The heterodimeric amino acid transporter 4F2hc/LAT1 is associated in Xenopus oocytes with a non-selective cation channel that is regulated by the serine/threonine kinase sgk-1 (original) (raw)
Related papers
1999
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 (2aminobicyclo-(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 ϳ286 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.
Journal of Pharmacological Sciences, 2012
Amino acid transport across the plasma membrane is mediated via amino acid transporters situated on the plasma membrane. Amino acid transporters were originally described as amino acid transport systems (1). Among them, system L, a Na +-independent neutral amino acid transport agency, is a major route for living cells to take up neutral amino acids including branched-chain or aromatic amino acids (1). By means of expression cloning, we isolated a cDNA encoding the first isoform of system L transporter named LAT1 (L-type amino acid transporter 1, SLC7A5) (2). Following LAT1, we furthermore identified the second isoform of system L transporter named LAT2 (L-type amino acid transporter 2, SLC7A8) (3). LAT1 or LAT2 forms heterodimers via a disulfide bond with the heavy chain of 4F2 antigen (4F2hc), which is essential for the proper targeting of LAT1 and LAT2 to the plasma membrane (4). In normal tissues, mRNA of LAT1 has been detected
Proceedings of the National Academy of Sciences, 2014
Heteromeric amino acid transporters (HATs) are the unique example, known in all kingdoms of life, of solute transporters composed of two subunits linked by a conserved disulfide bridge. In metazoans, the heavy subunit is responsible for the trafficking of the heterodimer to the plasma membrane, and the light subunit is the transporter. HATs are involved in human pathologies such as amino acidurias, tumor growth and invasion, viral infection and cocaine addiction. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, transmission electron microscopy and single-particle analysis of purified human 4F2hc/L-type amino acid transporter 2 (LAT2) heterodimers overexpressed in the yeast Pichia pastoris, together with docking analysis and crosslinking experiments, reveal that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. 4F2hc increases the stability of the light subunit LAT2 in detergent-solubilized Pichia membranes, allowing functional reconstitution of the heterodimer into proteoliposomes. Moreover, the extracellular domain of 4F2hc suffices to stabilize solubilized LAT2. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs.
The Biochemical journal, 1998
Expression of the type II membrane proteins of the rbAT/4F2hc family in Xenopus laevis oocytes results in the induction of amino acid transport activity. To elucidate the mechanism of action, amino acid transport was investigated in oocytes expressing the surface antigen 4F2hc. Leucine transport was mediated by a Na+-independent and a Na+-dependent transport mechanism. Both systems could be further discriminated by their stereochemical constraints. Isoleucine, with a branch at the beta-position, shared only the Na+-independent transport system with leucine. Both transport systems were sensitive to inhibition by arginine, but only the Na+-independent system was sensitive to inhibition by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. When compared with known transport systems the two transport activities could be described as similar to, but not identical with, mammalian systems b0,+ and y+L. The Na+-independent b0,+-like transport system was found both in rbAT and 4F2hc expressing ...
SLC36A4 (hPAT4) Is a High Affinity Amino Acid Transporter When Expressed in Xenopus laevis Oocytes
Journal of Biological Chemistry, 2010
The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [ 3 H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 M. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 M). Other amino acids that inhibited [ 3 H]proline were isoleucine (K i 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K i >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K i 1.09 mM), whereas ␥-aminobutyric acid (GABA), -alanine, L-Dopa, D-serine, and ␦-aminolevulinic acid were without effect on [ 3 H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.
The amino acid transport system y/L/4F2hc is a heteromultimeric complex
4F2hc is an almost ubiquitous transmembrane protein in mammalian cells; upon expression in Xenopus laevis oocytes, it induces amino acid transport with characteristics of system y / L. Indirect evidence fostered speculation that function requires the association of 4F2hc with another protein endogenous to oocytes and native tissues. We show that expression of system y / L-like amino acid transport activity by 4F2hc in oocytes is limited by an endogenous factor and that direct covalent modification of external cysteine residue(s) of an oocyte membrane protein blocks system y / L/4F2hc transport activity, based on the following. 1) Induction of system y / Llike activity saturates at very low doses of human 4F2hc cRNA (0.1 ng/oocyte). This saturation occurs with very low expression of 4F2hc at the oocyte surface, and further increased expression of the protein at the cell surface does not result in higher induction of system y / L-like activity. 2) Human 4F2hc contains only two cysteine residues (C109 and C330). We mutated these residues, singly and in combination, to serine (C109S; CS1, C330S; CS2 and C109S-C330S, Cys-less). Mutation CS2 had no effect on the expressed system y / L-like transport activity, whereas C109S-containing mutants (CS1 and Cys-less) retained only partial y / L-like transport activity (30 to 50% of wild type). 3) Hg 2/ , the organic mercury compounds pCMB, and the membrane-impermeant p-CMBS almost completely inactivated system y / L-like induced by human 4F2hc wild type and all the mutants studied. This was reversed by b-mercaptoethanol, indicating that external cysteine residue(s) are the target of this inactivation. 4) Sensitivity to Hg 2/ inactivation is increased by pretreatment of oocytes with b-mercaptoethanol or in the C109S-containing mutants (CS1 and Cys-less). The increased Hg 2/ reactivity of C109S-containing mutants supports the possibility that C109 may be linked by a disulfide bond to the Hg 2/ -targeted cysteine residue of the associated protein. These results indicate that 4F2hc is intimately associated with a membrane oocyte protein for the expression of system y / L amino acid transport activity. To our knowledge, this is the first direct evidence for a heteromultimeric protein structure of an organic solute carrier in mammals.-Estévez, R., Camps, M., Rojas, A. M., Testar, X., Devés, R., Hediger, M. A., Zorzano, A., Palacín, M. The amino acid transport system y / L/4F2hc is a heteromultimeric complex. FASEB J. 12, 1319-1329 (1998)
Regulation and Genetics of Amino Acid Transport
Annals of the New York Academy of Sciences, 1985
The major transport systems for the uptake of neutral amino acids in mammalian cells have been designated A, ASC, and L.',' The transport systems and their regulation have been characterized in Chinese hamster ovary (CHO) System A is sodium-dependent, subject to trans-inhibition, and serves for the uptake of amino acids with short, polar, or linear side chains. System ASC is also sodium dependent and has a strong preference for alanine, serine, and cysteine. In the C H O cell, the ASC system shows a somewhat broader specificity than that found in the Ehrlich cell.* Unlike System A, System ASC does not tolerate N-methylated substrates such as 2-methylaminoisobutyric acid (MeAIB). System L is sodium-independent and serves for the uptake of branched-chain and aromatic amino acids. We operationally define the systems as follows: System A can be represented by the sodium-dependent uptake of 0.2 m M 2-arninoisobutyric acid (AIB) that is inhibited by 25 m M MeAIB; System ASC, the sodium-dependent uptake of 0.2 m M L-alanine that is not inhibited by 25 m M MeAIB; and System L, the sodium-independent uptake of 0.2 m M L-leucine that is inhibited by 10 mM 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH).3 Although the systems have a preferred set of substrates, they do have overlapping specificities. FIGURE 1 shows the contributions of these systems to the uptake of individual amino acids in CHO-K1 cells. This overlap makes the study of transport systems in isolation difficult. The availability of mutations in one or more of the transport systems would greatly facilitate the study of the function and regulation of the transport systems. We are currently combining genetic approaches with kinetic studies using C H O cells because of the relative ease with which mutants can be obtained from these cells. Furthermore, C H O cells can be used to form interspecies hybrids with human cells.' The hamster-human hybrid cells preferentially segregate the human chromosomes, permitting the assignment of a phenotype to a particular chromosome. In the present study, we have isolated and characterized C H O mutants defective in the regulation of System L6 and mutants with reduced System L transport activity. We have also used hamster-human hybrids to map System L transport activity to human chromosome 20.' 404 OXENDER et al.: AMINO-ACID TRANSPORT 405 MATERIALS AND METHODS Cell Lines and Culture Methods The CHO-K1 and CHO-tsH1 cell lines were obtained from Dr. L. H. Thompson of the Lawrence Livermore Laboratory, Livermore, California. These cell lines were maintained as described previo~sly.~" The temperature-resistant cell lines C11, C11 B6, D10, and F10 were isolated in our lab and maintained as described previously.6 The transport mutant cell lines C5, C5F6, C9, and D3 were maintained in Eagle's minimal essential medium (MEM) containing Earle's salts and nonessential amino acids, supplemented with 5% (vol/vol) fetal calf serum (KC Biological, Lenexa, KS),