Saccharomyces cerevisiae mutant with a partial defect in the synthesis of CDP-diacylglycerol and altered regulation of phospholipid biosynthesis

et al. 2004

In the yeast Saccharomyces cerevisiae, the most abundant phospholipid phosphatidylcholine is synthesized by the complementary CDP-diacylglycerol and Kennedy pathways. Using a cki1⌬ eki1⌬ mutant defective in choline kinase and ethanolamine kinase, we examined the consequences of a block in the Kennedy pathway on the regulation of phosphatidylcholine synthesis by the CDP-diacylglycerol pathway. The cki1⌬ eki1⌬ mutant exhibited increases in the synthesis of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine via the CDP-diacylglycerol pathway. The increase in phospholipid synthesis correlated with increased activity levels of the CDPdiacylglycerol pathway enzymes phosphatidylserine synthase, phosphatidylserine decarboxylase, phosphatidylethanolamine methyltransferase, and phospholipid methyltransferase. However, other enzyme activities, including phosphatidylinositol synthase and phosphatidate phosphatase, were not affected in the cki1⌬ eki1⌬ mutant. For phosphatidylserine synthase, the enzyme catalyzing the committed step in the pathway, activity was regulated by increases in the levels of mRNA and protein. Decay analysis of CHO1 mRNA indicated that a dramatic increase in transcript stability was a major component responsible for the elevated level of phosphatidylserine synthase. These results revealed a novel mechanism that controls phospholipid synthesis in yeast.PC 1 is the most abundant phospholipid in the membranes of eukaryotic cells (1-4). It serves as a structural component of the membrane and as a source of bioactive lipids (e.g. lyso-PC, PA, DAG) (1-5). The importance of PC is underscored by the fact that alterations in its metabolism are linked to apoptosis (6 -9) and oncogenic transformation (10 -12). In the model eukaryote Saccharomyces cerevisiae, PC is synthesized by complementary pathways that are common to those found in mammalian cells 2 ( Fig. 1) (1,4,(13)(14)(15)(16)(17). In the CDP-DAG pathway, PC is synthesized from CDP-DAG via the reactions catalyzed by PS synthase (18 -20), PS decarboxylase (21-23), PE methyltransferase (24, 25), and phospholipid methyltransferase (24,26). In the CDP-choline branch of the Kennedy pathway, PC is synthesized from choline via the reactions catalyzed by choline kinase (27), phosphocholine cytidylyltransferase (28), and choline phosphotransferase (29,30). Analyses of mutations in S. cerevisiae (4, 31, 32), as well as in mammalian cells (33,34) indicate that the physiological role(s) of PC synthesized by the two pathways are different.The contribution of the CDP-DAG and Kennedy pathways for PC synthesis in wild type S. cerevisiae is dependent on the exogenous supply of choline (35). When grown in the presence of choline, yeast primarily synthesizes PC via the Kennedy pathway (35). On the other hand, when cells are grown in the absence of choline, PC is primarily synthesized via the CDP-DAG pathway (35). Yet, even under this growth condition, the Kennedy pathway still contributes to the synthesis of PC (36 -41). The choline required for the Kenne...

Section: Discussionmentioning

confidence: 99%

Regulation of Phospholipid Synthesis in the Yeast cki1Δ eki1Δ Mutant Defective in the Kennedy Pathway

Choi

Sreenivas

et al. 2004

“…The addition of inositol to the growth medium of wild type cells causes an increase in the level of PI and a decrease in the levels of PS, PE, and PC (28,52). The decreased levels of PS, PE, and PC are primarily the result of a repression mechanism that involves the positive transcription factors Ino2p and Ino4p, the negative transcription factor Opi1p, and a UAS INO element found in the promoter of genes (i.e.…”

Section: Discussionmentioning

confidence: 99%

“…Preparation of Cell Extracts and Protein Determination-Cell extracts were prepared as described previously (52). Cells were suspended in 50 mM Tris-maleate buffer, pH 7.0, containing 1 mM EDTA, 0.3 M sucrose, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 g/ml aprotinin, 5 g/ml leupeptin, and 5 g/ml pepstatin.…”

Section: Methodsmentioning

confidence: 99%

Regulation of the PIS1-encoded Phosphatidylinositol Synthase in Saccharomyces cerevisiae by Zinc

Iwanyshyn

et al. 2005

279, 21976 -21983).We examined the effects of zinc depletion on the regulation of the PIS1-encoded phosphatidylinositol synthase, the enzyme that catalyzes the formation of phosphatidylinositol from CDP-diacylglycerol and inositol. Phosphatidylinositol synthase activity increased when zinc was depleted from the growth medium. Analysis of a zrt1⌬ zrt2⌬ mutant defective in plasma membrane zinc transport indicated that the cytoplasmic levels of zinc were responsible for the regulation of phosphatidylinositol synthase. PIS1 mRNA, its encoded protein Pis1p, and the ␤-galactosidase activity driven by the P PIS1 -lacZ reporter gene were elevated in zinc-depleted cells. This indicated that the increase in phosphatidylinositol synthase activity was the result of a transcriptional mechanism. The zinc-mediated induction of the P PIS1 -lacZ reporter gene, Pis1p, and phosphatidylinositol synthase activity was lost in zap1⌬ mutant cells. These data indicated that the regulation of PIS1 gene expression by zinc depletion was mediated by the zinc-regulated transcription factor Zap1p. Direct interaction between glutathione S-transferase (GST)-Zap1p 687-880 and a putative upstream activating sequence (UAS) zinc-responsive element in the PIS1 promoter was demonstrated by electrophoretic mobility shift assays. Mutations in the UAS zinc-responsive element in the PIS1 promoter abolished the GSTZap1p 687-880 -DNA interaction in vitro and abolished the zinc-mediated regulation of the PIS1 gene in vivo. This work advances understanding of phospholipid synthesis regulation by zinc and the transcription control of the PIS1 gene. Phosphatidylinositol (PI)1 is the third most abundant phospholipid in the cellular membranes of the yeast Saccharomyces cerevisiae (1-3), and it is essential for the growth and metabolism of this model eukaryote (4 -6). In addition to being a major structural component of the membrane, PI serves as the precursor for sphingolipids (7,8), the D-3, D-4, and D-5 phosphoinositides (3, 9 -12), and glycosylphosphatidylinositol anchors (13, 14) (Fig. 1). Several of these PI-derived lipids and their metabolic products are prominent signaling molecules in S. cerevisiae and in higher eukaryotes that contribute to essential physiological functions (12,(15)(16)(17)(18)(19)(20).The enzyme responsible for the synthesis of PI in S. cerevisiae is the essential PIS1-encoded PI synthase (CDP-diacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) (6, 21-23). This endoplasmic reticulum-associated (24) enzyme catalyzes the formation of PI and CMP from CDP-diacylglycerol and inositol (25) (Fig. 1). The regulation of PI synthase activity in vivo is largely governed by the availability of its substrates inositol and CDP-diacylglycerol (26 -28). Cellular inositol levels are controlled by expression of the INO1 gene encoding inositol-3-phosphate synthase and by inositol supplementation (26 -28). The levels of CDP-diacylglycerol are controlled through its utilization by the PI synthase enzyme itself and the competing activity of ...

“…PS synthase activity was measured in cell extracts derived from inositol-supplemented cells that were grown in the presence and absence of zinc. As described previously (67,103), the addition of inositol to zinc-containing growth medium resulted in a 57% reduction in PS synthase activity (Fig. 6).…”

Section: Effect Of Inositol On the Zinc-mediated Regulation Of Ps Synmentioning

confidence: 99%

“…Preparation of Cell Extracts and Protein Determination-Cell extracts were prepared as described previously (67,68). Cells were suspended in 50 mM Tris maleate buffer (pH 7.0) containing 1 mM EDTA, 0.3 M sucrose, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 g/ml aprotinin, 5 g/ml leupeptin, and 5 g/ml pepstatin.…”

Section: Methodsmentioning

confidence: 99%

Regulation of Phospholipid Synthesis in Saccharomyces cerevisiae by Zinc

Iwanyshyn

Carman

2004

156

Zinc is an essential nutrient required for the growth and metabolism of eukaryotic cells. In this work, we examined the effects of zinc depletion on the regulation of phospholipid synthesis in the yeast Saccharomyces cerevisiae. Zinc depletion resulted in a decrease in the activity levels of the CDP-diacylglycerol pathway enzymes phosphatidylserine synthase, phosphatidylserine decarboxylase, phosphatidylethanolamine methyltransferase, and phospholipid methyltransferase. In contrast, the activity of phosphatidylinositol synthase was elevated in response to zinc depletion. The level of Aut7p, a marker for the induction of autophagy, was also elevated in zinc-depleted cells. For the CHO1-encoded phosphatidylserine synthase, the reduction in activity in response to zinc depletion was controlled at the level of transcription. This regulation was mediated through the UAS INO element and by the transcription factors Ino2p, Ino4p, and Opi1p that are responsible for the inositol-mediated regulation of UAS INO -containing genes involved in phospholipid synthesis. Analysis of the cellular composition of the major membrane phospholipids showed that zinc depletion resulted in a 66% decrease in phosphatidylethanolamine and a 29% increase in phosphatidylinositol. A zrt1⌬ zrt2⌬ mutant (defective in the plasma membrane zinc transporters Zrt1p and Zrt2p) grown in the presence of zinc exhibited a phospholipid composition similar to that of wild type cells depleted for zinc. These results indicated that a decrease in the cytoplasmic levels of zinc was responsible for the alterations in phospholipid composition.