Yeast cells synthesize a typically eukaryotic mixture of phospholipids, using pathways and enzymes that are largely similar to those in higher eukaryotes, ensuring that insights generated using this model system will be relevant to human metabolism and health. Experiments are proposed that exploit the rapid and dramatic changes that occur in the synthesis and turnover of phospholipids, sphingolipids and storage lipids when inositol, a precursor of phospholipids in eukaryotes, is added or removed from the growth medium of actively dividing yeast cells. Many of the lipids that respond to inositol availability i yeast are implicated in stress response signaling in eukaryotic cells. Rapid changes occurring in such lipids in response to abrupt changes in inositol availability will be correlated to changes in signaling and gene expression. This strategy will be used to define the specific signaling roles of the lipids; phosphatidic acid, diacylglycerol, and phosphatidylinositol 4- phosphate and sphingolipids in the endoplasmic reticulum and the plasma membrane. Phosphatidic acid not only serves as a signaling lipid but is also a key precursor of phospholipids essential for membrane biogenesis. Alternatively, phosphatidic acid serves as precursor via dephosphorylation for the production of diacylgycerol, which in turn serves as a precursor in the synthesis of both phospholipids and triacylglycerol, a major component of lipid droplets. The metabolic regulation controlling the synthesis of triacylglycerol is highly relevant to understanding factors underlying human health related conditions, such as chronic obesity. In yeast cells starved for inositol, phospholipid synthesis decreases and triacylglycerol levels increase, mimicking metabolic changes leading to obesity. Experiments designed to probe the mechanism of this important regulation are proposed. Lipids and metabolites derived from them have been implicated in many of the complex signaling pathways that regulate membrane biogenesis, cell growth and proliferation in higher eukaryotes, including humans. However, the specific roles of individual lipids and related metabolites in these complex cellular processes are often obscured by the complexity of the metabolism involved and by the fact that many potential signaling lipids serve as precursors to other such lipids. Moreover, many of the signals generated during ongoing lipid metabolism are transient and the correct identification of the specific signaling molecule may depend on being able to measure it accurately and observe its flux in the context of active metabolism. The experiments described in this proposal exploit the advantages of the yeast model system and the rapidity of changes in correlated lipid metabolism and signaling to overcome such barriers.
An understanding of the regulation of lipid metabolism is critical in dealing with chronic conditions such as obesity, diabetes, and atherosclerosis. Yeast is an excellent model system for such studies since the relevant pathways are common to yeast and mammals and the major enzymes are homologous, ensuring that insights derived from yeast will generate knowledge relevant to human health.
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