The ability to adapt to osmotic stress is an essential property of all living organisms. Phylogenetically diverse organisms adapt to osmotic stress through the accumulation of osmolytes that stabilize cellular components and prevent the loss of cellular water. Cellular adaptation to osmotic stress is also important for proper renal cell function in higher organisms, including humans. For example, mammalian renal cells are known to accumulate glycerophosphocholine (GPC), glycine betaine, sorbitol, inositol and urea during anti-diuresis. The yeast Saccharomyces cerevisiae has been used as a model to study the physiological responses to osmotic stress (principally glycerol synthesis and signal transduction). The long-term goal of the proposed research is to reveal the function and metabolism of low molecular weight osmolytes in yeast. Preliminary data show that hypersaline stress induces the rapid turnover of phosphatidylcholine (PC) to generate GPC. In addition, these events can occur in the absence of de novo protein synthesis and are dependent on Nte1 (neuropathy targeted esterase). Structural features within Nte1 suggest that its activity is regulated by phosphorylation by protein kinase A (PKA). ? ? The central hypothesis of this proposal is that PC deacylation that is coupled to GPC synthesis is regulated by phosphorylation of Nte1 in response to osmotic stress. To test our hypothesis, we will use Saccharomyces cerevisiae as our model microorganism to address the following specific aims: (1) examine the in vivo phenotypes in yeast strains with nte-deletion mutations; (2) examine the effects that amino acid substitutions in Nte1 and other regulatory mutations have on PC turnover/GPC synthesis in vivo, as well as esterase/ phospholipase activity in cell extracts; (3) examine the effects of organophosphate on cell growth, PC turnover/GPC synthesis, and Nte activity; (4) develop collaborative ties with a senior scientist (Dr. George Carman); (5) augment the research capabilities of the PI's laboratory, enhance student training and research opportunities. ? ? Relevance to Public Health: The results from this study are applicable to other areas of research that depend on a greater understanding of PC turnover/GPC synthesis, such as: neuropathy and paralysis by organophosphate pesticides, the role of NTE in embryonic, placental and brain development, synthesis of platelet activating factor and arachidonic acid, and GPC metabolism in individuals suffering from Alzheimers. ? ? ?
