Identification of Amino Acid Residues in System xc- that are Important in Regulating its Cell Surface Expression
Dr. Leah Chase
System xCT is an antiporter crucial for the production of glutathione by controlling cellular levels of cysteine, the limiting reagent in glutathione synthesis (Kim, et al., 2001). Glutathione is a key component in the antioxidant cascade that exists to protect neurons and glia from oxidative stress (Kim, et al., 2001). Previous research in the Chase laboratory found that membrane levels of xCT increase 2- to 3-fold in response to oxidative stress. Therefore, we sought to determine the events which regulate cell surface expression of xCT. Studies of other membrane proteins have demonstrated that their cell surface expression and trafficking are controlled through the ubiquitination of lysine residues on the membrane-bound protein (Traub and Lukacs, 2007). Therefore, in our study, we induced lysine to arginine mutations on the seven intracellular xCT lysine residues in order to determine whether these residues are ubiquitinated in vivo and whether these residues are involved in xCT trafficking. We performed biotinylation assays and immunocytochemistry on transfected cells to measure the membrane and cellular expressions of mutated xCT proteins. Ubiquitination assays were also performed. Since only lysine and not arginine residues can be ubiquitinated, we hypothesize that the mutation of key lysine residues to arginine will result in increased membrane xCT levels and a reduction in xCT ubiquitination. Furthermore, we hypothesize that this mutation will only affect membrane trafficking and not overall cellular expression of xCT.
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