All living organisms deal with a wide variety of toxic compounds of both endogenous and exogenous origin. Although many organs and metabolic systems are involved in this process, the mammalian liver provides the key elements to detoxification. Some of the most pernicious toxic compounds are lipophyllic in nature; enzymatic conjugation with glutathione, glucuronide or sulfate produces a more polar adduct which can be excreted into bile. In vitro analysis identified an ATP-dependent non-bile acid organic anion transporter (NBAOAT) in canalicular membrane vesicles. Although characterization of the transport activity suggested that an ABC- type protein was responsible, difficulties in purification of the low abundance membrane transport protein have not permitted characterization of the protein at the molecular level. Recent reports indicate that MRP1 (mdr1-related-protein) is a GSH conjugate transporter. However, the mRNA expression pattern suggests that MRP1 does not encode the canalicular NBAOAT. Previously, we had reported that two species of yeast, Saccharomyces cerevisiae and Schizosaccharomyces pombe, contain an ATP- dependent GSH conjugate transport activity that is strikingly similar to that of mammals. We have now shown that the ycf1 gene encodes this activity in S. cerevisae. ATP-dependent transport of the dinitrophenyl-GSH (DNP-SO) adduct is severely reduced in secretory vesicles and vacuoles purified from the delta-ycf1 knockout mutant. Transformation of this strain with a plasmid expressing YCF1 restores activity, indicating that the ycf1 gene product mediates GSH-conjugate transport. The main goal of the proposed research is the isolation and characterization of the gene encoding the liver ATP-dependent NBAOAT. Cloning strategies include screening of Lambda libraries at low stringency with ycf1 and mrp1 probes. PCR of liver mRNA using degenerate oligonucleotide primers generated by reverse translation of aa sequence motifs conserved in YCF1 and MRP1 will also be tried. A third alternative relies on the hypersensitivity delta- ycf1 yeast strains to toxic compounds which are bound or conjugated with GSH, such as Cd, diamide and hexachlorobutadiene. Restoration of tolerance to these compounds by complementation with a liver cDNA clone may identify the NBAOAT gene. Cloning of the mammalian gene would allow production of the tools, antibodies and DNA probes, enabling the characterization of the NBAOAT at the molecular level in vivo. Additionally, S. cerevisiae provides an ideal system to undertake structure-function studies on a membrane transport protein. Carrying out the experiments in parallel on the NBAOAT and YCF1 proteins should allow identification of essential features of a GSH-conjugate transporter.
| Ortiz, D F; Li, S; Iyer, R et al. (1999) MRP3, a new ATP-binding cassette protein localized to the canalicular domain of the hepatocyte. Am J Physiol 276:G1493-500 |
| Ortiz, D F; St Pierre, M V; Abdulmessih, A et al. (1997) A yeast ATP-binding cassette-type protein mediating ATP-dependent bile acid transport. J Biol Chem 272:15358-65 |
