Physiology of Osmotic Stress in Saccharmyces cerevisiae

描述（由申请人提供）：适应渗透胁迫的能力是所有生物的基本特性。系统发育多样的生物体通过积累渗透物来适应渗透胁迫，渗透物稳定细胞成分，防止细胞水分流失。细胞对渗透应激的适应对于包括人类在内的高等生物的正常肾细胞功能也很重要。例如，已知哺乳动物肾细胞在抗利尿过程中积累甘油酰胆碱（GPC）、甘氨酸甜菜碱、山梨醇、肌醇和尿素。酿酒酵母已被用作研究渗透胁迫生理反应（主要是甘油合成和信号转导）的模型。本研究的长期目标是揭示酵母中低分子量渗透物的功能和代谢。初步数据表明，高盐胁迫诱导磷脂酰胆碱（PC）快速转化生成GPC。此外，这些事件可能在缺乏从头蛋白合成的情况下发生，并依赖于Nte1（神经病变靶向酯酶）。Nte1的结构特征表明其活性受蛋白激酶A （PKA）的磷酸化调控。

DESCRIPTION (provided by applicant): 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.