FUNCTIONAL EXPRESSION OF P-GLYCOPROTEIN ENCODED BY THE MOUSE MDR3 GENE IN YEAST-CELLS

We have expressed P-glycoprotein (P-gp) encoded by the mouse mdr3 gene in the yeast Saccharomyces cerevisiae and have developed an experimen tal protocol to isolate and purify inside-out plasma membrane vesicles (IOVs) from these cells. Biochemical characterization of IOVs from co ntrol and P-gp-expressing cells isolated by this procedure show that t hey are greatly enriched for plasma membrane markers, are tightly seal ed, and are competent for D-glucose transport. P-gp expression in thes e vesicles results in the appearance of a specific ATP-dependent and t emperature-sensitive transport of the drugs colchicine and vinblastine that is osmotically sensitive. P-gp-mediated drug transport into thes e IOVs is inhibited by a known P-gp modulator, verapamil, and can be a brogated by prior incubation of the IOVs with an anti-P-gp antibody. A Ser-939 --> Phe mutation within the predicted transmembrane domain 11 of P-gp, which is known to modulate its function in mammalian cells, drastically reduces drug transport in IOVs obtained from yeast cells e xpressing the mutant protein. The successful demonstration of active d rug transport into IOVs from P-gp-expressing yeast cells indicates tha t P-gp can mediate both chemotherapeutic drugs and a-pheromone transpo rt in yeast cells.