Expression cloning of a rat hepatic reduced glutathione transporter with canalicular characteristics.
Using the Xenopus oocyte expression system, we have previously identified an approximately 4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein-glutathione (BSP-GSH)-insensitive reduced glutathione (GSH) transport (Fernandez-Checa, J., J. R. Yi, C. Garcia-Ruiz, Z. Knezic, S. Tahara, and N. Kaplowitz. 1993. J. Biol. Chem. 268:2324-2328). Starting with a cDNA library constructed from this fraction, we have now isolated a single clone that expresses GSH transporter activity. The cDNA for the rat canalicular GSH transporter (RcGshT) is 4.05 kb with an open reading frame of 2,505 nucleotides encoding for a polypeptide of 835 amino acids (95,785 daltons). No identifiable homologies were found in searching various databases. An approximately 96-kD protein is generated in in vitro translation of cRNA for RcGshT. Northern blot analysis reveals a single 4-kb transcript in liver, kidney, intestine, lung, and brain. The abundance of mRNA for RcGshT in rat liver increased 3, 6, and 12 h after a single dose of phenobarbital. Insensitivity to BSP-GSH and induction by phenobarbital, unique characteristics of canalicular GSH secretion, suggest that RcGshT encodes for the canalicular GSH transporter.
Yi
J R
JR
Department of Medicine, University of Southern California School of Medicine, Los Angeles 90033.
Lu
S
S
Fernandez-Checa
J
J
Kaplowitz
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D005978:Glutathione / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D014981:Xenopus
D002352:Carrier Proteins; D018076:DNA, Complementary; D026901:Membrane Transport Proteins; C086860:glutathione transporter; D005978:Glutathione