The studies in this proposal will determine the molecular mechanisms that induce a mammalian amino acid transporter gene during the cellular stress of amino acid limitation and the physiological significance of this process. Amino acids are essential nutrients for cell growth and maintenance. Transfer of amino acids across the plasma membrane is mediated by proteins that recognize, bind and transport these amino acids from the extracellular medium into the cell and vice versa. The essential cationic amino acids, arginine and lysine, are mainly transported via the cat-1 (cationic amino acid transporter 1) protein of the Y+ system. Mammalian cells have developed an adaptive response to changes in amino acid availability. When the amino acid supply is limited, protein synthesis decreases and there are increases in catabolism of cellular proteins, amino acid biosynthesis, and amino acid transport across the plasma membrane. Together these responses provide the amino acids that are essential for cell survival. A significant part of this adaptive response is the increased expression of the cat-1 gene. We discovered a novel mechanism used for the synthesis of the cat-1 protein during limited amino acid supply, when global protein synthesis is inhibited. This involves translation initiation via an internal ribosomal entry sequence (IRES), with common features with both viral (IRES-mediated) and bacterial (translation within a leader peptide) protein synthesis pointing to the evolutionary conserved mechanisms in amino acid metabolism. Our hypothesis is that the IRES is activated by the binding of translation initiation factors and IRES-specific proteins (ITAFs) which recruit the ribosome and position it at the correct translation initiation codon. The proposed studies aim to identify these proteins and characterize how they activate the IRES. This is the first report on the regulation of the activity of the IRES by nutrients. ? ? ?
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