Identification of Trafficking Motifs Involved in Constitutive and Regulated Trafficking of System xc-
Dr. Leah Chase, Hope College
System xc-, a sodium independent plasma membrane transport system that exchanges intracellular glutamate for extracellular cystine, has been shown to constitutively traffic to and from the plasma membrane, enabling a rapid response to oxidative stress via regulated trafficking. 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 mechanisms of these transporters. Thus, in an attempt to further elucidate the mechanism(s) by which system xc- traffics, this study sought to identify amino acids of xCT, the function-specific protein of system xc-, that are involved in its trafficking. Carboxyl-terminal truncations and point mutants of xCT were constructed in a FLAG-tagged cDNA vector and were transfected into PC12 cells, a rat pheochromocytoma cell line. Sites for mutation were selected based on known trafficking motifs of other proteins that traffic. The trafficking behavior of the mutant constructs was assayed using both fluorescence microscopy and a biotinylation protocol in which a membrane impermeable biotin was used to tag and separate membrane-localized proteins from intracellular proteins. Loss of the carboxyl-terminus appears to significantly reduce cell surface expression. Preliminary analysis of the point mutants suggests that we may have identified putative membrane trafficking motifs.
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