Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Feb 28;92(5):1495–1499. doi: 10.1073/pnas.92.5.1495

Expression cloning of the cDNA for a polypeptide associated with rat hepatic sinusoidal reduced glutathione transport: characteristics and comparison with the canalicular transporter.

J R Yi 1, S Lu 1, J Fernández-Checa 1, N Kaplowitz 1
PMCID: PMC42546  PMID: 7878007

Abstract

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.

Full text

PDF
1497

Images in this article

1498Fig. 3
on p.1498

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aw T. Y., Ookhtens M., Kaplowitz N. Inhibition of glutathione efflux from isolated rat hepatocytes by methionine. J Biol Chem. 1984 Aug 10;259(15):9355–9358. [PubMed] [Google Scholar]
  2. Aw T. Y., Ookhtens M., Kaplowitz N. Mechanism of inhibition of glutathione efflux by methionine from isolated rat hepatocytes. Am J Physiol. 1986 Sep;251(3 Pt 1):G354–G361. doi: 10.1152/ajpgi.1986.251.3.G354. [DOI] [PubMed] [Google Scholar]
  3. Ballatori N., Dutczak W. J. Identification and characterization of high and low affinity transport systems for reduced glutathione in liver cell canalicular membranes. J Biol Chem. 1994 Aug 5;269(31):19731–19737. [PubMed] [Google Scholar]
  4. Bertran J., Werner A., Moore M. L., Stange G., Markovich D., Biber J., Testar X., Zorzano A., Palacin M., Murer H. Expression cloning of a cDNA from rabbit kidney cortex that induces a single transport system for cystine and dibasic and neutral amino acids. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5601–5605. doi: 10.1073/pnas.89.12.5601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burgunder J. M., Lauterburg B. H. Decreased production of glutathione in patients with cirrhosis. Eur J Clin Invest. 1987 Oct;17(5):408–414. doi: 10.1111/j.1365-2362.1987.tb01135.x. [DOI] [PubMed] [Google Scholar]
  6. Canessa C. M., Horisberger J. D., Rossier B. C. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature. 1993 Feb 4;361(6411):467–470. doi: 10.1038/361467a0. [DOI] [PubMed] [Google Scholar]
  7. Dröge W., Eck H. P., Näher H., Pekar U., Daniel V. Abnormal amino-acid concentrations in the blood of patients with acquired immunodeficiency syndrome (AIDS) may contribute to the immunological defect. Biol Chem Hoppe Seyler. 1988 Mar;369(3):143–148. doi: 10.1515/bchm3.1988.369.1.143. [DOI] [PubMed] [Google Scholar]
  8. Dutczak W. J., Ballatori N. Transport of the glutathione-methylmercury complex across liver canalicular membranes on reduced glutathione carriers. J Biol Chem. 1994 Apr 1;269(13):9746–9751. [PubMed] [Google Scholar]
  9. Fernandez-Checa J. C., Maddatu T., Ookhtens M., Kaplowitz N. Inhibition of GSH efflux from rat liver by methionine: effects of GSH synthesis in cells and perfused organ. Am J Physiol. 1990 Jun;258(6 Pt 1):G967–G973. doi: 10.1152/ajpgi.1990.258.6.G967. [DOI] [PubMed] [Google Scholar]
  10. Fernández-Checa J. C., Ookhtens M., Kaplowitz N. Selective induction by phenobarbital of the electrogenic transport of glutathione and organic anions in rat liver canalicular membrane vesicles. J Biol Chem. 1993 May 25;268(15):10836–10841. [PubMed] [Google Scholar]
  11. Fernández-Checa J. C., Takikawa H., Horie T., Ookhtens M., Kaplowitz N. Canalicular transport of reduced glutathione in normal and mutant Eisai hyperbilirubinemic rats. J Biol Chem. 1992 Jan 25;267(3):1667–1673. [PubMed] [Google Scholar]
  12. Fernández-Checa J. C., Yi J. R., Garcia-Ruiz C., Knezic Z., Tahara S. M., Kaplowitz N. Expression of rat liver reduced glutathione transport in Xenopus laevis oocytes. J Biol Chem. 1993 Feb 5;268(4):2324–2328. [PubMed] [Google Scholar]
  13. García-Ruiz C., Fernández-Checa J. C., Kaplowitz N. Bidirectional mechanism of plasma membrane transport of reduced glutathione in intact rat hepatocytes and membrane vesicles. J Biol Chem. 1992 Nov 5;267(31):22256–22264. [PubMed] [Google Scholar]
  14. Ho K., Nichols C. G., Lederer W. J., Lytton J., Vassilev P. M., Kanazirska M. V., Hebert S. C. Cloning and expression of an inwardly rectifying ATP-regulated potassium channel. Nature. 1993 Mar 4;362(6415):31–38. doi: 10.1038/362031a0. [DOI] [PubMed] [Google Scholar]
  15. Jaeschke H. Enhanced sinusoidal glutathione efflux during endotoxin-induced oxidant stress in vivo. Am J Physiol. 1992 Jul;263(1 Pt 1):G60–G68. doi: 10.1152/ajpgi.1992.263.1.G60. [DOI] [PubMed] [Google Scholar]
  16. Kaplowitz N., Aw T. Y., Ookhtens M. The regulation of hepatic glutathione. Annu Rev Pharmacol Toxicol. 1985;25:715–744. doi: 10.1146/annurev.pa.25.040185.003435. [DOI] [PubMed] [Google Scholar]
  17. Kaplowitz N., Eberle D. E., Petrini J., Touloukian J., Corvasce M. C., Kuhlenkamp J. Factors influencing the efflux of hepatic glutathione into bile in rats. J Pharmacol Exp Ther. 1983 Jan;224(1):141–147. [PubMed] [Google Scholar]
  18. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kubo Y., Baldwin T. J., Jan Y. N., Jan L. Y. Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature. 1993 Mar 11;362(6416):127–133. doi: 10.1038/362127a0. [DOI] [PubMed] [Google Scholar]
  20. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  21. Meister A., Anderson M. E. Glutathione. Annu Rev Biochem. 1983;52:711–760. doi: 10.1146/annurev.bi.52.070183.003431. [DOI] [PubMed] [Google Scholar]
  22. Mosckovitz R., Udenfriend S., Felix A., Heimer E., Tate S. S. Membrane topology of the rat kidney neutral and basic amino acid transporter. FASEB J. 1994 Oct;8(13):1069–1074. doi: 10.1096/fasebj.8.13.7926373. [DOI] [PubMed] [Google Scholar]
  23. Ookhtens M., Hobdy K., Corvasce M. C., Aw T. Y., Kaplowitz N. Sinusoidal efflux of glutathione in the perfused rat liver. Evidence for a carrier-mediated process. J Clin Invest. 1985 Jan;75(1):258–265. doi: 10.1172/JCI111682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ookhtens M., Lyon I., Fernandez-Checa J., Kaplowitz N. Inhibition of glutathione efflux in the perfused rat liver and isolated hepatocytes by organic anions and bilirubin. Kinetics, sidedness, and molecular forms. J Clin Invest. 1988 Aug;82(2):608–616. doi: 10.1172/JCI113639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ookhtens M., Mittur A. V. Developmental changes in plasma thiol-disulfide turnover in rats: a multicompartmental approach. Am J Physiol. 1994 Aug;267(2 Pt 2):R415–R425. doi: 10.1152/ajpregu.1994.267.2.R415. [DOI] [PubMed] [Google Scholar]
  26. Ookhtens M., Mittur A. V., Erhart N. A. Changes in plasma glutathione concentrations, turnover, and disposal in developing rats. Am J Physiol. 1994 Mar;266(3 Pt 2):R979–R988. doi: 10.1152/ajpregu.1994.266.3.R979. [DOI] [PubMed] [Google Scholar]
  27. Sippel C. J., Suchy F. J., Ananthanarayanan M., Perlmutter D. H. The rat liver ecto-ATPase is also a canalicular bile acid transport protein. J Biol Chem. 1993 Jan 25;268(3):2083–2091. [PubMed] [Google Scholar]
  28. Snyder P. M., McDonald F. J., Stokes J. B., Welsh M. J. Membrane topology of the amiloride-sensitive epithelial sodium channel. J Biol Chem. 1994 Sep 30;269(39):24379–24383. [PubMed] [Google Scholar]
  29. Staal F. J., Ela S. W., Roederer M., Anderson M. T., Herzenberg L. A., Herzenberg L. A. Glutathione deficiency and human immunodeficiency virus infection. Lancet. 1992 Apr 11;339(8798):909–912. doi: 10.1016/0140-6736(92)90939-z. [DOI] [PubMed] [Google Scholar]
  30. Wells R. G., Hediger M. A. Cloning of a rat kidney cDNA that stimulates dibasic and neutral amino acid transport and has sequence similarity to glucosidases. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5596–5600. doi: 10.1073/pnas.89.12.5596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yi J. R., Lu S., Fernandez-Checa J., Kaplowitz N. Expression cloning of a rat hepatic reduced glutathione transporter with canalicular characteristics. J Clin Invest. 1994 Apr;93(4):1841–1845. doi: 10.1172/JCI117170. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES