The amino acid transport system b(o,+) and cystinuria

Amino acid transport in mammalian plasma membranes is mediated by a multiplicity of amino acid transport systems. Some of them (systems L, y+ L, x(c)- and b(o,+)) are the result of the activity of heteromeric amino acid transporters (HAT) (i.e. transport activity is elicited by the coexpression of a heavy and a light subunit). The two heavy subunits known today (HSHAT: rBAT and 4F2hc) were identified in 1992, and light subunits (LSHAT: LAT-1, LAT-2, asc-1, y+ LAT-1, y+ LAT-2, xCT and b(o,+)AT) have been cloned in the last 2 years. Defects in two genes of this family (SLC3A1, encoding rBAT and SLC7A9, encoding b(o,+)AT) are responsible for cystinuria, an inherited aminoaciduria of cystine and dibasic amino acids. This finding and functional studies of rBAT and b(o,+)AT suggested that these two proteins encompassed the high-affinity renal reabsorption system of cystine. In contrast to this view, immunofluorescence studies showed that rBAT is most abundant in the proximal straight tubule, and b(o,+)AT is most abundant in the proximal convoluted tubule of the nephron. The need for a new light subunit for rBAT and a heavy subunit for b(o,+)AT is discussed.     

Carrier subunit of plasma membrane transporter is required for oxidative folding of its helper subunit

We study the amino acid transport system b(0,+) as a model for folding, assembly, and early traffic of membrane protein complexes. System b(0,+) is made of two disulfide-linked membrane subunits: the carrier, b(0,+) amino acid transporter (b(0,+)AT), a polytopic protein, and the helper, related to b(0,+) amino acid transporter (rBAT), a type II glycoprotein. rBAT ectodomain mutants display folding/trafficking defects that lead to type I cystinuria. Here we show that, in the presence of b(0,+)AT, three disulfides were formed in the rBAT ectodomain. Disulfides Cys-242-Cys-273 and Cys-571-Cys-666 were essential for biogenesis. Cys-673-Cys-685 was dispensable, but the single mutants C673S, and C685S showed compromised stability and trafficking. Cys-242-Cys-273 likely was the first disulfide to form, and unpaired Cys-242 or Cys-273 disrupted oxidative folding. Strikingly, unassembled rBAT was found as an ensemble of different redox species, mainly monomeric. The ensemble did not change upon inhibition of rBAT degradation. Overall, these results indicated a b(0,+)AT-dependent oxidative folding of the rBAT ectodomain, with the initial and probably cotranslational formation of Cys-242-Cys-273, followed by the oxidation of Cys-571-Cys-666 and Cys-673-Cys-685, that was completed posttranslationally.     

The molecular bases of cystinuria and lysinuric protein intolerance

Cystinuria and lysinuric protein intolerance are inherited aminoacidurias caused by defective amino-acid transport activities linked to a family of heteromeric amino-acid transporters (HATs). HATs comprise two subunits: co-expression of subunits 4F2hc and y(+)LAT-1 induces the efflux of dibasic amino acids from cells, whereas co-expression of subunits rBAT and b(o,+)AT induces the renal reabsorption and intestinal absorption of cystine and dibasic amino acids at the brush border of epithelial cells. Recently, the role of b(o,+)AT (SLC7A9) in cystinuria (non Type I) and the role of y(+)LAT-1 (SLC7A7) in lysinuric protein intolerance have been demonstrated.     

Luminal heterodimeric amino acid transporter defective in cystinuria

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b(0,+) type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b(0,+)AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b(0,+) amino acid transporter complex. We demonstrate its b(0,+)-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 b(0,+)AT protein is the product of the gene defective in non-type I cystinuria.     

Differential influence of the 4F2 heavy chain and the protein related to b(0,+) amino acid transport on substrate affinity of the heteromeric b(0,+) amino acid transporter

We provide evidence here that b(0,+) amino acid transporter (b(0, +)AT) interacts with 4F2 heavy chain (4F2hc) as well as with the protein related to b(0,+) amino acid transporter (rBAT) to constitute functionally competent b(0,+)-like amino acid transport systems. This evidence has been obtained by co-expression of b(0, +)AT and 4F2hc or b(0,+)AT and rBAT in human retinal pigment epithelial cells and in COS-1 cells. The ability to interact with 4F2hc and rBAT is demonstrable with mouse b(0,+)AT as well as with human b(0,+)AT. Even though both the 4F2hc x b(0,+)AT complex and the rBAT x b(0,+)AT complex exhibit substrate specificity that is characteristic of system b(0,+), these two complexes differ significantly in substrate affinity. The 4F2hc x b(0,+)AT complex has higher substrate affinity than the rBAT x b(0,+)AT complex. In situ hybridization studies demonstrate that the regional distribution pattern of mRNA in the kidney is identical for b(0,+)AT and 4F2hc. The pattern of rBAT mRNA expression is different from that of b(0,+)AT mRNA and 4F2hc mRNA, but there are regions in the kidney where b(0,+)AT mRNA expression overlaps with rBAT mRNA expression as well as with 4F2hc mRNA expression.     

Expression of the activity of cystine/glutamate exchange transporter,system x(c)(-), by xCT and rBAT

The expression of the activity of cystine/glutamate exchange transporter, designated system x(c)(-), requires two components, xCT and 4F2 heavy chain (4F2hc) in Xenopus oocytes. rBAT (related to b(0,+) amino acid transporter) has a significant homology to 4F2hc and is known to be located in the apical membrane of epithelial cells. To determine whether xCT can associate with rBAT and express the activity of system x(c)(-), xCT, and rBAT were co-expressed in Xenopus oocytes and in mammalian cultured cells. In the oocytes injected with rBAT cRNA alone, the activities of cystine and arginine transport were induced, indicating that the system b(0,+)-like transporter was expressed by associating the exogenous rBAT with an endogenous b(0,+)AT-like factor as reported previously. In the oocytes injected with xCT and rBAT cRNAs, the activity of cystine transport was further induced. This induced activity of cystine transport was partially inhibited by glutamate or arginine and completely inhibited by adding both amino acids. In these oocytes, the activity of glutamate transport was also induced and it was strongly inhibited by cystine. In NIH3T3 cells transfected with xCT cDNA alone, the activity of cystine transport was significantly increased, and in the cells transfected with both xCT and rBAT cDNAs, the activity of cystine transport was further enhanced. The enhanced activity was Na(+)-independent and was inhibited by glutamate and homocysteate. These results indicate that rBAT can replace 4F2hc in the expression of the activity of system x(c)(-) and suggest that system x(c)(-) activity could be expressed in the apical membrane of epithelial cells.     

Identification of an amino acid transporter associated with the cystinuria-related type II membrane glycoprotein.

We identified an amino acid transporter that is associated with the cystinuria-related type II membrane glycoprotein, rBAT (related to b(0,+) amino acid transporter). The transporter designated BAT1 (b(0, +)-type amino acid transporter 1) from rat kidney was found to be structurally related to recently identified amino acid transporters for system L, system y(+)L, and system x(-)C, which are linked, via a disulfide bond, to the other type II membrane glycoprotein, 4F2hc (4F2 heavy chain). In the nonreducing condition, a 125-kDa band, which seems to correspond to the heterodimeric complex of BAT1 and rBAT, was detected in rat kidney with anti-BAT1 antibody. The band was shifted to 41 kDa in the reducing condition, confirming that BAT1 and rBAT are linked via a disulfide bond. The BAT1 and rBAT proteins were shown to be colocalized in the apical membrane of the renal proximal tubules where massive cystine transport had been proposed. When expressed in COS-7 cells with rBAT, but not with 4F2hc, BAT1 exhibited a Na(+)-independent transport of cystine as well as basic and neutral amino acids with the properties of system b(0,+). The results from the present investigation were used to establish a family of amino acid transporters associated with type II membrane glycoproteins.     

Functional cooperation of epithelial heteromeric amino acid transporters expressed in madin-darby canine kidney cells

The heteromeric amino acid transporters b(0,+)AT-rBAT (apical), y(+)LAT1-4F2hc, and possibly LAT2-4F2hc (basolateral) participate to the (re)absorption of cationic and neutral amino acids in the small intestine and kidney proximal tubule. We show now by immunofluorescence that their expression levels follow the same axial gradient along the kidney proximal tubule (S1>S2S3). We reconstituted their co-expression in MDCK cell epithelia and verified their polarized localization by immunofluorescence. Expression of b(0,+)AT-rBAT alone led to a net reabsorption of l-Arg (given together with l-Leu). Coexpression of basolateral y(+)LAT1-4F2hc increased l-Arg reabsorption and reversed l-Leu transport from (re)absorption to secretion. Similarly, l-cystine was (re)absorbed when b(0,+)AT-rBAT was expressed alone. This net transport was further increased by the coexpression of 4F2hc, due to the mobilization of LAT2 (exogenous and/or endogenous) to the basolateral membrane. In summary, apical b(0,+)AT-rBAT cooperates with y(+)LAT1-4F2hc or LAT2-4F2hc for the transepithelial reabsorption of cationic amino acids and cystine, respectively. The fact that the reabsorption of l-Arg led to the secretion of l-Leu demonstrates that the implicated heteromeric amino acid transporters function in epithelia as exchangers coupled in series and supports the notion that the parallel activity of unidirectional neutral amino acid transporters is required to drive net amino acid reabsorption.     

The amino acid transport system bo,+ and cystinuria

Amino acid transport in mammalian plasma membranes is mediated by a multiplicity of amino acid transport systems. Some of them (systems L, y⁺L, x_c^- and b^o,+) are the result of the activity of heteromeric amino acid transporters (HAT) (i.e. transport activity is elicited by the coexpression of a heavy and a light subunit). The two heavy subunits known today (HSHAT: rBAT and 4F2hc) were identified in 1992, and light subunits (LSHAT: LAT-1, LAT-2, asc-1, y⁺LAT-1, y⁺LAT-2, xCT and b^o,+AT) have been cloned in the last 2 years. Defects in two genes of this family (SLC3A1, encoding rBAT and SLC7A9, encoding b^o,+AT) are responsible for cystinuria, an inherited aminoaciduria of cystine and dibasic amino acids. This finding and functional studies of rBAT and b^o,+AT suggested that these two proteins encompassed the high-affinity renal reabsorption system of cystine. In contrast to this view, immunofluorescence studies showed that rBAT is most abundant in the proximal straight tubule, and b^o,+AT is most abundant in the proximal convoluted tubule of the nephron. The need for a newlight subunit for rBAT and a heavy subunit for b^o,+AT is discussed.     

Distribution and abundance of nutrient transporter mRNA in the intestinal tract of the black bear, Ursus americanus

End products of digestion are absorbed by the body through the action of transporter proteins expressed on the apical membrane of intestinal epithelial cells. We investigated the mRNA abundance and distribution of a peptide transporter (PepT1), a glucose transporter (SGLT1), two amino acid transporters (NBAT and b(o,+)AT), and a digestive enzyme, aminopeptidase N (APN), in the intestinal tract of black bears (Ursus americanus). Intestinal total RNA was isolated from 10 bears and abundance of PepT1, SGLT1, NBAT, b(o,+)AT, and APN mRNA were determined by Northern blots. Abundance of PepT1 (P<0.05), APN (P<0.05), and SGLT1 (P<0.0001) changed quadratically from the proximal to distal intestine with abundance being greatest in the midregion. Abundance of b(o,+)AT mRNA increased linearly (P<0.05) from the proximal to distal intestine. The number of molecules of mRNA/ng of total RNA for each gene was determined using Real-Time PCR. PepT1 mRNA was present at 10-fold or greater levels than amino acid transporter mRNA in all segments of the intestine, suggesting that di- and tripeptides constitute a major form in which amino acids are absorbed in the black bear. The abundance of NBAT and b(o,+)AT mRNA was greater towards the distal intestine, suggesting a role in salvaging unabsorbed amino acids.     

Identification and characterization of a novel member of the heterodimeric amino acid transporter family presumed to be associated with an unknown heavy chain

We identified a novel amino acid transporter designated Asc-2 (for asc-type amino acid transporter 2). Asc-2 exhibited relatively low but significant sequence similarity to the members of the heterodimeric amino acid transporters. The cysteine residue responsible for the disulfide bond formation between transporters (light chains) and heavy chain subunits in the heterodimeric amino acid transporters is conserved for Asc-2. Asc-2 is, however, not colocalized with the already known heavy chains such as 4F2 heavy chain (4F2hc) or related to b(0,+) amino acid transporter (rBAT) in mouse kidney. Because Asc-2 solely expressed or coexpressed with 4F2hc or rBAT did not induce functional activity, we generated fusion proteins in which Asc-2 is connected with 4F2hc or rBAT. The fusion proteins were sorted to the plasma membrane and expressed the function corresponding to the Na(+)-independent small neutral amino acid transport system asc. Distinct from the already identified system asc transporter Asc-1 which is associated with 4F2hc, Asc-2-mediated transport is less stereoselective and did not accept some of the high affinity substrates of Asc-1 such as alpha-aminoisobutyric acid and beta-alanine. Asc-2 message was detected in kidney, placenta, spleen, lung, and skeletal muscle. In kidney, Asc-2 protein was present in the epithelial cells lining collecting ducts. In the Western blot analysis on mouse erythrocytes and kidney, Asc-2 was detected as multiple bands in the nonreducing condition, whereas the bands shifted to a single band at lower molecular weight, suggesting the association of Asc-2 with other protein(s) via a disulfide bond. The finding of Asc-2 would lead to the establishment of a new subgroup of heterodimeric amino acid transporter family which includes transporters associated not with 4F2hc or rBAT but with other unknown heavy chains.     

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.     

Expression of human heteromeric amino acid transporters in the yeast Pichia pastoris

Human heteromeric amino acid transporters (HATs) play key roles in renal and intestinal re-absorption, cell redox balance and tumor growth. These transporters are composed of a heavy and a light subunit, which are connected by a disulphide bridge. Heavy subunits are the two type II membrane N-glycoproteins rBAT and 4F2hc, while L-type amino acid transporters (LATs) are the light and catalytic subunits of HATs. We tested the expression of human 4F2hc and rBAT as well as seven light subunits in the methylotrophic yeast Pichia pastoris. 4F2hc and the light subunit LAT2 showed the highest expression levels and yields after detergent solubilization. Co-transformation of both subunits in Pichia cells resulted in overexpression of the disulphide bridge-linked 4F2hc/LAT2 heterodimer. Two sequential affinity chromatography steps were applied to purify detergent-solubilized heterodimers yielding ～1 mg of HAT from 2 l of cell culture. Our results indicate that P. pastoris is a convenient system for the expression and purification of human 4F2hc/LAT2 for structural studies.     

The molecular basis of cystinuria: the role of the rBAT gene

Summary The cDNAs of mammalian amino acid transporters already identified could be grouped into four families. One of these protein families is composed of the protein rBAT and the heavy chain of the cell surface antigen 4F2 (4F2hc). The cRNAs of rBAT and 4F2hc induce amino acid transport activity via systems b^0,+ -like and y⁺L -like inXenopus oocytes respectively. Surprisingly, neither rBAT nor 4F2hc is very hydrophobic, and they seem to be unable to form a pore in the plasma membrane. This prompted the hypothesis that rBAT and 4F2hc are subunits or modulators of the corresponding amino acid transporters. The association of rBAT with a light subunit of ~40kDa has been suggested, and such an association has been demonstrated for 4F2hc.The b^0,+-like system expressed in oocytes by rBAT cRNA transports L-cystine, L-dibasic and L-neutral amino acids with high-affinity. This transport system shows exchange of amino acids through the plasma membrane ofXenopus oocytes, suggesting a tertiary active transport mechanism. The rBAT gene is mainly expressed in the outer stripe of the outer medulla of the kidney and in the mucosa of the small intestine. The protein localizes to the microvilli of the proximal straight tubules (S3 segment) of the nephron and the mucosa of the small intestine. All this suggested the participation of rBAT in a high-affinity reabsorption system of cystine and dibasic amino acids in kidney and intestine, and indicated rBAT (named SLC3A1 in Gene Data Bank) as a good candidate gene for cystinuria. This is an inherited aminoaciduria due to defective renal and intestinal reabsorption of cystine and dibasic amino acids. The poor solubility of cystine causes the formation of renal cystine calculi. Mutational analysis of the rBAT gene of patients with cystinuria is revealing a growing number (~20) of cystinuria-specific mutations, including missense, nonsense, deletions and insertions. Mutations M467T (substitution of methionine 467 residue for threonine) and R270X (stop codon at arginine residue 270) represent approximately half of the cystinuric chromosomes where mutations have been found. Mutation M467T reduces transport activity of rBAT in oocytes. All this demonstrates that mutations in the rBAT gene cause cystinuria.Three types of cystinuria (types, I, II and III) have been described on the basis of the genetic, biochemical and clinical manifestations of the disease. Type I cystinuria has a complete recessive inheritance; type I heterozygotes are totally silent. In contrast, type II and III heterozygotes show, respectively, high or moderate hyperaminoaciduria of cystine and dibasic amino acids. Type III homozygotes show moderate, if any, alteration of intestinal absorption of cystine and dibasic amino acids; type II homozygotes clearly show defective intestinal absorption of these amino acids. To date, all the rBAT cystinuria-specific mutations we have found are associated with type I cystinuria (~70% of the chromosomes studied) but not to types II or III. This strongly suggests genetic heterogeneity for cystinuria. Genetic linkage analysis with markers of the genomic region of rBAT in chromosome 2 (G band 2p16.3) and intragenic markers of rBAT have demonstrated genetic heterogeneity for cystinuria; the rBAT gene is linked to type I cystinuria, but not to type III. Biochemical, genetic and clinical studies are needed to identify the additional cystinuria genes; a low-affinity cystine reabsortion system and the putative light subunit of rBAT are additional candidate genes for cystinuria.     

rBAT is an Amino Acid Exchanger with Variable Stoichiometry

The rBAT protein, when expressed in Xenopus oocytes, was previously shown to reproduce the selectivity of the Na⁺-independent neutral and basic amino acid transport system called b^o,+. More recently, the capacity of rBAT to generate a transmembrane current was demonstrated when addition of neutral amino acids stimulated the efflux of cations (presumably basic amino acids) in rBAT-injected oocytes. In the present paper, aminoisobutyric acid (AIB), a neutral amino acid analogue, was shown to induce outward currents (efflux of basic amino acids) through rBAT similar to those caused by alanine in terms of affinity, maximal currents and I-V curves. Despite generating similar currents, the AIB transport rate was more than 30 times lower than that of alanine, thus challenging the assumption that rBAT functions as a classical exchanger. Experiments using a cut-open oocyte voltage clamp demonstrated that AIB was capable of stimulating rBAT-mediated currents from either side of the membrane. AIB, like alanine, was able to stimulate the efflux of radiolabeled alanine and arginine while no rBAT-mediated efflux was measurable in the absence of external rBAT substrates. These results demonstrate that (i) the presence of amino acids is required on both sides of the membrane for rBAT to mediate amino acid flux and thus rBAT must be some type of exchanger but (ii) rBAT-mediated amino acid influx is not stoichiometrically related to the efflux. A model of a ``double gated pore' is proposed to account for these properties of rBAT, which contravene standard models of exchangers and other transporters. Received: 15 June 1995/Revised: 21 September 1995     

The structural and functional units of heteromeric amino acid transporters. The heavy subunit rBAT dictates oligomerization of the heteromeric amino acid transporters

Heteromeric amino acid transporters are composed of a catalytic light subunit and a heavy subunit linked by a disulfide bridge. We analyzed the structural and functional units of systems b0,+ and xC-, formed by the heterodimers b0,+ AT-rBAT and xCT-4F2hc, respectively. Blue Native gel electrophoresis, cross-linking, and fluorescence resonance energy transfer in vivo indicate that system b0,+ is a heterotetramer [b0,+ AT-rBAT]2, whereas xCT-4F2hc seems not to stably or efficiently oligomerize. However, substitution of the heavy subunit 4F2hc for rBAT was sufficient to form a heterotetrameric [xCT-rBAT]2 structure. The functional expression of concatamers of two light subunits (which differ only in their sensitivity to inactivation by a sulfhydryl reagent) suggests that a single heterodimer is the functional unit of systems b0,+ and xC-.     

Trimeric subunit stoichiometry of the glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus

Catalysis of glutamate transport across cell membranes and coupling of the concentrative transport to sodium, proton, and potassium gradients are processes fundamental to organisms in all kingdoms of life. In bacteria, glutamate transporters participate in nutrient uptake, while in eukaryotic organisms, the transporters clear glutamate from the synaptic cleft. Even though glutamate transporters are crucial to the viability of many life forms, little is known about their structure and quaternary organization. In particular, the subunit stoichiometry of these polytopic integral membrane proteins has not been unequivocally defined. Determination of the native molecular mass of membrane proteins is complicated by their lability in detergent micelles and by their association with detergent and/or lipid molecules. Here we report the purification of glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus in a monodisperse, detergent-solubilized state. Characterization of both transporters either by chemical cross-linking and mass spectrometry or by size-exclusion chromatography and in-line laser light scattering, refractive index, and ultraviolet absorption measurements shows that the transporters have a trimeric quaternary structure. Limited proteolysis further defines regions of primary structure that are exposed to aqueous solution. Together, our results define the subunit stoichiometry of high-affinity glutamate transporters from B. caldotenax and B. stearothermophilus and localize exposed and accessible elements of primary structure. Because of the close amino acid sequence relationship between bacterial and eukaryotic transporters, our results are germane to prokaryotic and eukaryotic glutamate and neutral amino acid transporters.     

Functional characterization of Caenorhabditis elegans heteromeric amino acid transporters

Mammalian heteromeric amino acid transporters (HATs) are composed of a multi-transmembrane spanning catalytic protein covalently associated with a type II glycoprotein (e.g. 4F2hc, rBAT) through a disulfide bond. Caenorhabditis elegans has nine genes encoding close homologues of the HAT catalytic proteins. Three of these genes (designated AAT-1 to AAT-3) have a much higher degree of similarity to the mammalian homologues than the other six, including the presence of a cysteine residue at the position known to form a disulfide bridge to the glycoprotein partner in mammalian HATs. C. elegans also has two genes encoding homologues of the heteromeric amino acid transporter type II glycoprotein subunits (designated ATG-1 and ATG-2). Both ATG, and/or AAT-1, -2, -3 proteins were expressed in Xenopus oocytes and tested for amino acid transport function. This screen revealed that AAT-1 and AAT-3 facilitate amino acid transport when expressed together with ATG-2 but not with ATG-1 or the mammalian type II glycoproteins 4F2hc and rBAT. AAT-1 and AAT-3 covalently bind to both C. elegans ATG glycoproteins, but only the pairs with ATG-2 traffic to the oocyte surface. Both of these functional, surface-expressed C. elegans HATs transport most neutral amino acids and display the highest transport rate for l-Ala and l-Ser (apparent K(m) 100 microm range). Similar to their mammalian counterparts, the C. elegans HATs function as (near) obligatory amino acid exchangers. Taken together, this study demonstrates that the heteromeric structure and the amino acid exchange function of HATs have been conserved throughout the evolution of nematodes to mammals.     

New Glycoprotein-Associated Amino Acid Transporters

The L-type amino acid transporter LAT1 has recently been identified as being a disulfide-linked ``light chain' of the ubiquitously expressed glycoprotein 4F2hc/CD98. Several LAT1-related transporters have been identified, which share the same putative 12-transmembrane segment topology and also associate with the single transmembrane domain 4F2hc protein. They display differing amino acid substrate specificities, transport kinetics and localizations such as, for instance, y⁺LAT1 which is localized at the basolateral membrane of transporting epithelia, and the defect of which causes lysinuric protein intolerance. The b^0,+AT transporter which associates with the 4F2hc-related rBAT protein to form the luminal high-affinity diamino acid transporter defective in cystinuria, belongs to the same family of glycoprotein-associated amino acid transporters (gpaATs). These glycoprotein-associated transporters function as amino acid exchangers. They extend the specificity range of vectorial amino acid transport when located in the same membrane as carriers that unidirectionally transport one of the exchanged substrates. gpaATs belong to a phylogenetic cluster within the amino acid/polyamine/choline (APC) superfamily of transporters. This cluster, which we designate the LAT family (named after its first vertebrate member), includes some members from nematodes, yeast and bacteria. The latter of these proteins presumably lack association with a second subunit. In this review, we focus on the animal members of the LAT cluster that form, together with some of the nematode members, the family of glycoprotein-associated amino acid transporters (gpaAT family). Received: 20 July 1999/Revised: 7 September 1999