Molecular identification of cystine / glutamate exchanger

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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.