Identification of Trafficking Proteins that Interact with the C-terminus of System xc-
Dr. Leah Chase
System xc- is a transport system that plays an important role in protecting cells from oxidative stress. Previous studies in our lab have demonstrated that its activity is regulated by oxidants such as hydrogen peroxide. Specifically, oxidants cause System xc- to localize to the plasma membrane where the transporter functions to import cystine, the rate limiting reagent for the synthesis of the antioxidant, glutathione. In the Chase lab, we are interested in understanding the factors that regulate System xc-. Often, the C-terminus of membrane proteins offers binding sites for proteins that regulate trafficking the cell. We hypothesize that a binding site(s) for adaptor protein-2 (AP-2) exists within the C-terminus of System xc-, thus allowing System xc- to be trafficked from the plasma membrane to the endosomes through clathrin-mediated endocytosis. To test this hypothesis, we have expressed the C-terminus of System xc- in bacterial cells. We have developed a purification procedure so that we can use the C-terminus of System xc- in a cellular pull down assay. If the C-terminus of System xc- interacts with AP-2, we should be able to co-purify AP-2 with the C-terminus. Our initial results suggest that higher levels of our C-terminus will be required to test our hypothesis. We are currently working on increasing our yield in our protein purification.
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