Regulation of DGK1-encoded diacylglycerol kinase upon resumption of growth from stationary phase in Saccharomyces cerevisiae.

摘要

Studies in our laboratory are focused on the mobilization of triacylglycerol for phospholipid synthesis when yeast resumes growth from the stationary phase. This metabolic process includes the hydrolysis of triacylglycerol by lipase enzymes to generate free fatty acid and diacylglycerol. Fatty acids can be incorporated via multiple steps into the phospholipid precursor phosphatidic acid. Recent studies in our laboratory have uncovered a novel enzyme that can convert diacylglycerol and CTP into phosphatidic acid. This enzyme, known as diacylglycerol kinase, is encoded by the DGK1 gene. In this study, we addressed the hypothesis that diacylglycerol kinase plays an important role in growth resumption from stationary phase. Experimentally, wild type yeast and yeast without the DGK1 gene (e.g., dgk1Delta mutant) were first grown to the stationary phase, and then cultured in fresh medium to allow for growth resumption. The fatty acid synthesis inhibitor cerulenin was included in the medium to accentuate the need for triacylglycerol mobilization. While wild type yeast resumed growth (measured spectrophotometrically) from the stationary phase, the dgk1Delta mutant cells did not resume growth. If dgk1Delta mutant cells expressed the wild type DGK1 gene on a plasmid, cells resumed growth from the stationary phase. However, if dgk1Delta cells expressed the catalytic dead version of DGK1 (D177A), cells did not resume growth. These data indicated that growth resumption required diacylglycerol kinase activity. In wild type cells, diacylglycerol kinase protein (measured by immunoblotting with anti-diacylglycerol kinase antibodies) and activity (measured by following the incorporation of radioactive CTP into diacylglycerol to form phosphatidic acid) increased as cells resumed growth. This work advanced our understanding of the metabolic processes involved in the mobilization of triacylglycerol for membrane phospholipid synthesis and growth resumption of stationary phase yeast.