Metabolite movement across the schistosome surface

Intravascular schistosome parasites are covered by an unusual double lipid bilayer. Nutrients, such as glucose and amino acids, as well as other metabolites, are known to be transported across this surface via specific transporter proteins. For instance, the glucose transporter protein SGTP4 is found in the host-interactive tegumental membranes. A second glucose transporter, SGTP1, localizes to the tegumental basal membrane (and internal tissues). Following expression in Xenopus oocytes, SGTP1 and SGTP4 both function as facilitated-diffusion sugar transporters. Suppressing the expression of SGTP1 and SGTP4 in juvenile schistosomes using RNA interference (RNAi) impairs the parasite's ability to import glucose and severely decreases worm viability. Amino acids can also be imported into schistosomes across their surface and an amino acid transporter (SPRM1lc) has been localized in the parasite surface membranes (as well as internally). In Xenopus oocytes, SPRM1lc can import the basic amino acids arginine, lysine and histidine as well as leucine, phenylalanine, methionine and glutamine. To function, this protein requires the assistance of a heavy-chain partner (SPRM1hc) which acts as a chaperone. Water is transported across the tegument of schistosomes via the aquaporin protein SmAQP. Suppressing SmAQP gene expression makes the parasites less able to osmoregulate and decreases their viability. In addition, SmAQP-suppressed adult parasites have been shown to be impaired in their ability to excrete lactate. Analysis of tegumental transporter proteins, as described in this report, is designed to generate a comprehensive understanding of the role of such proteins in promoting parasite survival by controlling the movement of metabolites into and out of the worms.     

On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells

Monoclonal antibodies (Mab) are heterotetramers consisting of an equimolar ratio of heavy chain (HC) and light chain (LC) polypeptides. Accordingly, most recombinant Mab expression systems utilize an equimolar ratio of heavy chain (hc) to light chain (lc) genes encoded on either one or two plasmids. However, there is no evidence to suggest that this gene ratio is optimal for stable or transient production of recombinant Mab. In this study we have determined the optimal ratio of hc:lc genes for production of a recombinant IgG4 Mab, cB72.3, by Chinese hamster ovary (CHO) cells using both empirical and mathematical modeling approaches. Polyethyleneimine-mediated transient expression of cB72.3 at varying ratios of hc:lc genes encoded on separate plasmids yielded an optimal Mab titer at a hc:lc gene ratio of 3:2; a conclusion confirmed by separate mathematical modeling of the Mab folding and assembly process using transient expression data. On the basis of this information, we hypothesized that utilization of hc genes at low hc:lc gene ratios is more efficient. To confirm this, cB72.3 Mab was transiently produced by CHO cells at constant hc and varying lc gene dose. Under these conditions, Mab yield was increased with a concomitant increase in lc gene dose. To determine if the above findings also apply to stably transfected CHO cells producing recombinant Mab, we compared the intra- and extracellular ratios of HC and LC polypeptides for three GS-CHO cells lines transfected with a 1:1 ratio of hc:lc genes and selected for stable expression of the same recombinant Mab, cB72.3. Intra- and extracellular HC:LC polypeptide ratios ranged from 1:2 to 1:5, less than that observed on transient expression of the same Mab in parental CHO cells using the same vector. In conclusion, our data suggest that the optimal ratio of hc:lc genes used for transient and stable expression of Mab differ. In the case of the latter, we infer that optimal Mab production by stably transfected cells represents a compromise between HC abundance limiting productivity and the requirement for excess LC to render Mab folding and assembly more efficient.     

Leucine aminopeptidase of the human blood flukes, Schistosoma mansoni and Schistosoma japonicum

An array of schistosome endoproteases involved in the digestion of host hemoglobin to absorbable peptides has been described, but the exoprotease responsible for catabolising these peptides to amino acids has yet to be identified. By searching the public databases we found that Schistosoma mansoni and Schistosoma japonicum express a gene encoding a member of the M17 family of leucine aminopeptidases (LAPs). A functional recombinant S. mansoni LAP produced in insect cells shared biochemical properties, including pH optimum for activity, substrate specificity and reliance on metal cations for activity, with the major aminopeptidase activity in soluble extracts of adult worms. The pH range in which the enzyme functions and the lack of a signal peptide indicate that the enzyme functions intracellularly. Immunolocalisation studies showed that the S. mansoni LAP is synthesised in the gastrodermal cells surrounding the gut lumen. Accordingly, we propose that peptides generated in the lumen of the schistosome gut are absorbed into the gastrodermal cells and are cleaved by LAP to free amino acids before being distributed to the internal tissues of the parasite. Since LAP was also localised to the surface tegument it may play an additional role in surface membrane re-modelling.     

Identification of a membrane protein, LAT-2, that Co-expresses with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small and large zwitterionic amino acids.

We have identified a new human cDNA, L-amino acid transporter-2 (LAT-2), that induces a system L transport activity with 4F2hc (the heavy chain of the surface antigen 4F2, also named CD98) in oocytes. Human LAT-2 is the fourth member of the family of amino acid transporters that are subunits of 4F2hc. The amino acid transport activity induced by the co-expression of 4F2hc and LAT-2 was sodium-independent and showed broad specificity for small and large zwitterionic amino acids, as well as bulky analogs (e.g. BCH (2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid)). This transport activity was highly trans-stimulated, suggesting an exchanger mechanism of transport. Expression of tagged N-myc-LAT-2 alone in oocytes did not induce amino acid transport, and the protein had an intracellular location. Co-expression of N-myc-LAT-2 and 4F2hc gave amino acid transport induction and expression of N-myc-LAT-2 at the plasma membrane of the oocytes. These data suggest that LAT-2 is an additional member of the family of 4F2 light chain subunits, which associates with 4F2hc to express a system L transport activity with broad specificity for zwitterionic amino acids. Human LAT-2 mRNA is expressed in kidney > placenta > brain, liver > spleen, skeletal muscle, heart, small intestine, and lung. Human LAT-2 gene localizes at chromosome 14q11.2-13 (13 cR or approximately 286 kb from marker D14S1349). The high expression of LAT-2 mRNA in epithelial cells of proximal tubules, the basolateral location of 4F2hc in these cells, and the amino acid transport activity of LAT-2 suggest that this transporter contributes to the renal reabsorption of neutral amino acids in the basolateral domain of epithelial proximal tubule cells.     

Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication

Xenopus egg extracts provide a powerful system for in vitro reconstitution of nuclei and analysis of nuclear transport. Such cell- free extracts contain three major N-acetylglucosaminylated proteins: p200, p97, and p60. Both p200 and p60 have been found to be components of the nuclear pore. Here, the role of p97 has been investigated. Xenopus p97 was isolated and antisera were raised and affinity purified. Immunolocalization experiments indicate that p97 is present in a punctate pattern on the nuclear envelope and also in the nuclear interior. Peptide sequence analysis reveals that p97 contains a GLFG motif which defines a family of yeast nuclear pore proteins, as well as a peptide that is identical at 11/15 amino acids to a specific member of the GLFG family, NUP116. An additional peptide is highly homologous to a second sequence found in NUP116 and other members of the yeast GLFG family. A monoclonal antibody to the GLFG domain cross-reacts with a major Xenopus protein of 97 kD and polyclonal antiserum to p97 recognizes the yeast GLFG nucleoporin family. The p97 antiserum was used to immunodeplete Xenopus egg cytosol and p97-deficient nuclei were reconstituted. The p97-depleted nuclei remained largely competent for nuclear protein import. However, in contrast to control nuclei, nuclei deficient in p97 fail to grow in size over time and do not replicate their chromosomal DNA. ssDNA replication in such extracts remains unaffected. Addition of the N-acetylglucosaminylated nuclear proteins of Xenopus or rat reverses these replication and growth defects. The possible role(s) of p97 in these nuclear functions is discussed.     

Cloning and expression of a b(0,+)-like amino acid transporter functioning as a heterodimer with 4F2hc instead of rBAT. A new candidate gene for cystinuria.

We have cloned a transporter protein from rabbit small intestine, which, when coexpressed with the 4F2 heavy chain (4F2hc) in mammalian cells, induces a b(0,+)-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 b(0,+)-like amino acid transport activity previously shown to be associated with rBAT (protein related to b(0,+) 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 b(0,+)-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.     

Conditional deletion of CD98hc inhibits osteoclast development

The CD98 heavy chain (CD98hc) regulates virus-induced cell fusion and monocyte fusion, and is involved in amino acid transportation. Here, we examined the role that CD98hc plays in the formation of osteoclasts using CD98hc^flox/floxLysM-cre peritoneal macrophages (CD98hc-defect macrophages). Peritoneal macrophages were stimulated with co-cultured with osteoblasts in the presence of 1,25(OH)² vitamin D₃, and thereafter stained with tartrate-resistant acid phosphatase staining solution. The multinucleated osteoclast formation was severely impaired in the peritoneal macrophages isolated from the CD98hc^-defect mice compared with those from wild-type mice. CD98hc mediates integrin signaling and amino acid transport through the CD98 light chain (CD98lc). In integrin signaling, suppression of the M-CSF-RANKL-induced phosphorylation of ERK, Akt, JNK and p130Cas were observed at the triggering phase in the CD98h-defect peritoneal macrophages. Moreover, we showed that the general control non-derepressible (GCN) pathway, which was activated by amino acid starvation, was induced by the CD98hc-defect peritoneal macrophages stimulated with RANKL. These results indicate that CD98 plays two important roles in osteoclast formation through integrin signaling and amino acid transport.     

Identification of a novel Na+-independent acidic amino acid transporter with structural similarity to the member of a heterodimeric amino acid transporter family associated with unknown heavy chains

We identified a novel Na(+)-independent acidic amino acid transporter designated AGT1 (aspartate/glutamate transporter 1). AGT1 exhibits the highest sequence similarity (48% identity) to the Na(+)-independent small neutral amino acid transporter Asc (asc-type amino acid transporter)-2 a member of the heterodimeric amino acid transporter family presumed to be associated with unknown heavy chains (Chairoungdua, A., Kanai, Y., Matsuo, H., Inatomi, J., Kim, D. K., and Endou, H. (2001) J. Biol. Chem. 276, 49390-49399). The cysteine residue responsible for the disulfide bond formation between transporters (light chains) and heavy chain subunits of the heterodimeric amino acid transporter family is conserved for AGT1. Because AGT1 solely expressed or coexpressed with already known heavy chain 4F2hc (4F2 heavy chain) or rBAT (related to b(0,+)-amino acid transporter) did not induce functional activity, we generated fusion proteins in which AGT1 was connected with 4F2hc or rBAT. The fusion proteins were sorted to the plasma membrane and expressed the Na(+)-independent transport activity for acidic amino acids. Distinct from the Na(+)-independent cystine/glutamate transporter xCT structurally related to AGT1, AGT1 did not accept cystine, homocysteate, and l-alpha-aminoadipate and exhibited high affinity to aspartate as well as glutamate, suggesting that the negative charge recognition site in the side chain-binding site of AGT1 would be closer to the alpha-carbon binding site compared with that of xCT. The AGT1 message was predominantly expressed in kidney. In mouse kidney, AGT1 protein was present in the basolateral membrane of the proximal straight tubules and distal convoluted tubules. In the Western blot analysis, AGT1 was detected as a high molecular mass band in the nonreducing condition, whereas the band shifted to a 40-kDa band corresponding to the AGT1 monomer in the reducing condition, suggesting the association of AGT1 with other protein via a disulfide bond. The finding of AGT1 and Asc-2 has established a new subgroup of the heterodimeric amino acid transporter family whose members associate not with 4F2hc or rBAT but with other unknown heavy chains.     

Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines

System L is a major nutrient transport system responsible for the transport of large neutral amino acids including several essential amino acids. We previously identified a transporter (L-type amino acid transporter 1: LAT1) subserving system L in C6 rat glioma cells and demonstrated that LAT1 requires 4F2 heavy chain (4F2hc) for its functional expression. Since its oncofetal expression was suggested in the rat liver, it has been proposed that LAT1 plays a critical role in cell growth and proliferation. In the present study, we have examined the function of human LAT1 (hLAT1) and its expression in human tissues and tumor cell lines. When expressed in Xenopus oocytes with human 4F2hc (h4F2hc), hLAT1 transports large neutral amino acids with high affinity (K(m)= approximately 15- approximately 50 microM) and L-glutamine and L-asparagine with low affinity (K(m)= approximately 1.5- approximately 2 mM). hLAT1 also transports D-amino acids such as D-leucine and D-phenylalanine. In addition, we show that hLAT1 accepts an amino acid-related anti-cancer agent melphalan. When loaded intracellularly, L-leucine and L-glutamine but not L-alanine are effluxed by extracellular substrates, confirming that hLAT1 mediates an amino acid exchange. hLAT1 mRNA is highly expressed in the human fetal liver, bone marrow, placenta, testis and brain. We have found that, while all the tumor cell lines examined express hLAT1 messages, the expression of h4F2hc is varied particularly in leukemia cell lines. In Western blot analysis, hLAT1 and h4F2hc have been confirmed to be linked to each other via a disulfide bond in T24 human bladder carcinoma cells. Finally, in in vitro translation, we show that hLAT1 is not a glycosylated protein even though an N-glycosylation site has been predicted in its extracellular loop, consistent with the property of the classical 4F2 light chain. The properties of the hLAT1/h4F2hc complex would support the roles of this transporter in providing cells with essential amino acids for cell growth and cellular responses, and in distributing amino acid-related compounds.     

SmSak, the second Polo-like kinase of the helminth parasite Schistosoma mansoni: conserved and unexpected roles in meiosis

Polo-like kinases (Plks) are a family of conserved regulators of a variety of events throughout the cell cycle, expanded from one Plk in yeast to five Plks in mammals (Plk1-5). Plk1 is the best characterized member of the Plk family, homolog to the founding member Polo of Drosophila, and plays a major role in cell cycle progression by triggering G2/M transition. Plk4/Sak (for Snk (Serum-inducible kinase) akin kinase) is a unique member of the family, structurally distinct from other Plk members, with essential functions in centriole duplication. The genome of the trematode parasite Schistosoma mansoni contains only two Plk genes encoding SmPlk1 and SmSak. SmPlk1 has been shown already to be required for gametogenesis and parasite reproduction. In this work, in situ hybridization indicated that the structurally conserved Plk4 protein, SmSak, was largely expressed in schistosome female ovary and vitellarium. Expression of SmSak in Xenopus oocytes confirmed its Plk4 conserved function in centriole amplification. Moreover, analysis of the function of SmSak in meiosis progression of G2-blocked Xenopus oocytes indicated that, in contrast to SmPlk1, SmSak cannot induce G2/M transition in the absence of endogenous Plk1 (Plx1). Unexpectedly, meiosis progression was spontaneously observed in Plx1-depleted oocytes co-expressing SmSak and SmPlk1. Molecular interaction between SmSak and SmPlk1 was confirmed by co-immunoprecipitation of both proteins. These data indicate that Plk1 and Plk4 proteins have the potential to interact and cross-activate in cells, thus attributing for the first time a potential role of Plk4 proteins in meiosis/mitosis entry. This unexpected role of SmSak in meiosis could be relevant to further consider the function of this novel Plk in schistosome reproduction.     

Evidence for distinct substrate specificities of importin alpha family members in nuclear protein import.

Importin alpha plays a pivotal role in the classical nuclear protein import pathway. Importin alpha shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin beta-dependent import pathway. In contrast to what is found for importin beta, several isoforms of importin alpha, which can be grouped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin alpha7. To analyze specific functions of the distinct importin alpha proteins, we recombinantly expressed and purified five human importin alpha's along with importin alpha from Xenopus and Saccharomyces cerevisiae. Binding affinity studies showed that all importin alpha proteins from humans or Xenopus bind their import receptor (importin beta) and their export receptor (CAS) with only marginal differences. Using an in vitro import assay based on permeabilized HeLa cells, we compared the import substrate specificities of the various importin alpha proteins. When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin alpha3, whereas most of the other substrates were imported by all importin alpha proteins with similar efficiencies. However, strikingly different substrate preferences of the various importin alpha proteins were revealed when two substrates were offered simultaneously.     

Activation of system L heterodimeric amino acid exchangers by intracellular substrates

System L-type transport of large neutral amino acids is mediated by ubiquitous LAT1-4F2hc and epithelial LAT2-4F2hc. These heterodimers are thought to function as obligatory exchangers, but only influx properties have been studied in some detail up until now. Here we measured their intracellular substrate selectivity, affinity and exchange stoichiometry using the Xenopus oocyte expression system. Quantification of amino acid influx and efflux by HPLC demonstrated an obligatory amino acid exchange with 1:1 stoichiometry. Strong, differential trans-stimulations of amino acid influx by injected amino acids showed that the intracellular substrate availability limits the transport rate and that the efflux selectivity range resembles that of influx. Compared with high extracellular apparent affinities, LAT1- and LAT2-4F2hc displayed much lower intracellular apparent affinities (apparent K(m) in the millimolar range). Thus, the two system L amino acid transporters that are implicated in cell growth (LAT1-4F2hc) and transcellular transport (LAT2-4F2hc) are obligatory exchangers with relatively symmetrical substrate selectivities but strongly asymmetrical substrate affinities such that the intracellular amino acid concentration controls their activity.     

4F2hc stabilizes GLUT1 protein and increases glucose transport activity

Glucose transporter 1 (GLUT1) is widely distributed throughout various tissues and contributes to insulin-independent basal glucose uptake. Using a split-ubiquitin membrane yeast two-hybrid system, we newly identified 4F2 heavy chain (4F2hc) as a membrane protein interacting with GLUT1. Though 4F2hc reportedly forms heterodimeric complexes between amino acid transporters, such as LAT1 and LAT2, and regulates amino acid uptake, we investigated the effects of 4F2hc on GLUT1 expression and the associated glucose uptake. First, FLAG-tagged 4F2hc and hemagglutinin-tagged GLUT1 were overexpressed in human embryonic kidney 293 cells and their association was confirmed by coimmunoprecipitation. The green fluorescent protein-tagged 4F2hc and DsRed-tagged GLUT1 showed significant, but incomplete, colocalization at the plasma membrane. In addition, an endogenous association between GLUT1 and 4F2hc was demonstrated using mouse brain tissue and HeLa cells. Interestingly, overexpression of 4F2hc increased the amount of GLUT1 protein in HeLa and HepG2 cells with increased glucose uptake. In contrast, small interfering RNA (siRNA)-mediated 4F2hc gene suppression markedly reduced GLUT1 protein in both cell types, with reduced glucose uptake. While GLUT1 mRNA levels were not affected by overexpression or gene silencing of 4F2hc, GLUT1 degradation after the addition of cycloheximide was significantly suppressed by 4F2hc overexpression and increased by 4F2hc siRNA treatment. Taken together, these observations indicate that 4F2hc is likely to be involved in GLUT1 stabilization and to contribute to the regulation of not only amino acid but also glucose metabolism.     

Amino acid transport of y+L-type by heterodimers of 4F2hc/CD98 and members of the glycoprotein-associated amino acid transporter family.

Amino acid transport across cellular membranes is mediated by multiple transporters with overlapping specificities. We recently have identified the vertebrate proteins which mediate Na+-independent exchange of large neutral amino acids corresponding to transport system L. This transporter consists of a novel amino acid permease-related protein (LAT1 or AmAT-L-lc) which for surface expression and function requires formation of disulfide-linked heterodimers with the glycosylated heavy chain of the h4F2/CD98 surface antigen. We show that h4F2hc also associates with other mammalian light chains, e.g. y+LAT1 from mouse and human which are approximately 48% identical with LAT1 and thus belong to the same family of glycoprotein-associated amino acid transporters. The novel heterodimers form exchangers which mediate the cellular efflux of cationic amino acids and the Na+-dependent uptake of large neutral amino acids. These transport characteristics and kinetic and pharmacological fingerprints identify them as y+L-type transport systems. The mRNA encoding my+LAT1 is detectable in most adult tissues and expressed at high levels in kidney cortex and intestine. This suggests that the y+LAT1-4F2hc heterodimer, besides participating in amino acid uptake/secretion in many cell types, is the basolateral amino acid exchanger involved in transepithelial reabsorption of cationic amino acids; hence, its defect might be the cause of the human genetic disease lysinuric protein intolerance.     

The cellular distribution and stage-specific expression of two dynein light chains from the human blood fluke Schistosoma japonicum

Schistosomes are pathogenic helminth parasites of human portal veins. Their body wall is a highly active syncytial tegument involved in an array of host interactions. The cytoskeletal organization and dynamics of this syncytium are poorly understood, but predominant motor components are the LC8 class of cytoplasmic dynein light chains (DLC). Four LC8 members occur in schistosomes, two of which are expressed in the tegument. Here, we describe the cytoplasmic distribution, stage-specific expression and cellular location of two diverse LC8 molecules of Schistosoma japonicum. SjDLC1 was detected in surface-membrane specific extracts of adult worms and was shown by quantitative immuno-electron microscopy to predominate along heptalaminate membranes of the worm surface. SjDLC3 also occurs in the tegument, but was shown to be present in basal layers of the tegument and did not preferentially co-localize with particular membrane components. SjDLC3 was also detected in the gastrodermis. SjDLC1 is expressed only in mammalian-parasitic stages, whereas SjDLC3 is expressed throughout the life-cycle. The data suggest that SjDLC1 is preferentially located to the host-interactive distal parasite membrane, and plays a role in surface membrane dynamics, while SjDLC3 is a ubiquitous motor component of schistosome epithelia of all stages.     

Basolateral aromatic amino acid transporter TAT1 (Slc16a10) functions as an efflux pathway

Basolateral efflux is a necessary step in transepithelial (re)absorption of amino acids from small intestine and kidney proximal tubule. The best characterized basolateral amino acid transporters are y+LAT1-4F2hc and LAT2-4F2hc that function as obligatory exchangers and thus, do not contribute to net amino acid (re)absorption. The aromatic amino acid transporter TAT1 was shown previously to localize basolaterally in rat's small intestine and to mediate the efflux of L-Trp in the absence of exchange substrate, upon expression in Xenopus oocytes. We compared here the amino acid influx and efflux via mouse TAT1 in Xenopus oocytes. The results show that mTAT1 functions as facilitated diffusion pathway for aromatic amino acids and that its properties are symmetrical in terms of selectivity and apparent affinity. We show by real-time RT-PCR that its mRNA is highly expressed in mouse small intestine mucosa, kidney, liver, and skeletal muscle as well as present in all other tested tissues. We show that mTAT1 is not N-glycosylated and that it localizes to the mouse kidney proximal tubule. This expression is characterized by an axial gradient similar to that of the luminal neutral amino acid transporter B0AT1 and shows the same basolateral localization as 4F2hc. mTAT1 also localizes to the basolateral membrane of small intestine enterocytes and to the sinusoidal side of perivenous hepatocytes. In summary, we show that TAT1 is a basolateral epithelial transporter and that it can function as a net efflux pathway for aromatic amino acids. We propose that it, thereby, may supply parallel exchangers with recycling uptake substrates that could drive the efflux of other amino acids.     

LAT2, a new basolateral 4F2hc/CD98-associated amino acid transporter of kidney and intestine

Glycoprotein-associated amino acid transporters (gpaAT) are permease-related proteins that require heterodimerization to express their function. So far, four vertebrate gpaATs have been shown to associate with 4F2hc/CD98 for functional expression, whereas one gpaAT specifically associates with rBAT. In this study, we characterized a novel gpaAT, LAT2, for which mouse and human cDNAs were identified by expressed sequence tag data base searches. The encoded ortholog proteins are 531 and 535 amino acids long and 92% identical. They share 52 and 48% residues with the gpaATs LAT1 and y(+)LAT1, respectively. When mouse LAT2 and human 4F2hc cRNAs were co-injected into Xenopus oocytes, disulfide-linked heterodimers were formed, and an L-type amino acid uptake was induced, which differed slightly from that produced by LAT1-4F2hc: the apparent affinity for L-phenylalanine was higher, and L-alanine was transported at physiological concentrations. In the presence of an external amino acid substrate, LAT2-4F2hc also mediated amino acid efflux. LAT2 mRNA is expressed mainly in kidney and intestine, whereas LAT1 mRNA is expressed widely. Immunofluorescence experiments showed colocalization of 4F2hc and LAT2 at the basolateral membrane of kidney proximal tubules and small intestine epithelia. In conclusion, LAT2 forms with LAT1 a subfamily of L-type gpaATs. We propose that LAT1 is involved in cellular amino acid uptake, whereas LAT2 plays a role in epithelial amino acid (re)absorption.