RUI: Constitutive and Regulated Trafficking of the Cystine/Glutamate Exchanger, System xc-

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This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Intellectual merit. System xc- is a cell transport system that allows for the exchange of cystine, an amino acid, on the outside of the cell for glutamate, an amino acid and neurotransmitter, on the inside in of the cell in a variety of cell types. The transporter is found in the brain, retina, kidney and small intestine. System xc- has been shown to play an important role in regulating the production of the antioxidant, glutathione, and thus protecting cells from damage as a result of oxidative stress. The principal investigator (PI) and her students have recently shown that oxidants such as hydrogen peroxide appear to acutely (within minutes) regulate System xc- by redistributing the transporter from the inside of the cell to the cell membrane where it is active. These exciting findings suggest a novel form of regulation of System xc- that may serve as an important component of the cellular defense system in protecting cells from oxidative damage and may serve as a model for the regulation of other transport systems. To confirm and extend these findings, this project will use biochemical and cell biological techniques to elucidate the mechanism by which System xc- activity is regulated by insertion and removal from the membrane (membrane trafficking). Specifically, the experiments outlined in this project will allow for the identification of the membrane trafficking pathways of the protein components of System xc- under basal conditions in cultured human cells. The research will also investigate the mechanism by which hydrogen peroxide acutely regulates the cell membrane expression and activity of System xc-. Finally, this project will examine the basal and regulated trafficking of System xc- in distinctly different cell types in order to determine if the acute regulation of System xc- by oxidants is common to a variety of cells. Thus, when completed, this project will lead to significant advancements in our fundamental understanding of the basal and regulated trafficking of System xc-. Since previous studies of this transporter have focused only on the production of the transporter, which is under genetic control, this study will provide insight into a novel form of regulation of System xc- which allows for acute modulation of transporter activity. The fact that oxidants appear to acutely regulate this cystine/glutamate exchanger is intriguing since the transporter provides cells with the cystine required to synthesize the antioxidant glutathione. Therefore, these studies may reveal an important early defense mechanism utilized by cells immediately after exposure to an oxidative agent. Broader impacts. In addition to improving our understanding of the regulation of System xc-, this project provides research training for nine undergraduate students and six high school students, most of whom will be from groups underrepresented in science. The majority of the research proposed in this project will be completed by these students as a part of their comprehensive education in the sciences. Students will work collaboratively in teams on the proposed project side-by-side with the PI, learning to develop hypotheses, to design and conduct experiments, to analyze data, and to report their findings. In the PI's experience (41 students mentored in 8 years), engagement in original research projects leads to significant gains in students' understanding of the scientific process, ability to think critically about difficult and complex problems, and ability to communicate about science. Thus, this project will train students who will be better prepared for future careers in science. Additionally, this research experience could inspire the PI's students to pursue scientific careers, and thus, to contribute to increasing the diversity of individuals in science. The impact of this project will be extended by publishing the scientific findings in journals such as the Journal of Biological Chemistry and by presentations made at national scientific meetings such as Experimental Biology or the Annual Society for Neuroscience Meeting.