Functional expression of P-glycoproteins in secretory vesicles.
We expressed P-glycoproteins (P-gps) encoded by the three mouse mdr genes in the membranes of secretory vesicles (SV) accumulating in the yeast mutant strain sec 6-4. Expression of the Mdr1 and Mdr3 isoforms in SV membranes caused a significant increased accumulation of the drug vinblastine (VBL) over background levels measured in control SV. The Mdr1/Mdr3-mediated increased drug accumulation could be completely abolished by the P-gp modulator verapamil. By contrast, overexpression of Mdr2 in these vesicles failed to increase intravesicular VBL accumulation over background levels. Mdr3-mediated VBL transport was not affected by changes in the membrane potential, since identical rates of VBL uptake were measured in the presence or absence of the endogenous proton-translocating PMA1 H(+)-ATPase responsible for the strong electrochemical membrane potential across SV membranes. Moreover, in the presence of a delta micro-H+ across the SV membranes (inside positive) of almost 90 mV, we detected in Mdr3-expressing SV an enhanced accumulation of the lipophilic cation and P-gp substrate tetraphenylphosphonium, suggesting that P-gp-mediated uptake of this cation occurs against an intravesicular depolarized membrane. Likewise, VBL transport in Mdr3-expressing SV was not affected by the presence or absence of a steep proton gradient (inside acid) and was independent of any proton movements, excluding a proton synport or antiport mechanism for P-gp-mediated drug transport. Finally, we could demonstrate that colchicine accumulation in Mdr3-expressing SV occurred against a significant substrate concentration gradient, reaching a 7-fold increase in intravesicular colchicine concentration above the extravesicular medium drug concentration. Our studies show that SV isolated from the temperature-sensitive yeast sec 6-4 mutants are an ideal tool to express and to functionally characterize heterologous membrane proteins, in general and P-gps, in particular.
Ruetz
S
S
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Gros
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P
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000378:metabolism; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000502:physiology; D004351:Drug Resistance / Q000235:genetics*; D015870:Gene Expression; D007425:Intracellular Membranes / Q000378:metabolism / Q000502:physiology; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D009550:Nigericin / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D014747:Vinblastine / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D014747:Vinblastine; D006180:Proton-Translocating ATPases; D009550:Nigericin