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Expression Cloning of the cDNA for a Polypeptide Associated with Rat Hepatic Sinusoidal Reduced Glutathione Transport: Characteristics and Comparison with the Canalicular Transporter
Jian-Ri Yi, Shilun Lu, Jose Fernandez-Checa and Neil Kaplowitz
Proceedings of the National Academy of Sciences of the United States of America
Vol. 92, No. 5 (Feb. 28, 1995), pp. 1495-1499
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/2366809
Page Count: 5
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Using the Xenopus oocyte expression system, we previously identified an ≈4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein reduced glutathione S-conjugate (BSP-GSH)-insensitive and an ≈2.5-kb fraction expressing BSP-GSH-sensitive reduced glutathione (GSH) transport. From the former, a 4.05-kb cDNA was cloned and characterized as the putative rat canalicular GSH transporter. Starting with a cDNA library constructed from the ≈2.5-kb fraction, we have now isolated a single clone that leads to expression of a BSP-GSH- and cystathionine-inhibitable GSH transporter activity with Km ≈ 3 mM characteristic of the sinusoidal GSH transporter. The cDNA for the rat sinusoidal GSH transporter-associated polypeptide (RsGshT) is 2733 bases with an open reading frame of 1059 nucleotides encoding a polypeptide of 353 amino acids (39,968 Da) with two putative membrane-spanning domains. No identifiable homologies were found in searching various data bases. An ≈40-kDa protein is generated in in vitro translation of cRNA for RsGshT. Northern blot analysis revealed a single ≈2.8-kb transcript in rat and human liver with negligible hybridization signal in other organs. The abundance of mRNA for RsGshT did not increase with phenobarbital treatment. Cis-inhibition by BSP-GSH and trans-inhibition by cystathionine and lack of induction by phenobarbital are characteristic of sinusoidal GSH secretion and thus indicate that RsGshT either encodes the sinusoidal GSH transporter itself or a regulatory subunit of the transporter that determines its liver-specific activity.
Proceedings of the National Academy of Sciences of the United States of America © 1995 National Academy of Sciences