Expression cloning of the cDNA for a polypeptide associated with rat hepatic sinusoidal reduced glutathione transport: characteristics and comparison with the canalicular transporter.
Using the Xenopus oocyte expression system, we previously identified an approximately 4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein reduced glutathione S-conjugate (BSP-GSH)-insensitive and an approximately 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 approximately 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 approximately 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 approximately 40-kDa protein is generated in in vitro translation of cRNA for RsGshT. Northern blot analysis revealed a single approximately 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.
Yi
J R
JR
Department of Medicine, University of Southern California School of Medicine, Los Angeles 90033.
Lu
S
S
Fernández-Checa
J
J
Kaplowitz
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005978:Glutathione / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D014018:Tissue Distribution
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; C086860:glutathione transporter; D005978:Glutathione