Monitoring the kinetics and performance of yeast membrane ABC transporters by diS-C3(3) fluorescence.
Kinetic features (initial start-up phase, drug pumping velocity and efficiency as dependent on drug concentration and growth phase) of yeast plasma membrane multidrug resistance ABC pumps were studied by monitoring the uptake of the fluorescent potentiometric dye diS-C3(3), which has been found to be expelled from the cells by these pumps. The monitoring was done with Saccharomyces cerevisiae mutants AD1-8 and AD1-3 deleted in different ABC pumps, and in their pump-competent parent strain US50-18C overexpressing transcriptional activators Pdr1p and Pdr3p. On addition to the cells, diS-C3(3) is expelled by the Pdr5p, Yor1p and Snq2p pumps with overlapping substrate specificity. The pump action can be assessed as a difference between the dye uptake curve for pump-competent and pump-deleted cells. The pump-mediated dye efflux, which shows an initial lag of various lengths, maintains a certain residual intracellular dye level. In the absence of external glucose the dye efflux ability of the pumps depends on the growth phase; late exponential and stationary cells can maintain the export for tens of minutes, whereas exponential cells keep up the pump action for limited time periods. This may reflect an insufficient number of pump molecules in the membrane or an effect of insufficient pump energization from endogenous sources. This effect is not mediated by changes in membrane potential because lowered membrane potential caused by inhibition of the plasma membrane H+-ATPase does not affect the pump action.
Gásková
D
D
Institute of Physics, Charles University, Ke Karlovu 5, Prague, Czech Republic. gaskova@karlov.mff.cuni.cz
Cadek
R
R
Chaloupka
R
R
Vacata
V
V
Gebel
J
J
Sigler
K
K
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D002232:Carbocyanines / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
C091791:3,3'-dipropylthiacarbocyanine; D018528:ATP-Binding Cassette Transporters; D002232:Carbocyanines; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D008565:Membrane Proteins; C092438:PDR5 protein, S cerevisiae; C101797:SNQ2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C097077:YOR1 protein, S cerevisiae