FUNCTIONAL EXPRESSION OF P-GLYCOPROTEINS IN SECRETORY VESICLES

We expressed P glycoproteins (P-gps) encoded by the three mouse mdr ge nes in the membranes of secretory vesicles (SV) accumulating in the ye ast 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 M dr1/Mdr3-mediated increased drug accumulation could be completely abol ished by the P gp modulator verapamil. By contrast, overexpression of Mdr2 in these vesicles failed to increase intravesicular VBL accumulat ion over background levels. Mdr3-mediated VBL transport was not affect ed by changes in the membrane potential, since identical rates of VBL uptake were measured in the presence or absence of the endogenous prot on-translocating PMA1 H+-ATPase responsible for the strong electrochem ical membrane potential across SV membranes. Moreover, in the presence of a Delta<(mu)over bar>(H+) across the SV membranes (inside positive ) of almost 90 mV, we detected in Mdr3-expressing SV an enhanced accum ulation of the lipophilic cation and P-gp substrate tetraphenylphospho nium, suggesting that P gp-mediated uptake of this cation occurs again st an intravesicular depolarized membrane. Likewise, VBL transport in Mdr3-expressing SV was not affected by the presence or absence of a st eep proton gradient (inside acid) and was independent of any proton mo vements, excluding a proton synport or antiport mechanism for P-gp-med iated drug transport. Finally, we could demonstrate that colchicine ac cumulation in Mdr3-expressing SV occurred against a significant substr ate concentration gradient, reaching a 7-fold increase in intravesicul ar colchicine concentration above the extravesicular medium drug conce ntration. Our studies show that SV isolated from the temperature-sensi tive yeast sec 6-4 mutants are an ideal tool to express and to functio nally characterize heterologous membrane proteins, in general and P-gp s, in particular.