Does Mutation of a Putative Nedd 4-2 Binding Site Within the C-terminus of xCT Inhibit its Ubiquitination?

Faculty Mentor(s)

Dr. Leah Chase

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Glutamatergic synapses require precise chemical control in the surrounding space. System xc- is a membrane antiporter that regulates extracellular glutamate and intracellular cysteine in glial cells, ultimately regulating glutathione production. Glutathione is an endogenous reducing agent for mitigating oxidative stress. Thus, regulation of System xc- is important for regulation of glutamatergic signaling and the oxidative state of the glial cells. Previous studies in Dr. Chase’s lab have demonstrated that System xc- is regulated by its trafficking to and from the cell surface in response to oxidative stress. In addition, Dr. Chase and her students have also shown that xCT undergoes a change in ubiquitination state following exposure to hydrogen peroxide. While the specific factors which regulate the trafficking of the transporter are not well understood, we have identified several amino acid motifs in the carboxyl terminus that appear to regulate the trafficking of xCT. Specifically, we observed that mutating residues in a 462PAYYLFI468 motif to alanine led to an increase in cell surface expression of xCT. Studies of an epithelial sodium channel have shown that a similar amino acid motif serves as a binding site for the ubiquitinating enzyme, Nedd 4-2. Since loss of protein ubiquitination is known to increase the cell surface expression of other membrane proteins, we set out to determine if the mutants which increase cell surface expression of xCT also decreased the ubiquitination level of xCT. We used pulldown assays to determine if these mutations inhibited xCT ubiquitination. Specifically, COS-7 cells were transfected with either wild type or mutant xCT and His-ubiquitinated proteins were purified. The presence of ubiquitinated xCT was detected using western blot analysis. Once this study is completed, we will have a better understanding of the role the PAYYLFI motif plays in the regulation of cell surface expression and ubiquitination of xCT.

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