EXPRESSION CLONING OF THE CDNA FOR A POLYPEPTIDE ASSOCIATED WITH RAT HEPATIC SINUSOIDAL REDUCED GLUTATHIONE TRANSPORT - CHARACTERISTICS ANDCOMPARISON WITH THE CANALICULAR TRANSPORTER
Jr. Yi et al., EXPRESSION CLONING OF THE CDNA FOR A POLYPEPTIDE ASSOCIATED WITH RAT HEPATIC SINUSOIDAL REDUCED GLUTATHIONE TRANSPORT - CHARACTERISTICS ANDCOMPARISON WITH THE CANALICULAR TRANSPORTER, Proceedings of the National Academy of Sciences of the United Statesof America, 92(5), 1995, pp. 1495-1499
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.