Publications

• Markerless mutagenesis in Methanococcus maripaludis demonstrates roles for alanine dehydrogenase, alanine racemase, and alanine permease.

  Moore B.C., Leigh J.A.

  Among the archaea, Methanococcus maripaludis has the unusual ability to use L- or D-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in t ... >> More

  Among the archaea, Methanococcus maripaludis has the unusual ability to use L- or D-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in these genes, we devised a method for markerless mutagenesis that builds on previously established genetic tools for M. maripaludis. The technique uses a negative selection strategy that takes advantage of the ability of the M. maripaludis hpt gene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to the base analog 8-azahypoxanthine. In addition, we developed a negative selection method to stably incorporate constructs into the genome at the site of the upt gene encoding uracil phosphoribosyltransferase. Mutants with in-frame deletion mutations in the genes for alanine dehydrogenase and alanine permease lost the ability to grow on either isomer of alanine, while a mutant with an in-frame deletion mutation in the gene for alanine racemase lost only the ability to grow on D-alanine. The wild-type gene for alanine dehydrogenase, incorporated into the upt site, complemented the alanine dehydrogenase mutation. Hence, the permease is required for the transport of either isomer, the dehydrogenase is specific for the L isomer, and the racemase converts the D isomer to the L isomer. Phylogenetic analysis indicated that all three genes had been acquired by lateral gene transfer from the low-moles-percent G+C gram-positive bacteria. << Less

  J. Bacteriol. 187:972-979(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of a branched-chain amino-acid transporter SBAT1 (SLC6A15) that is expressed in human brain.

  Takanaga H., Mackenzie B., Peng J.B., Hediger M.A.

  The SLC6 gene family comprises membrane proteins that transport neurotransmitters, amino acids, or osmolytes. We report the first functional characterization of the human SLC6A15 gene, which codes for a sodium-coupled branched-chain amino-acid transporter 1 (SBAT1). SBAT1 expression is specific to ... >> More

  The SLC6 gene family comprises membrane proteins that transport neurotransmitters, amino acids, or osmolytes. We report the first functional characterization of the human SLC6A15 gene, which codes for a sodium-coupled branched-chain amino-acid transporter 1 (SBAT1). SBAT1 expression is specific to the brain. When expressed in Xenopus oocytes, SBAT1 mediated Na+-coupled transport of hydrophobic, zwitterionic alpha-amino and imino acids. SBAT1 exhibited a strong preference for branched-chain amino acids (BCAA) and methionine (K0.5 80-160 microM). SBAT1 excluded aromatic or charged amino acids, beta-amino acids, glycine, and GABA. SBAT1-mediated transport of amino or imino acids was extremely temperature-dependent (Q10=9) and was inhibited at acidic pH. PKC activation reduced the plasma-membrane population of SBAT1 protein. SBAT1-mediated transport of BCAA, particularly leucine, may be an important source of amino nitrogen for neurotransmitter synthesis in glutamatergic and GABAergic neurons. << Less

  Biochem. Biophys. Res. Commun. 337:892-900(2005) [PubMed] [EuropePMC]

  This publication is cited by 10 other entries.

• A novel system A isoform mediating Na+/neutral amino acid cotransport.

  Yao D., Mackenzie B., Ming H., Varoqui H., Zhu H., Hediger M.A., Erickson J.D.

  A cDNA clone encoding a plasma membrane alanine-preferring transporter (SAT2) has been isolated from glutamatergic neurons in culture and represents the second member of the system A family of neutral amino acid transporters. SAT2 displays a widespread distribution and is expressed in most tissues ... >> More

  A cDNA clone encoding a plasma membrane alanine-preferring transporter (SAT2) has been isolated from glutamatergic neurons in culture and represents the second member of the system A family of neutral amino acid transporters. SAT2 displays a widespread distribution and is expressed in most tissues, including heart, adrenal gland, skeletal muscle, stomach, fat, brain, spinal cord, colon, and lung, with lower levels detected in spleen. No signal is detected in liver or testis. In the central nervous system, SAT2 is expressed in neurons. SAT2 is significantly up-regulated during differentiation of cerebellar granule cells and is absent from astrocytes in primary culture. The functional properties of SAT2, examined using transfected fibroblasts and in cRNA-injected voltage-clamped Xenopus oocytes, show that small aliphatic neutral amino acids are preferred substrates and that transport is voltage- and Na(+)-dependent (1:1 stoichiometry), pH-sensitive, and inhibited by alpha-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of system A. Kinetic analyses of alanine and MeAIB uptake by SAT2 are saturable, with Michaelis constants (K(m)) of 200-500 microm. In addition to its ubiquitous role as a substrate for oxidative metabolism and a major vehicle of nitrogen transport, SAT2 may provide alanine to function as the amino group donor to alpha-ketoglutarate to provide an alternative source for neurotransmitter synthesis in glutamatergic neurons. << Less

  J. Biol. Chem. 275:22790-22797(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structure and function of ATA3, a new subtype of amino acid transport system A, primarily expressed in the liver and skeletal muscle.

  Sugawara M., Nakanishi T., Fei Y.-J., Martindale R.G., Ganapathy M.E., Leibach F.H., Ganapathy V.

  To date, two different transporters that are capable of transporting alpha-(methylamino)isobutyric acid, the specific substrate for amino acid transport system A, have been cloned. These two transporters are known as ATA1 and ATA2. We have cloned a third transporter that is able to transport the s ... >> More

  To date, two different transporters that are capable of transporting alpha-(methylamino)isobutyric acid, the specific substrate for amino acid transport system A, have been cloned. These two transporters are known as ATA1 and ATA2. We have cloned a third transporter that is able to transport the system A-specific substrate. This new transporter, cloned from rat skeletal muscle and designated rATA3, consists of 547 amino acids and has a high degree of homology to rat ATA1 (47% identity) and rat ATA2 (57% identity). rATA3 mRNA is present only in the liver and skeletal muscle. When expressed in Xenopus laevis oocytes, rATA3 mediates the transport of alpha-[(14)C](methylamino)isobutyric acid and [(3)H]alanine. With the two-microelectrode voltage clamp technique, we have shown that exposure of rATA3-expressing oocytes to neutral, short-chain aliphatic amino acids induces inward currents. The amino acid-induced current is Na(+)-dependent and pH-dependent. Analysis of the currents with alanine as the substrate has shown that the K(0. 5) for alanine (i.e., concentration of the amino acid yielding half-maximal current) is 4.2+/-0.1 mM and that the Na(+):alanine stoichiometry is 1:1. << Less

  Biochim. Biophys. Acta 1509:7-13(2000) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• The orphan transporter v7-3 (slc6a15) is a Na+-dependent neutral amino acid transporter (B0AT2).

  Broer A., Tietze N., Kowalczuk S., Chubb S., Munzinger M., Bak L.K., Broer S.

  Transporters of the SLC6 (solute carrier 6) family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. In the present study, we demonstrate that mouse v7-3 (slc6a15) encodes a transporter for neutral amino acids. The transport ... >> More

  Transporters of the SLC6 (solute carrier 6) family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. In the present study, we demonstrate that mouse v7-3 (slc6a15) encodes a transporter for neutral amino acids. The transporter is functionally and sequence related to B(0)AT1 (slc6a19) and was hence named B(0)AT2. Leucine, isoleucine, valine, proline and methionine were recognized by the transporter, with values of K(0.5) (half-saturation constant) ranging from 40 to 200 microM. Alanine, glutamine and phenylalanine were low-affinity substrates of the transporter, with K(0.5) values in the millimolar range. Transport of neutral amino acids via B(0)AT2 was Na+-dependent, Cl--independent and electrogenic. Superfusion of mouse B(0)AT2-expressing oocytes with amino acid substrates generated robust inward currents. Na+-activation kinetics of proline transport and uptake under voltage clamp suggested a 1:1 Na+/amino acid co-transport stoichiometry. Susbtrate and co-substrate influenced each other's K(0.5) values, suggesting that they share the same binding site. A mouse B(0)AT2-like transport activity was detected in synaptosomes and cultured neurons. A potential role of B(0)AT2 in transporting neurotransmitter precursors and neuromodulators is proposed. << Less

  Biochem. J. 393:421-430(2006) [PubMed] [EuropePMC]

  This publication is cited by 11 other entries.

• A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling.

  Fairweather S.J., Okada S., Gauthier-Coles G., Javed K., Broeer A., Broeer S.

  Amino acid transporters play a vital role in metabolism and nutrient signaling pathways. Typically, transport activity is investigated using single substrates and competing amounts of other amino acids. We used GC-MS and LC-MS for metabolic screening of <i>Xenopus laevis</i> oocytes expressing var ... >> More

  Amino acid transporters play a vital role in metabolism and nutrient signaling pathways. Typically, transport activity is investigated using single substrates and competing amounts of other amino acids. We used GC-MS and LC-MS for metabolic screening of <i>Xenopus laevis</i> oocytes expressing various human amino acid transporters incubated in complex media to establish their comprehensive substrate profiles. For most transporters, amino acid selectivity matched reported substrate profiles. However, we could not detect substantial accumulation of cationic amino acids by SNAT4 and ATB^0,+ in contrast to previous reports. In addition, comparative substrate profiles of two related sodium neutral amino acid transporters known as SNAT1 and SNAT2, revealed the latter as a significant leucine accumulator. As a consequence, SNAT2, but not SNAT1, was shown to be an effective activator of the eukaryotic cellular growth regulator mTORC1. We propose, that metabolomic profiling of membrane transporters in <i>Xe</i> <i>nopus laevis</i> oocytes can be used to test their substrate specificity and role in intracellular signaling pathways. << Less

  Front. Mol. Biosci. 8:646574-646574(2021) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Cloning and expression of a novel Na(+)-dependent neutral amino acid transporter structurally related to mammalian Na+/glutamate cotransporters.

  Shafqat S., Tamarappoo B.K., Kilberg M.S., Puranam R.S., McNamara J.O., Guadano-Ferraz A., Fremeau R.T. Jr.

  A cDNA has been isolated from human hippocampus that appears to encode a novel Na(+)-dependent, Cl(-)-independent, neutral amino acid transporter. The putative protein, designated SATT, is 529 amino acids long and exhibits significant amino acid sequence identity (39-44%) with mammalian L-glutamat ... >> More

  A cDNA has been isolated from human hippocampus that appears to encode a novel Na(+)-dependent, Cl(-)-independent, neutral amino acid transporter. The putative protein, designated SATT, is 529 amino acids long and exhibits significant amino acid sequence identity (39-44%) with mammalian L-glutamate transporters. Expression of SATT cDNA in HeLa cells induced stereospecific uptake of L-serine, L-alanine, and L-threonine that was not inhibited by excess (3 mM) 2-(methylamino)-isobutyric acid, a specific substrate for the System A amino acid transporter. SATT expression in HeLa cells did not induce the transport of radiolabeled L-cysteine, L-glutamate, or related dicarboxylates. Northern blot hybridization revealed high levels of SATT mRNA in human skeletal muscle, pancreas, and brain, intermediate levels in heart, and low levels in liver, placenta, lung, and kidney. SATT transport characteristics are similar to the Na(+)-dependent neutral amino acid transport activity designated System ASC, but important differences are noted. These include: 1) SATT's apparent low expression in ASC-containing tissues such as liver or placenta; 2) the lack of mutual inhibition between serine and cysteine; and 3) the lack of trans-stimulation. SATT may represent one of multiple activities that exhibit System ASC-like transport characteristics in diverse tissues and cell lines. << Less

  J. Biol. Chem. 268:15351-15355(1993) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• A novel human amino acid transporter, hNAT3: cDNA cloning, chromosomal mapping, genomic structure, expression, and functional characterization.

  Gu S., Adan-Rice D., Leach R.J., Jiang J.X.

  Amino acid transporters are proteins that transport amino acids across the membrane. We report here the isolation and characterization of a novel human cDNA clone encoding a protein of 547 amino acids. This protein shares approximately 50% amino acid sequence homology with the amino acid transport ... >> More

  Amino acid transporters are proteins that transport amino acids across the membrane. We report here the isolation and characterization of a novel human cDNA clone encoding a protein of 547 amino acids. This protein shares approximately 50% amino acid sequence homology with the amino acid transporters mouse mNAT and its orthologs, rat SN1 and human g17, and mouse GlnT/ATA1 and ATA2. Expression of this cRNA in Xenopus oocytes revealed that the strongest transport activities were specific for l-alanine. In addition, hNAT3 is a Na(+)- and pH-dependent, low-affinity transporter and partially tolerates substitution of Na(+) by Li(+). Since this protein has sequence and functional similarities to the previously identified system N amino acid transporters, we named this protein hNAT3. The genomic DNA sequence encoding the transcript of hNAT3 spans over 14 kb with 16 exons and 15 introns. Using fluorescence in situ hybridization, we mapped the hNAT3 gene to human chromosome 12q12-q13. By RT-PCR of embryonic and adult human tissues, hNAT3 was detected to be predominantly expressed in the liver and to a much lesser extent in the muscle, kidney, and pancreas. The data obtained in this study are likely to offer critical clues for identification of amino acid transporter-associated diseases. << Less

  Genomics 74:262-272(2001) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Functional properties and cellular distribution of the system A glutamine transporter SNAT1 support specialized roles in central neurons.

  Mackenzie B., Schaefer M.K.-H., Erickson J.D., Hediger M.A., Weihe E., Varoqui H.

  Glutamine, the preferred precursor for neurotransmitter glutamate and GABA, is likely to be the principal substrate for the neuronal System A transporter SNAT1 in vivo. We explored the functional properties of SNAT1 (the product of the rat Slc38a1 gene) by measuring radiotracer uptake and currents ... >> More

  Glutamine, the preferred precursor for neurotransmitter glutamate and GABA, is likely to be the principal substrate for the neuronal System A transporter SNAT1 in vivo. We explored the functional properties of SNAT1 (the product of the rat Slc38a1 gene) by measuring radiotracer uptake and currents associated with SNAT1 expression in Xenopus oocytes and determined the neuronal-phenotypic and cellular distribution of SNAT1 by confocal laser-scanning microscopy alongside other markers. We found that SNAT1 mediates transport of small, neutral, aliphatic amino acids including glutamine (K0.5 approximately 0.3 mm), alanine, and the System A-specific analogue 2-(methylamino)isobutyrate. Amino acid transport is driven by the Na+ electrochemical gradient. The voltage-dependent binding of Na+ precedes that of the amino acid in a simultaneous transport mechanism. Li+ (but not H+) can substitute for Na+ but results in reduced Vmax. In the absence of amino acid, SNAT1 mediates Na+-dependent presteady-state currents (Qmax approximately 9 nC) and a nonsaturable cation leak with selectivity Na+, Li+ >> H+, K+. Simultaneous flux and current measurements indicate coupling stoichiometry of 1 Na+ per 1 amino acid. SNAT1 protein was detected in somata and proximal dendrites but not nerve terminals of glutamatergic and GABAergic neurons throughout the adult CNS. We did not detect SNAT1 expression in astrocytes but detected its expression on the luminal membranes of the ependyma. The functional properties and cellular distribution of SNAT1 support a primary role for SNAT1 in glutamine transport serving the glutamate/GABA-glutamine cycle in central neurons. Localization of SNAT1 to certain dopaminergic neurons of the substantia nigra and cholinergic motoneurons suggests that SNAT1 may play additional specialized roles, providing metabolic fuel (via alpha-ketoglutarate) or precursors (cysteine, glycine) for glutathione synthesis. << Less

  J. Biol. Chem. 278:23720-23730(2003) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

• Mutations in SLC6A19, encoding B(0)AT1, cause Hartnup disorder.

  Kleta R., Romeo E., Ristic Z., Ohura T., Stuart C., Arcos-Burgos M., Dave M.H., Wagner C.A., Camargo S.R.M., Inoue S., Matsuura N., Helip-Wooley A., Bockenhauer D., Warth R., Bernardini I., Visser G., Eggermann T., Lee P., Chairoungdua A., Jutabha P., Babu E., Nilwarangkoon S., Anzai N., Kanai Y., Verrey F., Gahl W.A., Koizumi A.

  Hartnup disorder, an autosomal recessive defect named after an English family described in 1956 (ref. 1), results from impaired transport of neutral amino acids across epithelial cells in renal proximal tubules and intestinal mucosa. Symptoms include transient manifestations of pellagra (rashes), ... >> More

  Hartnup disorder, an autosomal recessive defect named after an English family described in 1956 (ref. 1), results from impaired transport of neutral amino acids across epithelial cells in renal proximal tubules and intestinal mucosa. Symptoms include transient manifestations of pellagra (rashes), cerebellar ataxia and psychosis. Using homozygosity mapping in the original family in whom Hartnup disorder was discovered, we confirmed that the critical region for one causative gene was located on chromosome 5p15 (ref. 3). This region is homologous to the area of mouse chromosome 13 that encodes the sodium-dependent amino acid transporter B(0)AT1 (ref. 4). We isolated the human homolog of B(0)AT1, called SLC6A19, and determined its size and molecular organization. We then identified mutations in SLC6A19 in members of the original family in whom Hartnup disorder was discovered and of three Japanese families. The protein product of SLC6A19, the Hartnup transporter, is expressed primarily in intestine and renal proximal tubule and functions as a neutral amino acid transporter. << Less

  Nat. Genet. 36:999-1002(2004) [PubMed] [EuropePMC]

  This publication is cited by 13 other entries.