title: Complete inhibition of the Pdr5p multidrug efflux pump ATPase activity by its transport substrate clotrimazole suggests that GTP as well as ATP may be used as an energy source.
abstract: The yeast Pdr5p transporter is a 160 kDa protein that effluxes a large variety of xenobiotic compounds. In this study, we characterize its ATPase activity and demonstrate that it has biochemical features reminiscent of those of other ATP-binding cassette multidrug transporters: a relatively high Km for ATP (1.9 mM), inhibition by orthovanadate, and the ability to specifically bind an azidoATP analogue at the nucleotide-binding domains. Pdr5p-specific ATPase activity shows complete, concentration-dependent inhibition by clotrimazole, which is also known to be a potent transport substrate. Our results indicate, however, that this inhibition is noncompetitive and caused by the interaction of clotrimazole with the transporter at a site that is distinct from the ATP-binding domains. Curiously, Pdr5p-mediated transport of clotrimazole continues at intracellular concentrations of substrate that should eliminate all ATPase activity. Significantly, however, we observed that the Pdr5p has GTPase and UTPase activities that are relatively resistant to clotrimazole. Furthermore, the Km(GTPase) roughly matches the intracellular concentrations of the nucleotide reported for yeast. Using purified plasma membrane vesicles, we demonstrate that Pdr5p can use GTP to fuel substrate transport. We propose that Pdr5p increases its multidrug transport substrate specificity by using more than one nucleotide as an energy source.
authors.affiliation: Department of Biology, The Catholic University of America, Washington, DC 20064, USA.
authors.forename: John
authors.initials: J
authors.lastname: Golin
authors.forename: Zachary N
authors.initials: ZN
authors.lastname: Kon
authors.forename: Chung-Pu
authors.initials: CP
authors.lastname: Wu
authors.forename: Justin
authors.initials: J
authors.lastname: Martello
authors.forename: Leanne
authors.initials: L
authors.lastname: Hanson
authors.forename: Sherry
authors.initials: S
authors.lastname: Supernavage
authors.forename: Suresh V
authors.initials: SV
authors.lastname: Ambudkar
authors.forename: Zuben E
authors.initials: ZE
authors.lastname: Sauna
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels; D001386:Azides; D002701:Chloramphenicol / Q000378:metabolism; D003022:Clotrimazole / Q000378:metabolism / Q000494:pharmacology*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors*; D014316:Tritium
chemical_list: D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels; D001386:Azides; D007093:Imidazoles; C092438:PDR5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014316:Tritium; C017040:8-azidoadenosine 5'-triphosphate; D002701:Chloramphenicol; C029899:imidazole; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D020558:GTP Phosphohydrolases; D003022:Clotrimazole