Structural and functional analysis of a cystine/glutamate-specific carrier protein of plasma membrane

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The amino acid transport activity designated System x_C^- is a Na^+-independent system and is highly specific for cystine and glutamate. In cell culture systems, the uptake of cystine mediated by this system is usually important in maintaining intracellular glutathione levels, because cystine and/or cysteine is the rate limiting precursor amino acid of glutathione synthesis. This transport activity can be induced in mouse peritoneal macrophages during culture by diethylmaleate, a sulfhydryl-reactive agent. We prepared mRNA from those macrophages and injected into Xenopus oocytes and demonstrated the expression of System x_C^-, i. e., a Na^+-independent, glutamate-inhibitable cystine transport system. The expressed cystine transport activity depended on the assay temperature, in that cystine uptake measured at 37ﾟC was several-fold higher than that measured at 20ﾟC. Injection of size- fractionated mRNA indicated that the System x_C^--transporter of the mouse macrophage is encoded by mRNA of 1.5 to 2.9 kb.In the next step, we constructed a lambdaZAPII cDNA library from the macrophage mRNA. Using a differential hybridization technique, we have isolated hundreds of cDNA clones which appeared to be induced under diethylmaleate treatment in macrophages. We are examining those selected cDNA clones expecting to find out a cDNA clone encoding cystine transporter.