Molecular Genetic Analysis of Pdr5p, a Major Yeast Multidrug Transporter

Catholic University Of America, Washington DC

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Abstract

DESCRIPTION (provided by applicant): The yeast Pdr5 multidrug efflux pump is a member of the ABC transport family of proteins. It is similar in structure and substrate specificity to those transporters found in pathogenic fungi such as Candida albicans and Cryptococcus neoformans. A major question in the study of all ABC transporters is how signals are communicated between the drug binding sites in the transmembrane domains from which xenobiotic compounds are transported and the nucleotide-binding domains where ATP is hydrolyzed to produce the energy for efflux. Work in our lab during the present funding period has started to define the residues required for interdomain cross talk. In this application for renewal, we propose a combination of genetic and biochemical experiments that should flesh out much of this interface which appears to extend from the N-terminal nucleotide-binding domain through the intracellular loops and into transmembrane-helix 2. We will use a combination of suppressor and site-directed mutations to identify more of the interface. Residues involved in communicating signal and those specifically required for drug binding will be distinguished using several assays including a well-established iodoarylazidoprazosin-binding assay. We will also begin to explore the details of the Pdr5-specific ATPase catalytic cycle as th relationship between drug binding and hydrolysis is poorly understood in this transporter. PUBLIC HEALTH RELEVANCE: Multidrug resistance to chemotherapeutic and microbial agents constitutes a major problem in the treatment of cancer and infectious diseases. Some of this is due to the overexpression of ABC efflux pumps: proteins that use the energy from ATP to drive the transport of a very broad range of xenobiotic compounds from the cell. This proposal will investigate the relationship between drug binding, ATP hydrolysis and drug efflux in the major yeast transporter Pdr5 using bioinformatic, genetic, and biochemical approaches.