Real Time Analysis of Protein xCT in System xc-

Faculty Mentor(s)

Dr. Leah Chase, Hope College

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System xc- is a plasma transport system that catalyzes the stoichiometric exchange of extracellular cystine for intracellular glutamate, which is then reduced to glutathione. Glutathione, an antioxidant, neutralizes reactive oxidant species (ROS) formed as a byproduct of cellular respiration. An imbalance of ROS in a cell causes oxidative stress, which can lead to cell death. Protein xCT functions in the unit of System xc- and preliminary data show oxidants increase the trafficking of xCT to the cellular membrane. Understanding how this trafficking event happens in real time will answer questions of how quickly cells begin to respond to an oxidative assault and how this response is mediated. To answer this question a fusion protein of GFP-xCT was transfected and is stably expressed in U138MG cells. Live cell imaging reveals xCT localized in intracellular vesicles in a non­oxidative stress treatment and shows traffick to the membrane when H2O2 is introduced to the cell media. Additionally Western blot analysis shows this construct protein trafficks the same as the native protein.

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