Abstract
A cDNA was isolated from rat C6 glioma cells by expression cloning which encodes a novel Na+-independent neutral amino acid transporter designated LAT1. For functional expression in Xenopus oocytes, LAT1 required the heavy chain of 4F2 cell surface antigen (CD98), a type II membrane glycoprotein. When co-expressed with 4F2 heavy chain, LAT1 transported neutral amino acids with branched or aromatic side chains and did not accept basic amino acids or acidic amino acids. The transport via LAT1 was Na+-independent and sensitive to a system L-specific inhibitor 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid. These functional properties correspond to those of the classically characterized amino acid transport system L, a major nutrient transporter. In in vitro translation, LAT1 was shown to be a nonglycosylated membrane protein consistent with the property of 4F2 light chain, suggesting LAT1 is at least one of the proteins formerly referred to as 4F2 light chain. LAT1 exhibits relatively low but significant amino acid sequence similarity to mammalian cationic amino acid transporters and amino acid permeases of bacteria and yeasts, indicating LAT1 is a new member of the APC superfamily. Because of highly regulated nature and high level of expression in tumor cell lines, LAT1 is thought to be up-regulated to support the high protein synthesis for cell growth and cell activation. The cloning of LAT1 is expected to facilitate the research on the protein-protein interaction in the transporter field and to provide a clue to the search for still unidentified transporters.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB015432
The function of 4F2 antigen has not been clarified, it has attracted investigators, because it is involved in variety of cellular activity such as cell activation, cell growth, and cell adhesion [5,6,7,8]. 4F2 heavy chain (4F2hc) is an integral membrane protein with a single membrane-spanning domain classified as type II membrane protein [9]
When 4F2hc was expressed in Xenopus oocytes, it induced the transport of neutral and basic amino acids with the property of system yϩL, which is in agreement with the fact that 4F2hc exhibits amino acid sequence similarity to the type II membrane protein D2/rBAT, a cystinuria-associated putative amino acid transport activator (10 –12)
Summary
Co-expression of 4F2hc and Poly(A)ϩ RNA—Xenopus laevis oocyte expression studies and uptake measurements were performed as described elsewhere [13, 14]. Defolliculated oocytes were injected with in vitro transcribed cRNA (5 ng) of 4F2hc (GenBankTM/EBI/DDBJ accession number AB015433) and poly(A)ϩ RNA (45 ng) obtained from C6 glioma cells. Expression Cloning—Expression cloning using the Xenopus oocyte expression system was performed as described [15,16,17]. Four-hundred g of C6 glioma poly(A)ϩ RNA was size-fractionated [17]. RNA from each fraction (45 ng) was co-expressed with 4F2hc cRNA (5 ng) in Xenopus oocytes. Positive fractions showing peak stimulation of [14C]Lleucine (50 M) uptake when co-expressed with 4F2hc were used to construct a directional cDNA library. Positive fractions showing peak stimulation of [14C]Lleucine (50 M) uptake when co-expressed with 4F2hc were used to construct a directional cDNA library. cRNA synthesized in vitro from
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