EXPRESSION CLONING OF A RAT HEPATIC REDUCED GLUTATHIONE TRANSPORTER WITH CANALICULAR CHARACTERISTICS

Using the Xenopus oocyte expression system, we have previously identif ied an similar to 4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein-glutathione (BSP-GSH)-insensitive reduced glutathi one (GSH) transport (Fernandez-Checa, J., J. R. Yi, C. Garcia-Ruiz, Z. Knezic, S. Tahara, and N. Kaplowitz. 1993. J. Biol. Chem. 268:2324-23 28). Starting with a cDNA library constructed from this fraction, we h ave now isolated a single clone that expresses GSH transporter activit y. The cDNA for the rat canalicular GSH transporter (RcGshT) is 4.05 k b with an open reading frame of 2,505 nucleotides encoding for a polyp eptide of 835 amino acids (95,785 daltons). No identifiable homologies were found in searching various databases. An similar to 96-kD protei n is generated in in vitro translation of cRNA for RcGshT. Northern bl ot analysis reveals a single 4-kb transcript in liver, kidney, intesti ne, lung, and brain. The abundance of mRNA for RcGshT in rat liver inc reased 3, 6, and 12 h after a single dose of phenobarbital. Insensitiv ity to BSP-GSH and induction by phenobarbital, unique characteristics of canalicular GSH secretion, suggest that RcGshT encodes for the cana licular GSH transporter.