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

氨基酸转运活性系统x_C^-是一个不依赖于Na~+的系统，对胱氨酸和谷氨酸具有高度专一性。在细胞培养系统中，半胱氨酸的摄取通常是维持细胞内谷胱甘肽水平的重要途径，因为半胱氨酸和/或半胱氨酸是谷胱甘肽合成的限速前体氨基酸。在培养过程中，这种转运活性可被巯基反应剂马来酸二乙酯诱导。我们从这些巨噬细胞中制备了mRNA，并将其注射到非洲爪哇卵母细胞中，证明了x_C^-系统的表达，即一个Na~+非依赖的、谷氨酸可抑制的胱氨酸运输系统。表达的半胱氨酸转运活性依赖于测定温度，37ﾟC下测得的半胱氨酸摄取量是20ﾟC时的数倍。注射不同大小的核糖核酸表明，小鼠巨噬细胞的x_C^-转运蛋白系统由1.5-2.9kb的基因编码。使用差异杂交技术，我们分离了数百个似乎是在马来酸二乙酯处理下诱导的巨噬细胞中的cDNA克隆。我们正在对这些选定的cDNA克隆进行检测，以期找到编码胱氨酸转运蛋白的cDNA克隆。

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.