It is a general principle of biochemistry that most proteins perform their cellular functions with high specificity. For instance, cells that respond to a particular hormone such as testosterone make a specific protein receptor. The binding of testosterone to this protein triggers a set of developmental events that result in major cellular changes. The testosterone receptor, however, will not interact significantly with other steroid hormones or growth factors when they are present in their normal cellular concentrations. In vivid contrast to the general behavior of most proteins, the family of ABC (ATP-binding cassette) multidrug transporters which are found in all organisms use chemical energy to "pump" many chemically distinct toxic compounds (xenobiotic drugs) out of the cell before they cause damage. These proteins, therefore, are highly non-specific and they give cells a modest resistance to literally thousands of xenobiotic drugs. These molecular pumps achieve this unusual capability by having a very large drug-binding pocket with multiple drug binding domains (unlike the testosterone receptor protein which generally has a single hormone binding site). In this work, the PI seeks to understand the arrangement of these drug-binding regions in the major multidrug transporter Pdr5. This protein is found in the membrane surrounding yeast cells, a superior model organism. Although yeast are easy to grow and are relatively simple to manipulate biochemically and genetically, the have a basic cell structure much like any higher organism (and proteins, such as Pdr5, are highly conserved and found in most eukaryotes, including humans). Students in the laboratory observed that by exposing cells to normally lethal concentrations of toxic compounds, mutations in Pdr5 appear that create even greater resistance to subsets of xenobiotic drugs that Pdr5 removes. Some of these mutations are, as expected, changes in the drug binding pocket and they will help identify which regions are critical for the transport of a particular group of drugs. Surprisingly, however, additional alterations are located in other regions of the very large Pdr5 protein. During the course of this research, a variety of genetic and biochemical approaches will be used to understand how these mutations increase drug resistance even though some are far removed from the actual drug- binding sites.
Broader Impact The easy, but extremely powerful genetic and biochemical methods that are used in this research makes it a superior training ground not only for graduate students, but also for undergraduates. Over 100 undergraduates have carried out independent research using Pdr5 as have about a dozen high school students. Eight of these students (seven undergraduates and one high school student) have published their results in prestigious journals such as The Journal of Biological Chemistry and Biochemistry. The vast majority of these students have gone on to graduate studies. During the present funding period, a postdoctoral fellow will be hired specifically to do research and to learn how to teach and mentor undergraduates.
