Molecular identification of cystine / glutamate exchanger

Cystin转运入细胞是细胞谷胱甘肽生物合成的限速步骤。胱氨酸/谷氨酸交换系统(运输系统x-c)负责这一过程。为了确定x-c系统的分子性质，我们从小鼠组织和培养细胞系中表达了Poly(A)^+RNA，并在非洲爪哇卵母细胞中进行了表达。经马来酸二乙酯处理的小鼠巨噬细胞株J774A.1的Poly(A)^+RNA在卵母细胞中表达时对^&lt；14&gt；C-胱氨酸的摄取率最高。半胱氨酸摄取不依赖于Na+，可被谷氨酸和L-α-氨基己二酸酯(一种x-c系统特异性抑制剂)抑制，但不受天冬氨酸的抑制。通过共表达的谷氨酸转运体EAAC1将谷氨酸预先加载到卵母细胞中，进一步提高了卵母细胞的摄取能力。这些传输特性与x-c系统的特性完全相同。用制备性凝胶电泳法对小鼠巨噬细胞系J774A.1的Poly(A)^+RNA进行了分级，并在卵母细胞中表达。确定了具有最高胱氨酸转运活性的组分。以胱氨酸转运活性最高的Poly(A)^+RNA组分为模板构建了表达文库。文库通过在非洲爪哇卵母细胞中表达和测定~(14)&gt；C-胱氨酸摄取量来筛选。在筛选的14,000个克隆中，我们未能鉴定出阳性克隆。使用其他小鼠巨噬细胞系RAW，我们进行了相同的克隆方法，然而，在进一步筛选的14,000个克隆中，我们没有找到阳性克隆。我们最近通过表达克隆的方法克隆了编码中性氨基酸转运系统L的基因，发现L转运系统由两个亚基组成。这表明存在一组氨基酸转运蛋白，需要额外的亚基才能发挥转运蛋白的功能。我们将为克隆系统x-c转运蛋白寻找可能的调节亚基。

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.