Real Time Trafficking of System xc– in COS-7 cells
(Meeting Abstract Supplement) 1017.8
Abstract
The cystine/glutamate exchanger, System xc–, is comprised of two proteins, xCT and CD98 and is known to play a significant role in the protective response of cells to oxidative insult. We have previously shown that System xc– activity is rapidly upregulated in the presence of oxidative agents as a result of an increase in trafficking of xCT to the plasma membrane, without an equivalent increase in CD98 membrane localization. Since CD98 has been suggested to be involved in the trafficking xCT, we sought out to monitor the association between xCT and CD98 during this trafficking event. To address this question, we have created three constructs: xCT-GFP, GFP-xCT, and mCherry-CD98 and expressed them in COS-7 cells. Addition of the GFP tag to either the N-terminus or the C-terminus of xCT does not disrupt the basal trafficking of xCT in COS-7 cells, as 34% of the GFP tagged xCT proteins are localized to the membrane under basal conditions, similar to endogenously expressed xCT. Using live cell fluorescence microscopy, we have observed that while some of GFP-xCT (or xCT-GFP) is co-localized with mCherry-CD98 on the membrane and within vesicular pools within the cytoplasm, distinct pools of intracellular mCherry-CD98 and GFP-xCT do not co-localize. We are currently monitoring the real time trafficking of xCT-GFP and mCherry-CD98 in response to oxidant exposure. This research is supported by NSF-RUI # 0843564.
