EXPRESSION CLONING OF THE CDNA FOR A POLYPEPTIDE ASSOCIATED WITH RAT HEPATIC SINUSOIDAL REDUCED GLUTATHIONE TRANSPORT - CHARACTERISTICS ANDCOMPARISON WITH THE CANALICULAR TRANSPORTER

Using the Xenopus oocyte expression system, we previously identified a n approximate to 4-kb fraction of mRNA from rat liver that expresses s ulfobromophthalein reduced glutathione S-conjugate (BSP-GSH)-insensiti ve and an approximate to 2.5-kb fraction expressing BSP-GSH-sensitive reduced glutathione (GSH) transport. From the former, a 4.05-kb cDNA w as cloned and characterized as the putative rat canalicular GSH transp orter. Starting with a cDNA library constructed from the approximate t o 2.5-kb fraction, we have now isolated a single clone that leads to e xpression of a BSP-GSH- and cystathionine-inhibitable GSH transporter activity with K-m approximate to 3 mM characteristic of the sinusoidal GSH transporter. The cDNA for the rat sinusoidal GSH transporter-asso ciated polypeptide (RsGshT) is 2733 bases with an open reading frame o f 1059 nucleotides encoding a polypeptide of 353 amino acids (39,968 D a) with two putative membrane-spanning domains. No identifiable homolo gies were found in searching various data bases. An approximate to 40- kDa protein is generated in in vitro translation of cRNA for RsGshT. N orthern blot analysis revealed a single approximate to 2.8-kb transcri pt in rat and human liver with negligible hybridization signal in othe r organs. The abundance of mRNA for RsGshT did not increase with pheno barbital treatment. Cis-inhibition by BSP-GSH and trans-inhibition by cystathionine and lack of induction by phenobarbital are characteristi c of sinusoidal GSH secretion and thus indicate that RsGshT either enc odes the sinusoidal GSH transporter itself or a regulatory subunit of the transporter that determines its liver-specific activity.