Molecular identification of cystine / glutamate exchanger

半胱氨酸转运细胞是细胞谷胱甘肽生物合成的限速步骤。胱氨酸/谷氨酸交换器（运输系统x-c）负责这一点。为了鉴定系统x-c的分子性质，我们在非洲爪蟾卵母细胞中表达了从小鼠组织和培养细胞系中提取的poly (A) ^+RNA。经马来酸二乙酯处理的小鼠巨噬细胞系J774A.1的Poly (A) ^+RNA在卵母细胞中表达时，对^<14> c -胱氨酸的摄取最高。胱氨酸的摄取与Na^+无关，受谷氨酸和l - α -氨基己二酸（一种系统x-c特异性抑制剂）的抑制，但不受天冬氨酸的抑制。通过共表达的谷氨酸转运蛋白EAAC1向卵母细胞预加载谷氨酸，进一步增强了摄取。这些输运特性与系统x-c完全相同。采用制备凝胶电泳法对小鼠巨噬细胞系J774A.1的poly (A) ^+RNA进行大小分离，并在卵母细胞中表达。鉴定出胱氨酸运输活性最高的部分。质粒表达文库由具有胱氨酸转运活性峰值的poly (A) ^+RNA片段构建而成。通过在非洲爪蟾卵母细胞中表达和测量^<14> c -胱氨酸摄取来筛选该文库。在筛选的14000个克隆中，我们无法识别出阳性的cDNA克隆。使用其他小鼠巨噬细胞系RAW，我们执行了相同的克隆方法，然而，我们无法在进一步筛选的14,000个克隆中找到阳性克隆。我们最近通过表达克隆分离到了一个编码中性氨基酸转运系统L的cDNA，发现系统L转运蛋白由两个亚基组成。这表明有一组氨基酸转运蛋白需要额外的亚基才能发挥作用。我们将为克隆系统x-c转运体寻找可能的调控亚基。

Cystinetransportinto cells is the rate-limiting step for cellular glutathione bio-synthesis. Cystine/glutamate exchanger (transport system x-c) is responsible for this. In order to identify molecular nature of system x-c, we have expressed poly (A) ^+RNA extracted from mouse tissues and culture cell lines in Xenopus laevis oocytes. Poly (A) ^+RNA from mouse macrophage cell line J774A.1 which had been treated with diethylmaleate exhibited the highest uptake of ^<14>C-cystine when expressed in oocytes. The cystine uptake was Na^+-independent, inhibited by glutamate and L-alpha-aminoadipate a system x-c specific inhibitor, but not by aspartate. The uptake was further enhanced by preloading oocytes with glutamate via coexpressed glutamate transporter EAAC1. Those characteristics of the transport were exactly the same as those of system x-c. The poly (A) ^+RNA from mouse macrophage cell line J774A.1 was size-fractionated by preparative gel electrophoresis, and each fraction was expressed in oocytes. The fractions which exhibited the highest cystine transport activity were identified. The plasmid expression library was constructed from the poly (A) ^+RNA fractions with peak cystine transport activity. The library was screened by expressing in Xenopus oocytes and measuring ^<14>C-cystine uptake. Out of 14,000 clones screened, we could not indentify positive cDNA clones. Using other mouse macrophage cell line RAW we have performed the same cloning approach, however, we could not find positive clones out of further 14,000 screened. We have recently isolated a cDNA encoding the neutral amino acid transport system L by expression cloning and found that system L transporter is composed of two subunits. This suggests that there are a group of amino acid transporters which requires additional subunits for the transporters to be functional. We are going to search for putative regulatory subunits for the cloning of system x-c transporter.