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Cloning and functional expression of the first eukaryotic Na+-tryptophan symporter, AgNAT6.

ABSTRACT: The nutrient amino acid transporter (NAT) subfamily of the neurotransmitter sodium symporter family (NSS, also known as the solute carrier family 6, SLC6) represents transport mechanisms with putative synergistic roles in the absorption of essential and conditionally essential neutral amino acids. It includes a large paralogous expansion of insect-specific genes, with seven genes from the genome of the malaria mosquito, Anopheles gambiae. One of the An. gambiae NATs, AgNAT8, was cloned, functionally expressed and characterized in X. laevis oocytes as a cation-coupled symporter of aromatic amino acids, preferably l-phenylalanine, l-tyrosine and l-DOPA. To explore an evolutionary trend of NAT-SLC6 phenotypes, we have cloned and characterized AgNAT6, which represents a counterpart of AgNAT8 descending from a recent gene duplication (53.1% pairwise sequence identity). In contrast to AgNAT8, which preferably mediates the absorption of phenol-branched substrates, AgNAT6 mediates the absorption of indole-branched substrates with highest apparent affinity to tryptophan (K(0.5)(Trp)=1.3 micromol l(-1) vs K(0.5)(Phe)=430 micromol l(-1)) and [2 or 1 Na(+) or K(+)]:[aromatic substrate] stoichiometry. AgNAT6 is highly transcribed in absorptive and secretory regions of the alimentary canal and specific neuronal structures, including the neuropile of ventral ganglia and sensory afferents. The alignment of AgNATs and LeuT(Aa), a bacterial NAT with a resolved 3D structure, reveals three amino acid differences in the substrate-binding pocket that may be responsible for the indole- vs phenol-branch selectivity of AgNAT6 vs AgNAT8. The identification of transporters with a narrow selectivity for essential amino acids suggests that basal expansions in the SLC6 family involved duplication and retention of NATs, improving the absorption and distribution of under-represented essential amino acids and related metabolites. The identified physiological and expression profiles suggest unique roles of AgNAT6 in the active absorption of indole-branched substrates that are used in the synthesis of the neurotransmitter serotonin as well as the key circadian hormone and potent free-radical scavenger melatonin.

SUBMITTER: Meleshkevitch EA

PROVIDER: S-EPMC2675963 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Cloning and functional expression of the first eukaryotic Na+-tryptophan symporter, AgNAT6.

Meleshkevitch Ella A EA Robinson Marvin M Popova Lyudmila B LB Miller Melissa M MM Harvey William R WR Boudko Dmitri Y DY

The Journal of experimental biology 20090501 Pt 10

The nutrient amino acid transporter (NAT) subfamily of the neurotransmitter sodium symporter family (NSS, also known as the solute carrier family 6, SLC6) represents transport mechanisms with putative synergistic roles in the absorption of essential and conditionally essential neutral amino acids. It includes a large paralogous expansion of insect-specific genes, with seven genes from the genome of the malaria mosquito, Anopheles gambiae. One of the An. gambiae NATs, AgNAT8, was cloned, function ...[more]

PMID: 19411550

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Project description:A 3-kb region, located downstream of the Lactobacillus brevis xylA gene (encoding D-xylose isomerase), was cloned in Escherichia coli TG1. The sequence revealed two open reading frames which could code for the D-xylulose kinase gene (xylB) and another gene (xylT) encoding a protein of 457 amino acids with significant similarity to the D-xylose-H+ symporters of E. coli, XylE (57%), and Bacillus megaterium, XylT (58%), to the D-xylose-Na+ symporter of Tetragenococcus halophila, XylE (57%), and to the L-arabinose-H+ symporter of E. coli, AraE (60%). The L. brevis xylABT genes showed an arrangement similar to that of the B. megaterium xylABT operon and the T. halophila xylABE operon. Southern hybridization performed with the Lactobacillus pentosus xylR gene (encoding the D-xylose repressor protein) as a probe revealed the existence of a xylR homologue in L. brevis which is not located with the xyABT locus. The existence of a functional XylR was further suggested by the presence of xylO sequences upstream of xylA and xylT and by the requirement of D-xylose for the induction of D-xylose isomerase, D-xylulose kinase, and D-xylose transport activities in L. brevis. When L. brevis was cultivated in a mixture of D-glucose and D-xylose, the D-xylose isomerase and D-xylulose kinase activities were reduced fourfold and the D-xylose transport activity was reduced by sixfold, suggesting catabolite repression by D-glucose of D-xylose assimilation. The xylT gene was functionally expressed in Lactobacillus plantarum 80, a strain which lacks proton motive force-linked D-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of D-xylose in L. plantarum 80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of D-glucose. The apparent affinity constant of XylT for D-xylose was approximately 215 microM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-D-glucose inhibited the transport of D-xylose by XylT competitively, with a Ki of 220 microM.

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Cloning and functional expression of the first eukaryotic Na+-tryptophan symporter, AgNAT6.

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Cloning and functional expression of the first eukaryotic Na+-tryptophan symporter, AgNAT6.

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