The basic amino acid transporter (system y+) is the primary mechanism for cellular uptake of arginine and lysine; it is the first mammalian amino acid transporter to be cloned, and the first to be expressed and studied by electrophysiological methods in Xenopus oocytes. The y+ molecule also functions as the receptor that mediates attachment and infection of ecotropic host-range murine retroviruses that produce leukemia.
The specific aims of this proposal involve structure-function studies on the y+ molecule and isolation and characterization of genes related to it.
Aim 1 is to express y+ in oocytes and to determine its transport functions using voltage clamp methods, both with microelectrodes and with the 'cut- open' oocyte technique. A biophysical of model time-, voltage-, and concentration-dependence of substrate uptake and efflux will be developed.
Aim 2 is to clone and express genes encoding other transporters. Using a partial-length cDNA encoding the y+-related gene Tea, the full length cDNA will be cloned and expressed, and its transport functions characterized. Other genes homologous to y+ will be identified by screening cDNA libraries and similarly tested. Another strategy for isolating amino acid transporter genes will utilize an oocyte expression cloning scheme to screen for expression of radiolabeled substrate uptake.
Aim 3 involves structure-function studies on the system y+ protein; comparisons with related transporters will allow chimera construction and site-directed mutagenesis to map functional domains in the y+ molecule.
In Aim 4 studies will be done to analyze the structural features of y+ important for its role in infection and in the natural resistance of non-murine species. Site-directed mutants and chimeric molecules made in aim 3 will be expressed in mammalian cells and analyzed for abilities to bind virus and to mediate infection. The results will be of significance in cell biology because they provide information about a critical class of transporter molecules, in virology because they will suggest how retroviruses invade cells, and in medicine because they promise to provide new understanding of common inherited diseases that result from abnormal amino acid transport.
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