Heteromeric Amino Acid Transporters Research Articles

Membrane Topology of System Xc- Light Subunit Reveals a Re-entrant Loop with Substrate-restricted Accessibility

Heteromeric amino acid transporters are composed of a heavy and a light subunit linked by a disulfide bridge. 4F2hc/xCT elicits sodium-independent exchange of anionic L-cysteine and L-glutamate (system x(c)(-)). Based on the accessibility of single cysteines to 3-(N-maleimidylpropionyl)biocytin, we propose a topological model for xCT of 12 transmembrane domains with the N and C termini located inside the cell. This location of N and C termini was confirmed by immunofluorescence. Studies of biotinylation and accessibility to sulfhydryl reagents revealed a re-entrant loop within intracellular loops 2 and 3. Residues His(110) and Thr(112), facing outside, are located at the apex of the re-entrant loop. Biotinylation of H110C was blocked by xCT substrates, by the nontransportable inhibitor (S)-4-carboxyphenylglycine, and by the impermeable reagent (2-sulfonatoethyl) methanethiosulfonate, which produced an inactivation of H110C that was protected by L-glutamate and L-cysteine with an IC(50) similar to the K(m). Protection was temperatureindependent. The data indicate that His(110) may lie close to the substrate binding/permeation pathway of xCT. The membrane topology of xCT could serve as a model for other light subunits of heteromeric amino acid transporters.

Expression of heteromeric amino acid transporters along the murine intestine

Members of the new heterodimeric amino acid transporter family are composed of two subunits, a catalytic multitransmembrane spanning protein (light chain) and a type II glycoprotein (heavy chain). These transporters function as exchangers and thereby extend the transmembrane amino acid transport selectivity to specific amino acids. The heavy chain rBAT associates with the light chain b degrees (,+)AT to form a cystine and cationic amino acid transporter. The other heavy chain, 4F2hc, can interact with seven different light chains to form various transporters corresponding to systems L, y(+)L, asc or x(-)(c). The importance of some of these transporters in intestinal and renal (re)absorption of amino acids is highlighted by the fact that mutations in either the rBAT or b degrees (,+)AT subunit result in cystinuria whereas a defect in the y(+)-LAT1 light chain causes lysinuric protein intolerance. Here we investigated the localization of these transporters in intestine since both diseases are also characterized by altered intestinal amino acid absorption. Real time PCR showed organ-specific expression patterns for all transporter subunit mRNAs along the intestine and Western blotting confirmed these findings on the protein level. Immunohistochemistry demonstrated basolateral coexpression of 4F2hc, LAT2 and y(+)-LAT1 in stomach and small intestine, whereas rBAT and b degrees (,+)AT were found colocalizing on the apical side of small intestine epithelium. In stomach, 4F2hc and LAT2 were localized in H(+)/K(+)-ATPase-expressing parietal cells. The abundant expression of several members of the heterodimeric transporter family along the murine small intestine suggests their involvement in amino acids absorption. Furthermore, strong expression of rBAT, b degrees (,+)AT and y(+)-LAT1 in the small intestine explains the reduced intestinal absorption of some amino acid in patients with cystinuria or lysinuric protein intolerance.

Lysinuric protein intolerance: mechanisms of pathophysiology

Heteromeric amino acid transporters (HATs) are composed of two subunits, a polytopic membrane protein (the light subunit) and a disulfide-linked type II membrane glycoprotein (the heavy subunit). HATs represent several of the classic mammalian amino acid transport systems (e.g., L isoforms, y +L isoforms, asc, x c −, and b 0,+). The light subunits confer the amino acid transport specificity to the HAT. Two transporters of this family are relevant for inherited aminoacidurias. Mutations in any of the two genes coding for the subunits of system b 0,+ (rBAT and b 0,+AT) lead to cystinuria (MIM 220100). Transport defects in a system y +L isoform, composed of 4F2hc and y +LAT-1, result in lysinuric protein intolerance (LPI) (MIM 222700). In this case, only mutations in the light subunit y +LAT-1, but not in the heavy chain 4F2hc, cause the disease. LPI, in addition to affecting intestinal and renal reabsorption of amino acids, is a multisystemic disease affecting the urea cycle and presents also with symptoms related to the immune system. The pathogenesis of these alterations is less well, or not understood at all.

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.

The ancillary proteins of HATs: SLC3 family of amino acid transporters.

The heteromeric amino acid transporters (HATs) are composed of a light and a heavy subunit linked by a disulfide bridge. The heavy subunits are the SLC3 members (rBAT and 4F2hc), whereas the light subunits are members of the SLC7 family of amino acid transporters. SLC3 proteins are type II membrane glycoproteins (i.e., one single transmembrane domain and the C-terminus located outside the cell) with a bulky extracellular domain that shows homology with alpha-glucosidases. rBAT heterodimerizes with b(0,+)AT (SLC7A9) constituting the amino acid transport b(0,+), the main system responsible for the apical reabsorption of cystine in kidney. The defect in this system causes cystinuria, the most common primary inherited aminoaciduria. 4F2hc subserves various amino acid transport systems by dimerization with different SLC7 proteins. The main role of SLC3 proteins is to help routing of the holotransporter to the plasma membrane. A working model for the biogenesis of HATs based on recent data on the rBAT/b(0,+)AT heterodimeric complex is presented. 4F2hc is a multifunctional protein, and in addition to its role in amino acid transport, it may be involved in other cellular functions. Studies on two SLC7 members (Asc-2 and AGT1) demonstrate heterodimerization with unknown heavy subunits.

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.

Five novel SLC7A7 variants and y+L gene-expression pattern in cultured lymphoblasts from Japanese patients with lysinuric protein intolerance.

Two distinct human light subunits of the heteromeric amino acid transporter, y+LAT-1 coded by SLC7A7 and y+LAT-2 coded by SLC7A6, are both known to induce transport system y+L activity. SLC7A7 has already been identified as the gene responsible for lysinuric protein intolerance (LPI). We successfully identified five novel SLC7A7 variants (S238F, S489P, 1630delC, 1673delG, and IVS3-IVS5del9.7kb) in Japanese patients with LPI by PCR amplification and direct DNA sequencing. In addition, we performed a semi-quantitative expression analysis of SLC7A7 and SLC7A6 in human tissue. In normal tissue, the gene-expression ratio of SLC7A6 to SLC7A7 was high in the brain, muscle, and cultured skin fibroblasts; low in the kidneys and small intestine; and at an intermediate level in peripheral blood leukocytes, the lungs, and cultured lymphoblasts. The gene-expression ratio of SLC7A6 to SLC7A7 in cultured lymphoblasts was significantly different between normal subjects and LPI patients with R410X and/or S238F, where the relative amount of SLC7A7 mRNA was significantly lower and the relative amount of SLC7A6 mRNA was statistically higher in affected lymphoblasts than in normal cells. Expression of SLC7A7 and SLC7A6 may thus be interrelated in cultured lymphoblasts.

The light subunit of system b(o,+) is fully functional in the absence of the heavy subunit.

The heteromeric amino acid transporters are composed of a type II glycoprotein and a non-glycosylated polytopic membrane protein. System b(o,+) exchanges dibasic for neutral amino acids. It is composed of rBAT and b(o,+)AT, the latter being the polytopic membrane subunit. Mutations in either of them cause malfunction of the system, leading to cystinuria. b(o,+)AT-reconstituted systems from HeLa or MDCK cells catalysed transport of arginine that was totally dependent on the presence of one of the b(o,+) substrates inside the liposomes. rBAT was essential for the cell surface expression of b(o,+)AT, but it was not required for reconstituted b(o,+)AT transport activity. No system b(o,+) transport was detected in liposomes derived from cells expressing rBAT alone. The reconstituted b(o,+)AT showed kinetic asymmetry. Expressing the cystinuria-specific mutant A354T of b(o,+)AT in HeLa cells together with rBAT resulted in defective arginine uptake in whole cells, which was paralleled by the reconstituted b(o,+)AT activity. Thus, subunit b(o,+)AT by itself is sufficient to catalyse transmembrane amino acid exchange. The polytopic subunits may also be the catalytic part in other heteromeric transporters.

Heteromeric amino acid transporters: biochemistry, genetics, and physiology.

The heteromeric amino acid transporters (HATs) are composed of two polypeptides: a heavy subunit (HSHAT) and a light subunit (LSHAT) linked by a disulfide bridge. HSHATs are N-glycosylated type II membrane glycoproteins, whereas LSHATs are nonglycosylated polytopic membrane proteins. The HSHATs have been known since 1992, and the LSHATs have been described in the last three years. HATs represent several of the classic mammalian amino acid transport systems (e.g., L isoforms, y(+)L isoforms, asc, x(c)(-), and b(0,+)). Members of the HAT family are the molecular bases of inherited primary aminoacidurias cystinuria and lysinuric protein intolerance. In addition to the role in amino acid transport, one HSHAT [the heavy subunit of the cell-surface antigen 4F2 (also named CD98)] is involved in other cell functions that might be related to integrin activation. This review covers the biochemistry, human genetics, and cell physiology of HATs, including the multifunctional character of CD98.

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 new light subunit for rBAT and a heavy subunit for b(o,+)AT is discussed.

Heteromeric amino acid transporters explain inherited aminoacidurias

In the past 5 years, the first genes responsible for aminoacidurias caused by defects in renal reabsorption transport mechanisms have been identified. These diseases are type I and non-type I cystinuria and lysinuric protein intolerance. This knowledge came from the molecular characterization of the first heteromeric amino acid transporters in mammals. In 1992, rBAT and 4F2hc (genes SLC3A1 and SLC3A2, respectively, in the nomenclature of the Human Genome Organization) were identified as putative heavy subunits of mammalian amino acid transporters. In 1994, it was demonstrated that mutations in SLC3A1 cause type I cystinuria. Very recently, several light subunits of the heteromeric amino acid transporters have been identified. In 1999, a putative light subunit of rBAT (the SLC7A9 gene; complementary DNA and protein termed amino acid transporter) and a light subunit of 4F2hc (the SLC7A7 gene; cDNA and protein termed y+LAT-1) were shown to be the non-type I cystinuria and lysinuric protein intolerance genes, respectively. In this review, the characteristics of these heteromeric amino acid transporters and their role in these inherited aminoacidurias is described.