Abstract
A cDNA was isolated from mouse testis which encodes a Na+-dependent neutral amino acid transporter. The encoded protein, designated ASCT2, showed amino acid sequence similarity to the mammalian glutamate transporters (40-44% identity), Na+-dependent neutral amino acid transporter ASCT1 (57% identity; Arriza, J. L., Kavanaugh, M. P., Fairman, W. A., Wu, Y.-N., Murdoch, G. H., North, R. A., and Amara, S. G.(1993) J. Biol. Chem. 268, 15329-15332; Shafqat, S., Tamarappoo, B. K., Kilberg, M. S., Puranam, R. S., McNamara, J. O., Guadano-Ferraz, A., and Fremeau, T., Jr. (1993) J. Biol. Chem. 268, 15351-15355) and a mouse adipocyte differentiation-associated gene product AAAT (94% identity; Liao, K., and Lane, D.(1995) Biochem. Biophys. Res. Commun. 208, 1008-1015). When expressed in Xenopus laevis oocytes, ASCT2 exhibited Na+-dependent uptakes of neutral amino acids such as L-alanine, L-serine, L-threonine, L-cysteine, and L-glutamine at high affinity with Km values around 20 microM. L-Methionine, L-leucine, L-glycine, and L-valine were also transported by ASCT2 but with lower affinity. The substrate selectivity of ASCT2 was typical of amino acid transport system ASC, which prefers neutral amino acids without bulky or branched side chains. ASCT2 also transported L-glutamate at low affinity (Km = 1.6 mM). L-Glutamate transport was enhanced by lowering extracellular pH, suggesting that L-glutamate was transported as protonated form. In contrast to electrogenic transport of glutamate transporters and the other ASC isoform ASCT1, ASCT2-mediated amino acid transport was electroneutral. Na+ dependence of L-alanine uptake fits to the Michaelis-Menten equation, suggesting a single Na+ cotransported with one amino acid, which was distinct from glutamate transporters coupled to two Na+. Northern blot hybridization revealed that ASCT2 was mainly expressed in kidney, large intestine, lung, skeletal muscle, testis, and adipose tissue. Functional characterization of ASCT2 provided fruitful information on the properties of substrate binding sites and the mechanisms of transport of Na+-dependent neutral and acidic amino acid transporter family, which would facilitate the structure-function analyses based on the comparison of the primary structures of ASCT2 and the other members of the family.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) D85044
We have searched for new members of glutamate transporter family by performing reverse transcription polymerase chain reaction (PCR) using degenerative oligonucleotide primers designed on well conserved amino acid sequences between family members
From mouse testis where massive amino acid transport is thought to be required for continuous cell division and proliferation, we have isolated a new system ASC-like Naϩ-dependent neutral amino acid transporter, which is structurally related to glutamate transporters yet exhibits distinct functional properties
Summary
MeAIB, ␣-(aminomethyl)isobutyric acid; PCR, polymerase chain reaction; PKC, protein kinase C; kb, kilobase pair(s); MES, 4-morpholineethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid. The comparison of the family members that have different substrate selectivity, ion coupling stoichiometry, or kinetic properties would provide important insights into the substrate binding sites and the mechanisms of the transport. Toward this end, we have searched for new members of glutamate transporter family by performing reverse transcription polymerase chain reaction (PCR) using degenerative oligonucleotide primers designed on well conserved amino acid sequences between family members. From mouse testis where massive amino acid transport is thought to be required for continuous cell division and proliferation, we have isolated a new system ASC-like Naϩ-dependent neutral amino acid transporter, which is structurally related to glutamate transporters yet exhibits distinct functional properties
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