Characterization of a functional bacterial homologue of sodium-dependent neurotransmitter transporters.
The tnaT gene of Symbiobacterium thermophilum encodes a protein homologous to sodium-dependent neurotransmitter transporters. Expression of the tnaT gene product in Escherichia coli conferred the ability to accumulate tryptophan from the medium and the ability to grow on tryptophan as a sole source of carbon. Transport was Na(+)-dependent and highly selective. The K(m) for tryptophan was approximately 145 nm, and tryptophan transport was unchanged in the presence of 100 microM concentrations of other amino acids. Tryptamine and serotonin were weak inhibitors with K(I) values of 200 and 440 microM, respectively. By using a T7 promoter-based system, TnaT with an N-terminal His(6) tag was expressed at high levels in the membrane and was purified to near-homogeneity in high yield.
Androutsellis-Theotokis
Andreas
A
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, USA.
Goldberg
Naomi R
NR
Ueda
Kenji
K
Beppu
Teruhiko
T
Beckman
Matthew L
ML
Das
Shonit
S
Javitch
Jonathan A
JA
Rudnick
Gary
G
D039903:Actinobacteria / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins; D012964:Sodium / Q000494:pharmacology; D014364:Tryptophan / Q000378:metabolism*
D002352:Carrier Proteins; D017931:DNA Primers; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; D012701:Serotonin; D014364:Tryptophan; D012964:Sodium