Abstract 1733 The ubiquitination of System xc- impacts the protein's abundance, turnover rate, and cell surface localization

System xc-imports cystine and exports glutamate.Its cell surface localization has been shown to increase in cultured cells acutely following brief oxidative insult.Ubiquitin, a small protein, is directly involved in the trafficking and degradation of numerous proteins within cells and has been shown to bind to System xc-.Moreover, upon oxidative insult, cellular protein ubiquitination increases as a result of inhibition of deubiquitinases.However, it is not understood how ubiquitination of the transporter impacts its abundance, localization, and activity.Therefore, the objective of this project is to directly assess how ubiquitination affects System xc-abundance, turnover rate, and cell surface localization in the context of oxidative stress.We expressed System xc-(heterodimer consisting of xCT and 4F2HC) in COS7 cells transfected with increasing levels of ubiquitin (1X, 2X or 4X) and observed a decrease in xCT expression and an increase in xCT turnover rate.When cells are transfected with xCT, 4F2HC, and ubiquitin (2X) and fixed 24 hours later, we see that xCT and ubiquitin are co-localized primarily to the membrane as observed by ICC.However, when we co-express xCT with ubiquitin (1X, 2X or 4X) for 48 hours, we observe an increase in transporter degradation quantified with Western blot.This may suggest that ubiquitination regulates the transporter in a complex way, potentially reflecting the difference between xCT monoubiquitination and polyubiquitination.Based on these preliminary results, we have developed a working model that ubiquitination of xCT has two distinct effects: 1) stabilizing of xCT on the plasma membrane and 2) targeting xCT for degradation.Ultimately, this work will allow us to better understand the mechanisms by which System xc-activity and cell surface localization is regulated under oxidative stress.