Mammalian cells contain several distinct transport systems for maintaining their levels of neutral amino acides. These internal levels are maintained by balancing the transport activity and the biosynthesis with the cellular requirements for protein synthesis and energy metabolism. The long-rang goal of this project is the under standing of the molecular basis of active transport of amino acids in mammalian cells. These amion acid transport systems have been characterized in Chinese hamster ovary (CHO) cells, and they include the Na+-dependent A, ASC, and P Systems, as well as the Na+-independent L System. We are applying genetic approaches and recombinant DNA techniques in an attempt to clone the human genes responsible for amion acid transport. The strategy being used is to obtain transport mutants in CHO cells, and then complement these mutants by transferring human DNA sequences that code for the amino acid transport activity. Using genetic approaches, CHO mutants have been isolated that are defective in system L transport and its regulation. These mutants are being characterized and used for identifying human transport genes. The transfer of human DNA will be carried out be either cell fusion techniques or direct transformation with a human genomic cosmid library. The human transport genes will be rescued from the transformants by in vitro packaging techniques. To identify the transpot components, we will attempt to increase their expression by gene amplification of the transport genes, and then sperating and identifying the proteins by two-dimensional gel electrophoresis and by fluorescent reagent labeling. We also plan to raise anti-idiotypic antibodies to the transport receptors to aid in the identification of the transporters.
