works with 4F2hc to form a new heterodimeric amino acid transport system. To establish the functional identity of 4F2-lc6, we expressed 4F2-lc6 cDNA either alone or together with human 4F2hc cDNA in HRPE cells using the vaccinia virus expression system and studied the transport of a neutral amino acid (alanine) in the presence or absence of Na ϩ ( Fig. 2A and B). In the presence of Na ϩ , neither 4F2-lc6 nor 4F2hc mediated alanine transport. The transport activity in either case was not different from the transport activity in control cells transfected with vector alone. Similarly, the transport activity remained essentially the same even when 4F2-lc6 was coexpressed with 4F2hc. In contrast to the results obtained in the presence of Na ϩ , an increase in alanine transport was seen in cells coexpressing the two proteins when the transport was measured in the absence of Na ϩ . Expression of either 4F2-lc6 or 4F2hc alone did not show any increase in alanine transport under similar conditions. These data demonstrate that the 4F2hc/4F2-lc6 heterodimeric complex mediates Na ϩ -independent transport of the neutral amino acid alanine. Na ϩ -independent alanine transport activity was also demonstrable with the heterodimeric complex consisting of 4F2-lc6 and rat 4F2hc (data not shown). It is of some interest that the heterodimer-mediated alanine transport could not be detected when measured in the presence of Na ϩ . A Na ϩ -independent transport process is expected to be detectable both in the presence and absence of Na ϩ . It is possible that the high endogenous alanine transport activity seen in the presence of Na ϩ masks the transport activity associated with the 4F2hc/4F2-lc6 complex in this experimental system.
We then tested whether 4F2-lc6 is capable of working with rBAT instead of 4F2hc to induce the Na ϩ -independent alanine transport activity (Fig. 2C). Human rBAT cDNA and 4F2-lc6 cDNA were expressed either independently or together in HRPE cells and alanine transport was measured in the absence of Na ϩ . These were the same experimental conditions in which the alanine transport activity of the 4F2hc/4F2-lc6 complex was demonstrable. The data given in Fig. 2C show that there was no alanine transport activity associated with rBAT and 4F2-lc6 when expressed either independently or together. Fig. 2D shows the time course of alanine transport, measured in the absence of Na ϩ , in cells transfected with pSPORT vector and in cells coexpressing 4F2hc and 4F2-lc6. The transport activity was 2-3-fold higher in cells coexpressing the two proteins than in vector-transfected cells at all time periods tested. When the transport activity specific for the 4F2hc/4F2-lc6 complex alone was considered, the transport was linear (r ϭ 0.98) at least up to 20 min. Table I describes the specificity of the transport system mediated by the 4F2hc/4F2-lc6 complex, as evidenced from direct measurements of the transport of radiolabeled amino acids and comparison of the transport activities between control cells transfected with pSPORT vector alone and cells coexpressing 4F2hc and 4F2-lc6. The 4F2hc/4F2-lc6-mediated transport was demonstrable for the neutral amino acids alanine, glutamine, cysteine, cystine, leucine, histidine, and phenylalanine and for the cationic amino acid arginine. The transport of the anionic amino acid glutamate and the transport of the system A-specific substrate MeAIB were not different between the control cells and the cells expressing the 4F2hc/4F2-lc6 complex. These results show that the 4F2hc/4F2-lc6 complex mediates Na ϩindependent transport of most of the neutral amino acids as well as the cationic amino acids.

FIG. 2. Characteristics of alanine transport associated with 4F2-lc6.
HRPE cells were transfected with pSPORT vector, human 4F2hc cDNA, human rBAT cDNA, and rabbit 4F2-lc6 cDNA either independently or together. Transport of 1 M [ 3 H]alanine was measured in these cells with a 15-min incubation in the presence (A) or absence (B and C) of Na ϩ . D, time course of alanine transport, measured in the absence of Na ϩ , in control cells transfected with vector alone (E) and in cells coexpressing 4F2hc and 4F2-lc6 (ⅷ). The 4F2hc/4F2-lc6 complex-specific transport is also shown (f). Data are means Ϯ S.E. from six to eight determinations made with two to three independent transfections. Table II describes the substrate specificity of the 4F2hc/4F2-lc6 complex as evidenced from competition studies. Na ϩ -independent transport of radiolabeled alanine was measured in control cells transfected with vector alone and in cells coexpressing 4F2hc and 4F2-lc6, and the ability of various unlabeled amino acids to inhibit the transport of radiolabeled alanine was assessed. The 4F2hc/4F2-lc6 complex-specific transport of [ 3 H]alanine was almost completely inhibited by neutral amino acids (leucine, tryptophan, phenylalanine, methionine, alanine, serine, cysteine, threonine, glutamine, asparagine, and glycine) as well as by cationic amino acids (lysine and arginine). The inhibition was also almost complete with cystine and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (an amino acid considered to be specific for system L). The imino acid proline, the system A-specific amino acid MeAIB and the anionic amino acids aspartate and glutamate showed little or no inhibition. Taken collectively, these data demonstrate that the 4F2hc/4F2-lc6 complex interacts with neutral amino acids, cationic amino acids, and cystine in a Na ϩ -independent manner. This substrate specificity is similar to that of the amino acid transport system b 0,ϩ . Previous studies have shown that rBAT associates with an hitherto unidentified light chain to form the amino acid transport system b 0,ϩ (2,11,12). The present studies provide the first evidence for the presence of a b 0,ϩ -like amino acid transport activity that is independent of rBAT.
We then studied the potency of the neutral amino acids alanine and leucine, the cationic amino acid lysine, and cystine to inhibit the Na ϩ -independent transport of [ 3 H]alanine in control cells transfected with vector alone and in cells coexpressing 4F2hc and 4F2-lc6. Fig. 3 describes the results with respect to the endogenous [ 3 H]alanine transport activity and the [ 3 H]alanine transport activity that is specific to the heter-ologously expressed 4F2hc/4F2-lc6 complex. The latter was determined by subtracting the endogenous activity from the activity measured in cells transfected with 4F2hc cDNA plus 4F2-lc6 cDNA. The endogenous [ 3 H]alanine transport activity was inhibited by alanine and leucine but not by lysine (Fig. 3A). Cystine showed a small, but significant, inhibition of the endogenous activity. In contrast, the 4F2hc/4F2-lc6 complex-specific [ 3 H]alanine transport was completely inhibitable by alanine, leucine, lysine, and cystine (Fig. 3B). The IC 50 values (i.e. the concentrations of unlabeled amino acids needed to cause 50% inhibition of [ 3 H]alanine transport) for these four amino acids were 25 Ϯ 2, 9 Ϯ 1, 12 Ϯ 3, and 27 Ϯ 3 M, respectively. Thus, the transport activity specific to the 4F2hc/4F2-lc6 complex exhibits high affinity toward neutral amino acids, cationic amino acids, and cystine. The IC 50 values for alanine and leucine to inhibit the endogenous [ 3 H]alanine transport were 28 Ϯ 3 and 10 Ϯ 2 M, respectively. Fig. 4 describes the saturation kinetics of Na ϩ -independent alanine transport in control cells transfected with vector alone and in cells coexpressing 4F2hc and 4F2-lc6. The transport was saturable over an alanine concentration range of 5-75 M in both cases. The Michaelis-Menten constant (K t ) for the 4F2hc/ 4F2-lc6 complex-specific transport activity was 41 Ϯ 6 M. This value was similar to the IC 50 value (25 Ϯ 2 M) determined from the dose-response relationship for the inhibition of [ 3 H]alanine transport by unlabeled alanine.
The tissue distribution of the expression of 4F2-lc6 in the TABLE II Substrate specificity of the transport process mediated by the 4F2hc/4F2-lc6 complex HRPE cells were transfected with either empty pSPORT vector or h4F2hc cDNA plus rabbit intestinal 4F2-lc6 cDNA. Transport of 1 M [ 3 H]alanine was measured for 15 min in a Na ϩ -free medium in the absence or presence of 2.5 mM unlabeled amino acids (concentration of cystine, 0.5 mM). The data represent means Ϯ S.E. for four determinations from two independent transfection experiments. Values in parentheses are percentages of control uptake measured in the absence of inhibitory amino acids for the 4F2hc/4F2-lc6 complex-specific transport activity (i.e. the difference between the transport activity in cells transfected with 4F2hc cDNA plus 4F2-lc6 cDNA and the transport activity in cells transfected with pSPORT vector alone.  . 3. A, inhibition of endogenous [ 3 H]alanine transport measured in cells transfected with vector alone. B, inhibition of [ 3 H]alanine transport mediated by the 4F2hc/4F2-lc6 complex. The 4F2hc/4F2-lc6 complex-specific transport was calculated by subtracting the transport measured in vector-transfected cells from the transport measured in cells coexpressing 4F2hc and 4F2-lc6. Transport was measured in the absence of Na ϩ . Data are means Ϯ S.E. from six determinations made with two independent transfections. q, lysine; E, cystine; , leucine; ƒ, alanine. rabbit was studied by Northern blot analysis (Fig. 5). The 4F2-lc6-specific mRNA (2.0 kilobases in size) was detectable only in the small intestine and the kidney. The other tissues tested (heart, lung, liver, and skeletal muscle) were negative. To confirm that the northern hybridization signal obtained with the kidney mRNA represents the 4F2-lc6-specific message, we performed RT-PCR and analyzed the product. With a pair of primers specific for the rabbit intestinal 4F2-lc6, mRNA samples from the rabbit intestine (positive control) and the rabbit kidney yielded RT-PCR products of expected size (980 bp) (data not shown). These products were subcloned and analyzed for their restriction pattern with three different restriction enzymes (BamHI, EcoRI, and StyI). Both products exhibited an identical restriction pattern, indicating that both products were identical. This was confirmed by sequence analysis of the products. These results show unequivocally that the small intestine and the kidney express 4F2-lc6 mRNA. Interestingly, the data from the sensitive RT-PCR technique showed that the 4F2-lc6 mRNA is also expressed in other tissues, although to a much lesser extent than in the intestine and kidney.
Since the ability of the 4F2hc/4F2-lc6 complex to mediate the transport of cystine is directly relevant to the genetic disorder cystinuria, we used the X. laevis oocyte expression system to confirm the 4F2hc/4F2-lc6-mediated cystine transport (Fig.  6A). 4F2hc and 4F2-lc6 were expressed in the oocytes either independently or together by microinjection of corresponding cRNAs, and the uptake of cystine in these oocytes was then studied. Expression of 4F2hc alone increased cystine uptake to a small extent, but expression of 4F2-lc6 alone did not. However, when both proteins were co-expressed, cystine uptake increased severalfold compared with water-injected oocytes or oocytes expressing the two proteins independently. We have also used the oocyte expression system to demonstrate that the human rBAT clone used in the present study is functionally active. As shown in Fig. 6B, the b 0,ϩ -like transport function of rBAT is evident from the increased uptake of cystine, leucine, and arginine in rBAT-expressing oocytes compared with uptake in water-injected oocytes. DISCUSSION This is the first report of the molecular identification of a transporter that is associated with a b 0,ϩ -like amino acid transport activity independent of rBAT. This transporter, designated 4F2-lc6, was cloned from the rabbit intestine. 4F2-lc6 interacts with the heavy chain of the cell surface antigen 4F2 to induce a b 0,ϩ -like amino acid transport activity. Thus, 4F2-lc6 represents the first light chain subunit to be identified at the molecular level that is associated with a b 0,ϩ -like amino acid transport activity. This is also the first report showing the involvement of the 4F2 heavy chain in a b 0,ϩ -like transport activity. Previous studies have shown that the 4F2 heavy chain is involved in the amino acid transport activities identified as systems L1, L2, y ϩ L1, y ϩ L2, and x c Ϫ (18 -25). To date, the only protein that has been shown to be involved in a b 0,ϩ -like amino acid transport activity is rBAT (7,8). This protein shows a significant homology to the 4F2 heavy chain and is believed to constitute one of the subunits of the b 0,ϩ -like transport system (2,11,12). However, the other putative subunit that associates with rBAT has not been identified. The 4F2-lc6 reported in this paper is a subunit of a b 0,ϩ -like transport system, but does not interact with rBAT.
The transport activity mediated by the 4F2hc/4F2-lc6 complex shows high affinity for neutral amino acids, cationic amino acids, and cystine. The 4F2-lc6-specific message is expressed predominantly in the intestine and the kidney in the rabbit. The transport characteristics and the tissue distribution pattern suggest that 4F2-lc6 is a candidate gene for cystinuria. Available evidence indicates that, in addition to the rBAT gene, there are other genes hitherto unidentified that are associated with this disease (12). This evidence includes the following: (a) mutations in the rBAT gene are responsible for some but not all cystinuria patients, (b) the genetic locus of the putative subunit that interacts with the rBAT gene has not been identified, (c) the rBAT-specific mRNA is detectable only in the proximal straight tubule of the nephron while the absorption of cystine and cationic amino acids occurs in the proximal convoluted tubule as well as in the proximal straight tubule and the proteins responsible for the absorption in the proximal convoluted tubule have not been identified, (d) cystinuria presents as different, phenotypically distinguishable, genetic variants, and (e) multiple genetic loci have been definitively identified that are associated with the disease. We suggest that 4F2-lc6 is a candidate gene for cystinuria.
Based on the present studies, there are at least two distinct b 0,ϩ -like amino acid transport activities. One of them consists of rBAT and a hitherto unidentified subunit, whereas the other consists of the 4F2 heavy chain and the newly cloned 4F2-lc6. The rBAT-associated b 0,ϩ -like transport process has been shown to be an obligatory amino acid exchanger (12). However, preliminary studies with the 4F2hc/4F2-lc6 complex-associated b 0,ϩ -like transport process in mammalian cells have indicated that the transport process induced by the complex is not an obligatory amino acid exchanger. Neither the influx nor the efflux of arginine or glutamine in 4F2hc/4F2-lc6-expressing cells was influenced by other 4F2hc/4F2-lc6-specific substrates on the trans-side. 2 Thus, there may be significant functional differences between the b 0,ϩ -like transport activities associated with rBAT and 4F2hc/4F2-lc6 complex. The ability of the 4F2hc/4F2-lc6 heterodimeric complex to induce a b 0,ϩ -like amino acid transport activity implies that mutations not only in the 4F2-lc6 gene but also in the 4F2hc gene may be associated with cystinuria. To date, there have been no reports of mutational analysis of the 4F2hc gene in patients with cystinuria. However, it must be noted that mutations in the 4F2hc gene are likely to affect not only the b 0,ϩ -like transport activity but also the transport activities identified as L1, L2, y ϩ L1, y ϩ L2, and x c Ϫ since 4F2hc is a common subunit of these transport systems. On the other hand, mutations in the 4F2-lc6 gene are likely to affect only the b 0,ϩ -like transport activity.