Effects of truncation of the COOH-terminal region of a Na+-independent neutral and basic amino acid transporter on amino acid transport in Xenopus oocytes.
To determine the role of a neutral and basic amino acid transporter (NBAT) in amino acid transport, we microinjected several COOH-terminal deletion mutants of NBAT cRNA into Xenopus oocytes and measured transport activity for arginine, leucine, and cystine in the presence and absence of sodium. Wild-type NBAT significantly stimulated the uptake of all three amino acids 10-20-fold compared with controls. On the other hand, no mutant, except a Delta511-685 mutant, stimulated the uptake of these amino acids. The Delta511-685 mutant significantly increased the uptake of arginine. In the presence of sodium, the Delta511-685 mutant also increased the uptake of leucine. The Delta511-685 mutant did not stimulate cystine uptake in the presence or absence of sodium. The stimulation of arginine uptake by the Delta511-685 mutant was inhibited by a 100-fold excess of unlabeled leucine in the presence of sodium. Inhibition of L-arginine uptake by L-homoserine was seen only in the presence of sodium, and an increase in the inhibition of L-arginine uptake by L-histidine was seen when the extracellular pH was decreased. Furthermore, an inward current in oocytes injected with the Delta511-685 mutant was recorded electrophysiologically when basic amino acids were applied. Homoserine was also taken up, but sodium was necessary for their transport. These properties of the Delta511-685 mutant correspond to those of the y+ amino acid transporter. If NBAT is a component of the b0,+-like amino acid transport system, it is unlikely that a mutant protein (Delta511-685) is able to stimulate an endogenous y+-like transport system. These results suggest that NBAT functions as a activator of the amino acid transport system in Xenopus oocytes.
Miyamoto
K
K
Department of Clinical Nutrition, School of Medicine, Tokushima University, Kuramoto-Cho 3, Tokushima 770, Japan.
Segawa
H
H
Tatsumi
S
S
Katai
K
K
Yamamoto
H
H
Taketani
Y
Y
Haga
H
H
Morita
K
K
Takeda
E
E
D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems; D026923:Amino Acid Transport Systems, Basic; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005260:Female; D006801:Humans; D007672:Kidney Cortex / Q000378:metabolism; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism / Q000502:physiology; D017384:Sequence Deletion; D012964:Sodium / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics
D026905:Amino Acid Transport Systems; D026923:Amino Acid Transport Systems, Basic; D000596:Amino Acids; D002352:Carrier Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D012964:Sodium