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Ling-Ing Lau, Parameswaran G. Sreekumar, Christine Spee, Ram Kannan, David R. Hinton; Gene and Protein Expression of Two Carrier Proteins for Mitochondrial Glutathione Transport in Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4771.
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Higher cellular glutathione (GSH) level has been associated with resistance to apoptosis in several cell types including retinal pigment epithelium (RPE). The precise contribution of cytosolic GSH (80-85%) versus mitochondrial GSH (10-15%) pools in arresting cell death is not fully delineated. Mitochondria do not possess enzymatic machinery for GSH biosynthesis and rely on GSH import from cytosol via specific carrier proteins. The aim of this study was to characterize the gene and protein expression of two specific mitochondrial GSH carrier proteins in human RPE cells.
Primary cultures of fetal human RPE cells (fRPE) and spontaneously immortalized ARPE-19 cells were used in the study. Mitochondria were isolated from RPE cells using the Mitochondria/Cytosol Fractionation Kit (BioVision Inc., Mountain View, CA) and the purity of mitochondria-enriched fraction was verified by cytochrome c oxidase IV expression. Gene expression of dicarboxylate carrier (DIC; Slc25a10) protein and 2-oxoglutarate carrier (OGC; Slc25a11) protein, was determined by reverse transcription-PCR and real time-PCR. Immunoblot analysis was performed using polyclonal antibodies for DIC and OGC (Abcam, Cambridge, MA). The cellular localization was studied by immunofluorescence staining with confocal microscopy using a mitotracker (MitoTracker Deep Red FM, Invitrogen, Eugene, Oregon).
Expression of DIC and OGC in RPE cells was identified by reverse transcription-PCR and real time-PCR. Western blot analysis revealed the expression of a 31 kDa band for DIC and a 34 kDa band for OGC in RPE mitochondrial and whole cell lysates, verified using positive controls. Immunofluorescence staining showed colocalization of the two carrier proteins with mitotracker in confocal microscopy.
We have shown for the first time expression of two recently identified mitochondrial GSH carrier proteins namely, DIC and OGC, in human RPE cells. Given the importance of mitochondrial GSH in prevention of RPE cell death, this finding may help in devising therapeutic strategies to restore mitochondrial GSH from depletion under pathological conditions.
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