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Functional expression of the multidrug resistance-associated protein in the yeast Saccharomyces cerevisiae
Abstract:
The multidrug resistance-associated protein (MRP) is a member of the ATP binding cassette superfamily of transporters which includes the mammalian P-glycoproteins (P-gp) family. In order to facilitate the biochemical and genetic analyses of MRP, we have expressed human MRP in the yeast Saccharomyces cerevisiae and have compared its functional properties to those of the mouse Mdr3 P-gp isoform. Expression of both MRP and Mdr3 in the anthracycline hypersensitive mutant VASY2563 restored cellular resistance to Adriamycin in this mutant. MRP and Mdr3 expression produced pleiotropic effects on drug resistance in this mutant, as corresponding VASY2563 transformants also acquired resistance to the anti-fungal agent FK506 and to the K+/H+ ionophore valinomycin. The appearance of increased cellular resistance to the toxic effect of Adriamycin (ADM) in MRP and Mdr3 transformants was concomitant with a reduced intracellular accumulation of [14C]ADM in spheroplasts prepared from these cells. Moreover, MRP and Mdr3, but not control spheroplasts, could mediate a time-dependent reduction in the overall cell-associated [14C]ADM from preloaded cells, suggesting the presence of an active ADM transport mechanism in MRP and Mdr3 transformants. Finally, human MRP was found to complement the biological activity of the yeast peptide pheromone transporter Ste6 and partially restored mating in a sterile ste6 null mutant. These findings suggest that despite their relatively low level of structural homology, MRP and P-gp share similar functional aspects, since both proteins can mediate transport of chemotherapeutic drugs and the a mating peptide pheromone in yeast.
Insights
Human multidrug resistance-associated protein (MRP) and mouse P-glycoprotein (P-gp) confer resistance to Adriamycin and other toxins when expressed in yeast. Both transporters also facilitate yeast mating, suggesting shared functional roles.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Multidrug resistance-associated protein (MRP) belongs to the ATP binding cassette superfamily, similar to mammalian P-glycoproteins (P-gp).
- Understanding MRP's function is crucial for biochemical and genetic analyses, particularly in drug resistance mechanisms.
Purpose of the Study:
- To express human MRP in yeast Saccharomyces cerevisiae and compare its functional properties with mouse Mdr3 P-gp.
- To investigate the roles of MRP and P-gp in cellular resistance to various compounds and in yeast mating.
Main Methods:
- Expression of human MRP and mouse Mdr3 in the yeast mutant VASY2563.
- Assessing cellular resistance to Adriamycin, FK506, and valinomycin in transformed yeast.
- Measuring intracellular accumulation of [14C]Adriamycin in spheroplasts.
- Evaluating the complementation of the yeast peptide pheromone transporter Ste6 activity.
Main Results:
- Expression of MRP and Mdr3 restored Adriamycin resistance in the VASY2563 mutant.
- Transformed yeast exhibited pleiotropic drug resistance, including to FK506 and valinomycin.
- Reduced intracellular Adriamycin accumulation and active efflux were observed in MRP and Mdr3 transformants.
- Human MRP complemented the Ste6 transporter function, partially restoring mating in a ste6 null mutant.
Conclusions:
- Despite low structural homology, MRP and P-gp share functional similarities in substrate transport.
- Both transporters can mediate the efflux of chemotherapeutic drugs and yeast mating pheromones.
- Yeast serves as a valuable model system for studying the functional aspects of mammalian transporters like MRP and P-gp.