Identification of a Novel System L Amino Acid Transporter Structurally Distinct from Heterodimeric Amino Acid Transporters*

A cDNA that encodes a novel Na+-independent neutral amino acid transporter was isolated from FLC4 human hepatocarcinoma cells by expression cloning. When expressed in Xenopus oocytes, the encoded protein designated LAT3 (L-type amino acid transporter 3) transported neutral amino acids such as l-leucine, l-isoleucine, l-valine, and l-phenylalanine. The LAT3-mediated transport was Na+-independent and inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with the properties of system L. Distinct from already known system L transporters LAT1 and LAT2, which form heterodimeric complex with 4F2 heavy chain, LAT3 was functional by itself in Xenopus oocytes. The deduced amino acid sequence of LAT3 was identical to the gene product of POV1 reported as a prostate cancer-up-regulated gene whose function was not determined, whereas it did not exhibit significant similarity to already identified transporters. The Eadie-Hofstee plots of LAT3-mediated transport were curvilinear, whereas the low affinity component is predominant at physiological plasma amino acid concentration. In addition to amino acid substrates, LAT3 recognized amino acid alcohols. The transport of l-leucine was electroneutral and mediated by a facilitated diffusion. In contrast, l-leucinol, l-valinol, and l-phenylalaninol, which have a net positive charge induced inward currents under voltage clamp, suggesting these compounds are transported by LAT3. LAT3-mediated transport was inhibited by the pretreatment with N-ethylmaleimide, consistent with the property of system L2 originally characterized in hepatocyte primary culture. Based on the substrate selectivity, affinity, and N-ethylmaleimide sensitivity, LAT3 is proposed to be a transporter subserving system L2. LAT3 should denote a new family of organic solute transporters.

System L is a plasma membrane amino acid transport system, which mediates Na ϩ -independent transport of large neutral amino acids (1). It was first characterized in Ehrlich ascites tumor cells as a transport system specifically inhibited by a bicyclic amino acid, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) 1 (1)(2)(3). Various subtypes with different characteristics in substrate selectivity and transport property have, subsequently, been described so far for system L (4 -9). System L is a major route to provide cells with branched-chain and aromatic amino acids. In addition, system L is present in the basolateral membrane of epithelial cells and plays important roles in the absorption of amino acids through the epithelial cells of small intestine and renal proximal tubules (1). System L is also pivotal in the permeation of amino acids through the blood-tissue barriers such as blood-brain barrier and placenta barrier (1).
By means of expression cloning, we identified the first isoform of system L amino acid transporter LAT1 (L-type amino acid transporter 1) in C6 rat glioma cells (10). LAT1 is a member of the SLC (solute carrier) 7 family with putative 12-membrane-spanning domains (11,12). LAT1 mediates a Na ϩ -independent amino acid exchange and prefers large neutral amino acids such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine, and histidine for its substrates (10,(13)(14)(15). We and others further demonstrated that a single-membrane-spanning protein, the heavy chain of 4F2 antigen (4F2hc), is essential for the functional expression of LAT1 in the plasma membrane (10,16). LAT1 and 4F2hc form a heterodimeric complex via a disulfide bond (13, 16 -18). Following the identification of LAT1, transporters structurally related to LAT1 have been found to be associated with 4F2hc or other single-membrane-spanning subunit rBAT (related to the b 0,ϩ amino acid transporter), establishing the heterodimeric amino acid transporter family (11,19). These transporters include systems asc, y ϩ L, x Ϫ C , and b 0,ϩ as well as the second system L isoform, LAT2 (11,19). LAT2 is more ubiquitously expressed than LAT1 and transports not only large neutral amino acids but also small neutral amino acids (20 -22).
Even after the finding of the heterodimeric amino acid transporters, some of the previously reported properties of system L still remain to be explained by the properties of LAT1 and LAT2. For example, system L2 characterized in hepatocyte primary culture exhibits narrower substrate selectivity in * This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Japan Society for the Promotion of Science, the Promotion and Mutual Aid Corporation for Private Schools of Japan, the Japan Health Sciences Foundation, Uehara Memorial Foundation, YASUDA Medical Research Foundation, and Health and Labor Sciences Research Grants for Research on Advanced Medical Technology: Toxicogenomics Project. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM  which it prefers leucine, isoleucine, and phenylalanine, distinct from that of LAT1 and LAT2 (5). Therefore, it has been proposed that still unidentified system L transporters should be present. During the search for culture cell lines with the amino acid transport properties distinct from those of LAT1 and LAT2, we have found that human hepatocarcinoma-derived cell line FLC4 (23, 24) exhibits a unique character in the leucine transport. In this study, we have performed expression cloning to identify the system L transporter of FLC4 cells and isolated a cDNA encoding a novel amino acid transporter. The transporter is structurally distinct from the heterodimeric transporters and exhibits the properties corresponding to system L2.
Expression Cloning-Expression cloning using a Xenopus oocyte expression system was performed as described previously (10,(27)(28)(29)(30). 400 g of poly(A) ϩ RNA obtained from FLC4 cells was size-fractionated by preparative gel electrophoresis (10,29). RNA from each fraction (50 ng) was expressed in Xenopus oocytes. Positive fractions showing peak stimulation of L-[ 14 C]leucine (100 M) were used to construct a directional cDNA library (10,29). cRNA synthesized in vitro from pools of ϳ500 clones was injected into Xenopus oocytes (10,29). A positive pool was sequentially subdivided and analyzed until a single clone (LAT3) was identified. The cDNA was sequenced in both direction by dye terminator cycle sequencing method (PerkinElmer Life Sciences). Transmembrane regions of proteins were predicted based on TopPred2 algorithm (31).
Functional Characterization-Xenopus oocytes were injected with 25 ng of LAT3 cRNA synthesized in vitro from the LAT3 cDNA in plasmid pSPORT1 (Invitrogen) linearized with NotI (25). Three days after injection, the uptake of 14 C-labeled amino acids was measured as described above in the regular uptake solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, 5 mM Tris, pH 7.4) or Na ϩ -free uptake solution in which NaCl in the regular uptake solution was replaced by choline chloride, containing 0.5-2.0 Ci/ml of radiolabeled compounds. For Cl Ϫ -free uptake solution, Cl Ϫ in the regular uptake solution was replaced by gluconate anion. To prepare uptake solution with varied pH for pH dependence experiments, MES-NaOH (pH 5.5 and 6.0), PIPES-NaOH (pH 6.5 and 7.0), HEPES-NaOH (pH 7.5 and 8.0), and Tris-HCl (pH 8.5) were used for buffer systems (21). Preliminary experiments to determine the time course of L-[ 14 C]leucine (3 mM) uptake into oocytes expressing LAT3 indicated that the uptake was linearly dependent on incubation time up to 30 min (data not shown), so for all the following experiments uptakes were measured for 30 min, and the values were expressed as picomoles/oocyte/min.
The effect of N-ethylmaleimide (NEM) on the transport was examined by measuring the L-[ 14 C]leucine uptake after 15-min preincubation of the oocytes expressing LAT3 or LAT1 with Na ϩ -free uptake solution containing 5 mM NEM at room temperature (32). After removing the solution for preincubation, the uptake of L-leucine by the oocytes was measured in the Na ϩ -free uptake solution containing 100 M L-[ 14 C]leucine with or without 5 mM NEM. The uptake values were compared with those measured without NEM preincubation. For the expression of LAT1, Xenopus oocytes were injected with 17.6 ng of human LAT1 cRNA and 7.4 ng of human 4F2hc cRNA at a final molar ratio of 1:1 (13).
Kinetic parameters of amino acid substrates were determined based on the LAT3-mediated amino acid uptakes measured at 1, 3, 10, 30, 60, 100, 200, 400, 1000, 2000, and 3000 M of substrates. LAT3-mediated amino acid uptakes were calculated as differences between the means of uptakes of the oocytes injected with LAT3 cRNA and those of the control oocytes injected with water. Because Eadie-Hofstee plots were curvilin-ear for the substrates of LAT3, apparent K m and V max were estimated by fitting the concentration-dependent substrate transport to the following equation for two-component kinetics using For the efflux measurement, 100 nl (3 nCi) of L-[ 14 C]leucine (100 M) was injected into oocytes with a fine-tipped glass micropipette as described elsewhere (30,34,35). The individual oocytes were incubated for 5 min in the ice-cold Na ϩ -free uptake solution and then transferred to the Na ϩ -free uptake solution kept at room temperature (18 -22°C). The radioactivity in the medium and the remaining radioactivity in the oocytes were measured. The values were expressed as percent radioactivity (radioactivity of medium/(radioactivity of medium ϩ radioactivity of oocytes) ϫ 100%) (30,34,35).
For the uptake and efflux measurements in the present study, six to ten oocytes were used for each data point. Each data point in the figures represents the mean Ϯ S.E. of uptake (n ϭ 6 -10). To confirm the reproducibility of the results, three separate experiments using different batches of oocytes and in vitro transcribed cRNA were performed for each measurement. Results from the representative experiments are shown in figures.
Electrophysiological Measurements-The electrical currents induced by 10 mM L-leucine, L-leucinol, L-valinol, and L-phenylalaninol in both control and LAT3-cRNA-injected Xenopus oocytes were recorded during voltage clamp at Ϫ60 mV by two-electrode voltage camping using Gene-Clamp 500 (Axon Instruments) in the standard bath solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, pH 7.4) (26,36,37). The baseline electrical current was first determined and then 10 mM of L-leucine, L-leucinol, L-valinol, or L-phenylalaninol was added to the bath medium for 2 min. The solution was then changed back to the standard uptake solution, and the record was continued until the electrical current was returned to the baseline level.
Northern Analysis-The cDNA fragment corresponding to 1790 -1936 bp of LAT3 cDNA (GenBank TM /EMBL/DDBJ accession number AB103033) was PCR-amplified and labeled with [ 32 P]dCTP using a T7 Quick prime kit (Amersham Biosciences) (25). Multiple Tissue Northern Blots (Clontech) were hybridized with the probe and processed following the manufacturer's instructions. FLC4 poly(A) ϩ RNA prepared as described above and human liver poly(A) ϩ RNA (Clontech) were separated on a 1% agarose gel in the presence of 2.2 M formaldehyde and blotted onto a nitrocellulose filter (Schleicher & Schuell) (25). Hybridization was performed for 20 h at 42°C as described elsewhere (25). The filters were washed in 0.1 ϫ SSC/0.1% SDS at 65°C.

RESULTS
When poly(A) ϩ RNA from FLC4 human hepatocarcinoma cells was expressed in Xenopus laevis oocytes, significant augmentation of L-[ 14 C]leucine uptake was detected. The size-fractionation of the FLC4 poly(A) ϩ RNA revealed that the fraction of 2.2-2.7 kb exhibited the peak activity of L-[ 14 C]leucine uptake (data not shown). From this fraction, a cDNA library was constructed and screened for L-[ 14 C]leucine uptake by expression in Xenopus oocytes. A 2.5-kb cDNA was isolated that encodes a protein designated LAT3 (L-type amino acid transporter 3).
When expressed in Xenopus oocytes, LAT3 induced L-[ 14 C]leucine transport, which was not dependent on Na ϩ or Cl Ϫ in the medium (Fig. 1, a and b). Although LAT3-mediated L-[ 14 C]leucine uptake was saturable, its Eadie-Hofstee plot was curvilinear, suggesting the presence of multiple components for the L-leucine uptake induced by the expression of a single protein LAT3 (Fig. 1c). The concentration-dependent substrate uptake was fit to the two-component kinetics (see "Experimental Procedures"). The apparent K m and V max were estimated by best fitting and shown in Table I.
The substrate selectivity of LAT3 was investigated by inhibition experiments in which L-[ 14 C]leucine (100 M) uptake was measured in the presence of 10 mM amino acids. The L-leucine uptake was highly inhibited by L-isomers of isoleucine, valine, phenylalanine, and methionine (Fig. 2a). Weaker inhibition was detected when the other neutral amino acids were used as inhibitors. Acidic amino acids, L-aspartate and L-glutamate, and basic amino acids, L-lysine and L-arginine, did not inhibit LAT3-mediated L-[ 14 C]leucine uptake (Fig. 2a). Although Damino acids less affected LAT3-mediated L-[ 14 C]leucine uptake, D-leucine, D-histidine, and D-methionine showed relatively strong inhibitory effects on the L-[ 14 C]leucine uptake was compared with that measured in the Na ϩ -free uptake solution (Na ϩ -free) and that measured in the Cl Ϫ -free uptake solution (Cl Ϫ -free). c, concentration dependence of LAT3-mediated L-[ 14 C]leucine uptake. LAT3-mediated L-[ 14 C]leucine uptake was measured at 1, 3, 10, 30, 60, 100, 200, 400, 1000, 2000, and 3000 M L-leucine in a Na ϩ -free uptake solution and plotted against the L-leucine concentration. LAT3-mediated L-[ 14 C]leucine uptake levels were calculated as differences between the means of uptake of the oocytes injected with LAT3 cRNA and those of the control oocytes injected with water. The L-leucine uptake was saturable and fit to the two-component kinetics  A inhibitor ␣-(aminomethyl)isobutyric acid had no inhibitory effect (data not shown).
Consistent with the results from the inhibition experiments, 14 C-labeled L-leucine, L-isoleucine, L-valine, L-phenylalanine, and L-methionine (100 M) were transported at relatively high rate by LAT3 (Fig. 2b). Among D-amino acids, D-leucine, for which a 14 C-labeled compound was available, was confirmed to be transported by LAT3 (Fig. 2b). As observed for L-leucine uptake, the Eadie-Hofstee plots for the uptake of L-isoleucine, L-valine, and L-phenylalanine were curvilinear (data not shown). Kinetic parameters of these amino acid substrates are listed in Table I.
To examine whether LAT3-mediated transport is electrogenic or not, we performed electrophysiological measurements. Under the voltage clamp at Ϫ60 mV, L-leucinol, L-valinol, and L-phenylalaninol (10 mM) induced significantly larger inward current in the Xenopus oocytes expressing LAT3 than that in the control oocytes, whereas the current induced by L-leucine (10 mM) was indistinguishable between LAT3-expressing oocytes and control oocytes (Fig. 4). In the same batch of oocytes expressing LAT3, 6.3 Ϯ 0.4 pmol/oocyte/min (mean Ϯ S.E., n ϭ 8) of L-[ 14 C]leucine uptake was detected for 10 mM L-leucine (data not shown).
The effect of pH on LAT3-mediated L-[ 14 C]leucine transport was examined by varying pH of the uptake solution.
To examine the mode of transport mediated by LAT3, the efflux of radioactivity from the oocytes preloaded with L-[ 14 C]leucine was measured. As shown in Fig. 5, time-dependent efflux of radioactivity was detected from the oocytes expressing LAT3. In contrast, only a low level of efflux of preloaded L-[ 14 C]leucine was detected in the control oocytes injected with water instead of LAT3 cRNA (Fig. 5).
Because it was once reported that the treatment with NEM affects the function of not all but at least part of system L transport activity (32), we examined the effect of NEM on LAT3-mediated L-[ 14 C]leucine transport. As shown in Fig. 6 were predicted on the LAT3 amino acid sequence by means of the TopPred2 algorithm. LAT3 protein contains a relatively long extracellular loop with putative N-linked glycosylation sites (Asn 212 and Asn 229 ) between transmembrane domains 1 and 2. A long intracellular loop was predicted between transmembrane domains 6 and 7, which contain putative protein kinase C-dependent phosphorylation sites (Thr 231 and Ser 262 ) and a tyrosine phosphorylation site (Tyr 251 ). An additional protein kinase C-dependent phosphorylation site was predicated at Ser 405 in the putative intracellular loop between transmembrane domains 8 and 9. A leucine zipper motif was predicted at Leu 149 -Leu 170 . The search of protein databases revealed that the amino acid sequence of LAT3 is identical to that of the gene product of POV1, which was identified as a prostate cancer-up-regulated gene whose function was not determined (38). Northern blot analysis using human Multiple Tissue Northern Blots indicated that a 2.5-kb LAT3 message was expressed at high level in pancreas, liver, skeletal muscle, and fetal liver. Weaker signals were also detected in heart, placenta, lung, kidney, spleen, prostate, testis, ovary, small intestine, colon, lymph node, and bone marrow (Fig. 7a). In pancreas, an additional 4.4 kb message was also detected (Fig. 7a). The expression of LAT3 in FLC4 from which LAT3 cDNA was isolated was confirmed by the Northern blot using FLC4 poly(A) ϩ RNA (Fig. 7b). DISCUSSION We previously showed that L-leucine uptake by T24 human bladder carcinoma cells is almost exclusively mediated by LAT1 (39). Most of the tumor cell lines exhibit similar properties to that of T24 cells. However, we found that FLC4 hepa-tocarcinoma-derived cells (23, 24) exhibited a somewhat different character in the leucine transport. L-[ 14 C]Leucine uptake by FLC4 cells was not inhibited by the compounds that inhibited T24 cell-mediated uptake, such as triiodothyronine and ␣-methyltyrosine (data not shown). LAT3 was identified as a transporter responsible for the L-leucine uptake by FLC4 cells. LAT3 proved to be a gene product of POV1 reported previously as a prostate cancer-up-regulated gene whose function was not determined (38). LAT3 did not show significant sequence similarity to already identified transporters and was not classified into any established solute carrier families SLC1ϳ41 (HUGO Gene Nomenclature Committee, www.gene.ucl.ac.uk/nomenclature/). LAT3, however, shows similar transmembrane topology to that of the members of organic cation/anion transporter family (SLC22) and facilitated glucose transporter family ]leucine uptake (100 M) was measured in the Na ϩ -free uptake solution in the presence of 10 mM non-radiolabeled compounds and expressed as a percentage of the control L-leucine uptake in the absence of inhibitors (Ϫ). R 1 -CH(NH 2 )COOH, L-leucine; R 1 -CH(NH 2 )CH 2 OH, L-leucinol; R 1 -CH 2 NH 2 , isopentylamine; R 1 -CH 2 COOH, 4-methylvaleric acid; R 1 -CH(NH 2 )CH 3 , 1,3-dimethyl-n-butylamine; R 1 -CH(NH 2 )CONH2, L-leucinamide; R 1 -CH(NH 2 )COOCH 3 , L-leucine methylester; R 1 -CH(NHCOCH 3 )COOH, N-acetyl-L-leucine; R 1 -CH(NHCH 3 )COOH, N-methyl-L-leucine; R 2 -CH(NH 2 )COOH, L-valine; R 2 -CH(NH 2 )CH 2 OH, L-valinol; R 2 -CH 2 NH 2 , isobutylamine; R 2 -CH 2 COOH, isovaleric acid; R 3 -CH(NH 2 )COOH, Lphenylalanine; R 3 -CH(NH 2 )CH 2 OH, L-phenylalaninol; R 3 -CH 2 NH 2 , 2-phenylethylamine; R 3 -CH 2 COOH, 3-phenylpropionic acid; R 4 -CH(NH 2 )COOH, L-tyrosine; R 4 -CH(NH 2 )CH 2 OH, L-tyrosinol; R 4 -CH 2 NH 2 , tyramine; R 4 -CH 2 COOH, 3-(p-hydroxyphenyl)propionic acid. The asterisks indicate statistical significance. *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001 (Student's unpaired t test).

FIG. 4. Electrophysiological measurement.
The current responses of control oocytes (left) and LAT3-expressing oocytes (right) to L-leucine, L-leucinol, L-valinol, and L-phenylalaninol. Membrane current was measured in the standard bath solution at a holding potential of Ϫ60 mV. Ten millimolar L-leucine and indicated amino acid alcohols were applied to the bath at moment in time indicated by the bars after stabilizing the membrane current.
(SLC2), in that LAT3 has a relatively long extracellular loop with predicted N-linked glycosylation sites between transmembrane domains 1 and 2 and a long intracellular loop with several putative phosphorylation sites between transmembrane domains 6 and 7 (40,41). LAT3 should, thus, denote a new solute carrier family.
LAT3-mediated transport was Na ϩ -independent and BCHsensitive, showing the properties of system L (1). Distinct from LAT1 and LAT2, which are the members of the SLC7 family and already assigned to system L (10, 16, 20 -22), LAT3 did not require 4F2hc for its functional expression. A single injection of LAT3 cRNA is sufficient for the functional expression of LAT3 in Xenopus oocytes. Substrate selectivity of LAT3 was also distinct from LAT1 and LAT2. LAT3 shows narrower substrate selectivity compared with LAT1 and LAT2 and mainly transports branched-chain amino acids and phenylalanine. LAT3 does not recognize triiodothyronine and ␣-methyltyrosine, consistent with the properties of leucine transport of FLC4 cells (data not shown). It has, thus, turned out that more than one families of transporters contribute to system L transport activity.
We previously showed that the transport mediated by TAT1, a Na ϩ -independent aromatic amino acid transporter for system T, was inhibited by N-acetyl-and N-methyl-derivatives of substrate amino acids but not by amino acid methylesters (30). Based on this observation, it was suggested that TAT1 recognizes amino acid substrates as anions, consistent with its structural similarity to H ϩ /monocarboxylate transporters of SLC16 family. In contrast, LAT3-mediated transport was not prominently inhibited by N-acetyl-derivatives, N-methyl-derivatives, or methylesters. We found that L-leucinol, 1,3-dimethyln-butylamine, L-valinol, and L-phenylalaninol exhibited relatively strong inhibitory effects on LAT3-mediated transport. Thus, it is suggested that ␣-carboxyl group can be substituted by hydroxymethyl group or methyl group for the recognition by LAT3, although hydroxymethyl and methyl groups appear less suited compared with the carboxyl group. Because N-methyl-L-leucine exhibited relatively strong inhibition, it seems that the ␣-amino group does not have to be intact for the interaction with LAT3. It is interesting that isopentylamine, isobutylamine, and 2-phenylethylamine, which possess side chains of L-leucine, L-valine, and L-phenylalanine, respectively, and lack ␣-carboxyl groups, exhibited weak but significant inhibitory effects, whereas 4-methylvaleric acid, isovaleric acid, and 3-phenylpropionic acid, which lack ␣-amino groups, had no inhibitory effects. Therefore, the ␣-amino group or its modified moieties seems to be indispensable for the interaction with the substrate binding site.
A remarkable feature of the transport kinetics of LAT3 is in its curvilinear Eadie-Hofstee plots. The expression of a transporter encoded by a single cDNA into heterologous expression systems usually results in the appearance of a transport function with a single-component kinetics (14,21,25,27,30,34,35,(42)(43)(44). We compared the concentration-dependent transport mediated by LAT3 with that mediated by system T transporter TAT1 (30) in the same batch of oocytes. We obtained linear Eadie-Hofstee plots for L-tryptophan transport by TAT1 in contrast to curvilinear Eadie-Hofstee plots for L-leucine transport by LAT3, confirming the peculiar nature of LAT3 (data not shown). In some transmembrane receptors, it has been reported that phosphorylation of the receptor proteins alter the affinity for ligand binding, so that producing heterogenic populations is receptor affinity-dependent on their phosphorylation state, which results in the apparent multicomponent kinetics (45)(46)(47). This might be considered also for LAT3, because it possesses putative phosphorylation sites, although the effect of phosphorylation on the substrate affinity is not determined yet. The low affinity component is assumed to be predominant at physiological plasma amino acid concentration based on the comparison of V max values ( Table I). The properties of LAT3-mediated L-leucine transport characterized by using 100 M L-[ 14 C]leucine as a tracer in the present study mainly reflect those of the low affinity component.
Recently it has been shown that many amino acid transporters mediate the exchange of substrates (10,14,15,34,35,48,49). As shown in Fig. 5, however, the efflux of preloaded L-[ 14 C]leucine was detected even in the absence of extracellular L-leucine, which is totally different from the properties of typical amino acid exchangers such as LAT1 and y ϩ LAT1 (y ϩ L-type amino acid transporter 1) (10,14,15,35). The L-leucine transport mediated by LAT3 was electroneutral and not dependent on Na ϩ , Cl Ϫ , and pH around physiological pH (6.5-8.0) (Figs. 1a, 1b, and 4), suggesting that LAT3-mediated transport is not coupled to the transport of inorganic ions, including H ϩ . These properties are similar to those of TAT1, which mediate an electroneutral facilitated diffusion of neutral amino acids (30). It is thus suggested that LAT3-mediated transport is due to the facilitated diffusion rather than the obligatory exchange of substrate amino acids.
We found that L-leucinol, L-valinol, and L-phenylalaninol, which have a net positive charge, induced inward current in the oocytes expressing LAT3 under the voltage clamp condition, although a neutral amino acid L-leucine did not generate significant currents (Fig. 4). This suggests that L-leucinol, Lvalinol, and L-phenylalaninol, which inhibit LAT3-mediated L-leucine uptake, are in fact transported by LAT3. Electrogenic facilitated transport of cationic compounds was once reported for cationic amino acid transporters (50). Because L-leucinol, L-valinol, and L-phenylalaninol are positively charged, the facilitated diffusion of these amino acid alcohols via LAT3 from outside to the inside of the oocytes generates inward currents.
In primary hepatocyte culture, it was shown that system L with low affinity and narrow substrate selectivity ("system L2") is predominant in freshly isolated hepatocytes. It is replaced by the other subtype of system L with high affinity and broader substrate selectivity ("system L1") over the initial 24 to 48 h of culture (1,5). It was reported that the uptake by system L1 is of high affinity (micromolar range) and substantially inhibited by cysteine, valine, isoleucine, leucine, methionine, histidine, tryptophan, tyrosine, phenylalanine, and BCH. In contrast, system L2-mediated transport exhibited low affinity (millimolar range) and narrower substrate selectivity. The uptake by system L2 was inhibited by isoleucine, leucine, phenylalanine, and BCH (5,32,51). It was noted that system L2 is sensitive to inhibition by NEM, whereas system L1 is not affected by NEM (32). In this study, we showed that L-leucine transport mediated by LAT3 was completely inhibited by NEM, whereas that mediated by LAT1 was not affected by NEM (Fig. 6). This NEM sensitivity of LAT3 is similar to that of system L2. Once the oocytes were pretreated with NEM, the presence or absence of NEM in the uptake solution with L-[ 14 C]leucine did not affect the results. This is consistent with NEM, a thiol reagent, inhibiting the functional activity of LAT3 by reacting covalently with exposed sulfhydryl groups (32) and not by competing with substrates at the binding site of LAT3. Considering the low affinity and narrower substrate selectivity together with the NEM sensitivity, we propose that LAT3 is a transporter subserving system L2. In contrast, the properties of LAT1 are consistent with those of system L1.
The Northern blot analysis of human tissues revealed the high level of LAT3 mRNA in pancreas and adult and fetal liver (Fig. 7a), consistent with the report by Cole et al. (38). Our Northern blot further showed strong signals in skeletal muscle and weaker signals in many other tissues (Fig. 7a). LAT3, the gene product of POV1, was reported to be up-regulated in prostate cancers (38). DNA microarray analyses revealed that POV1 gene is also up-regulated in testicular tumors, suggesting the possible involvement of LAT3 in a variety of malignant tumors (52). In prostate cancers, two spliced variants were reported for POV1 (38). The one that is identical to LAT3 and encoded by a shorter message (2.4 -2.6 kb) was reported to be predominantly expressed in normal tissues, whereas the other encoded by a longer message (4.4 -5 kb) was proposed to be primarily associated with fetal tissues and tumors (38). Because of this alternative splicing, putative transmembrane domains 11 and 12 and C terminus intracellular domains are altered. The functional significance of this alternative splicing and its roles in cancer cells remain to be clarified.
In summary, we have identified a novel Na ϩ -independent neutral amino acid transporter LAT3. LAT3 exhibits system L activity corresponding to the NEM-sensitive system L2. Because LAT3 does not exhibit structural similarity to already identified transporters, including system L transporters LAT1 and LAT2, LAT3 should denote a new family of organic solute transporters.