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 experimental protocol to isolate and purify inside-out plasma membrane vesicles (IOVs) from these cells. Biochemical characterization of IOVs from control and P-gp-expressing cells isolated by this procedure show that they are greatly enriched for plasma membrane markers, are tightly sealed, and are competent for D-glucose transport. P-gp expression in these vesicles results in the appearance of a specific ATP-dependent and temperature-sensitive transport of the drugs colchicine and vinblastine that is osmotically sensitive. P-gp-mediated drug transport into these IOVs is inhibited by a known P-gp modulator, verapamil, and can be abrogated 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 expressing the mutant protein. The successful demonstration of active drug transport into IOVs from P-gp-expressing yeast cells indicates that P-gp can mediate both chemotherapeutic drugs and a-pheromone transport in yeast cells.
Ruetz
S
S
Department of Biochemistry, McGill University, Montreal, PQ, Canada.
Raymond
M
M
Gros
P
P
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D001693:Biological Transport, Active; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D003078:Colchicine / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics*; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D051379:Mice / Q000235:genetics*; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013816:Thermodynamics; D014747:Vinblastine / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D014747:Vinblastine; D017301:Ca(2+) Mg(2+)-ATPase; D005947:Glucose; D003078:Colchicine