GENETIC ANALYSIS OF PLEIOTROPIC DRUG RESISTANCE

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Description (Adapted from the abstract). Multiple drug resistance refers to the acquisition of broad range of resistance phenotypes through genetic changes at a small number of loci. Multidrug resistance is a clinical problem in chemotherapeutic treatment of tumors and infectious disease. The applicant is studying pleiotropic drug resistance (Pdr) in Saccharomyces cerevisiae as a model of eukaryotic multiple drug resistance. Previous work has demonstrated tha the zinc finger transcription factor Pdr1p is a major contributor to the ability of cells to tolerate a range of otherwise toxic compounds. Pdr1p carries this function through transcriptional activation of several ATP binding cassette transporter encoding genes like PDR5 and YOR1. A gene that encodes a Hsp70 homolog was recently cloned that regulates the activity of Pdr1p. This Hsp70 homologue (Pdr13p) can up-regulate Pdr1p function and thereby increase expression of Pdr1p target genes and associated drug resistance. The goal of this proposal is to understand the molecular details behind Pdr13p modulation of Pdr1p activity. Antisera has been prepared against both Pdr13p and Pdr1p. The plan is use this sera to localize these factors within the cell to determine if these proteins are likely to directly interact. Along with its effect on Pdr1p, Pdr13p has other protein targets. These other target proteins will be identified using two hybrid sand co-immunoprecipitation approaches. An important functional domain in a Hsp70 protein is its ATPase domain. It is proposed to explore the role of Pdr13p-dependent ATPas activity in its biological function through construction and assay of mutations that are predicted ot lack this enzymatic function. The regions(s) of Pdr1p that are required to receive the positive regulatory signal from Pdr13p will be mapped by deletion mutagenesis of the Pdr1p coding sequence. The finding that the function of a Hsp70 protein is required for normal drug resistance has provided a unique opportunity to analyze the action of an eukaryotic Hsp70 in a genetically tractable organism. Additionally, continue progress in understanding the control of Pdr in S. cerevisiae will provide and important basic model for multidrug resistance in human tumor cells and a direct model for multidrug tolerance in pathogenic fungi like Candida albicans.