Identification of Trafficking Motifs in the C-terminus of the Cystine/Glutamate Exchanger, System xc-
Dr. Leah Chase
System xc- is a heterodimeric plasma membrane transporter, comprised of the proteins xCT and CD98, which facilitates the stoichiometric exchange of extracellular cystine for intracellular glutamate. System xc- serves to protect the cell from oxidative stress, and brief exposure of cells to oxidative agents leads to an increase in trafficking of xCT to the plasma membrane. However, little is known about the mechanisms which govern the constitutive and regulated trafficking of xCT. In the present study, we sought to identify trafficking motifs within the C-terminus of xCT that are important in regulating the delivery and internalization of xCT from the membrane. We used a mutagenesis approach combined with cell surface biotinylation assays to identify regions of the C-terminus that are involved in the constitutive trafficking of xCT. Specifically, using site-directed mutagenesis, we identified a tyrosine based motif between positions 464 -486 containing the sequence YYLFII that contributes to the constitutive trafficking of xCT to the plasma membrane. In addition, we created three truncation mutants at positions 469, 484, and 496 of xCT, and found that an endocytic motif(s) must also exist between position 469 and 484. While there are no canonical endocytic motifs within this region, there is a putative ubiquitination site. Since we have recently demonstrated that xCT is ubiquitinated in Cos-7 cells and ubiquitination of membrane proteins is known to regulate cell surface expression, we are currently using site-directed mutagenesis to test the hypothesis that ubiquitination of xCT in this region leads to the increased rate of internalization of xCT.
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