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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
2R15GM077211-02
Application #
7713234
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2006-04-01
Project End
2012-08-31
Budget Start
2009-09-01
Budget End
2012-08-31
Support Year
2
Fiscal Year
2009
Total Cost
$202,080
Indirect Cost
Name
Catholic University of America
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
041962788
City
Washington
State
DC
Country
United States
Zip Code
20064