Identification of Trafficking Motifs Involved in Constitutive and Regulated Trafficking of System xc−

Abstract

System xc−, a membrane transport system that exchanges glutamate for cystine, has been shown to traffic to and from the plasma membrane, enabling a rapid response to oxidative stress. Trafficking is a common form of regulation among transport proteins, and various amino acid motifs have been shown to play integral roles in the trafficking of these transporters. This study sought to identify amino acids within xCT that are involved in its trafficking. Carboxyl‐terminal truncations and point mutants of xCT were constructed in a FLAG‐tagged cDNA vector and transfected into PC12 cells. The trafficking behavior of the mutant constructs was assayed using both fluorescence microscopy and a biotinylation protocol to separate cell surface proteins from intracellular proteins. Preliminary analysis of the data suggests 1) loss of the carboxyl‐terminus results in significantly reduced cell surface expression of xCT and 2) disruption of a consensus phosphorylation site leads to greater membrane localization of xCT, providing a mechanism for rapid regulation of transporter trafficking and activity. This research was supported by the Arnold and Mable Beckman Foundation and NSF RUI # 0843564.