System x_c⁻ Regulation: Effects of Mutations on Membrane Expression and Ubiquitination Status

Abstract

System xc− exchanges intracellular glutamate for extracellular cystine across the membrane of many cell types, including astrocytes. Its activity directly regulates the synthesis of the antioxidant glutathione and the extracellular concentration of glutamate in some areas of the brain. Dysregulation of the transporter can lead to excessive glutamate release and excitotoxic cell death or the depletion of glutathione stores and the development of oxidative stress. We recently demonstrated that oxidants acutely upregulate System xc− activity by triggering the rapid redistribution of the transporter from intracellular compartments to the cell surface. Our current work suggests that the trafficking of the transporter may be regulated by ubiquitination and that oxidant exposure directly influences the ubiquitination of the transporter. Since increased ubiquitination tends to decrease the cell surface expression of many membrane transporters, we sought to test the hypothesis that System xc− is ubiquitinated and that the ubiquitination status of the transporter regulates both its cell surface expression and activity. We have used a mutagenesis approach to disrupt putative ubiquitination sites and a putative ubiquitin ligase binding site within a myc‐tagged System xc− construct so that we can understand the role ubiquitination plays in regulating the cell surface expression of System xc−. There are seven highly conserved lysine residues within xCT that are located on the cytoplasmic side of the membrane. These residues are located at positions 4, 37, 41, 43, 422, 472, and 473. We have created mutant forms of this construct containing single or multiple lysine to arginine mutations so that we could assess the effect of these mutations on cell surface expression of System xc‐. Using biotinylation assays and immunocytochemical analysis, we have demonstrated that mutation of the N‐terminal lysine residues increases the cell surface expression of the transporter. We are currently assessing the ubiquitination status of these mutant transporters to determine if the changes in ubiquitination of the transporter are associated with changes in the cell surface expression and activity of the transporter. In addition, we have identified a putative ubiquitin ligase binding site in the C‐terminus of the transporter. Disruption of this binding site also leads to an increase in cell surface expression of the transporter. Collectively, these data suggest that System xc− is regulated by changes in its ubiquitination status such that factors which lead to diminished ubiquitination will allow for increased cell surface expression of the transporter.Support or Funding InformationThis work was supported by RUI 0843564.