No abstract

The regulation of phosphatidylcholine degradation as a function of the route of phosphatidylcholine (PC) synthesis and changing environmental conditions has been investigated in the yeast Saccharomyces cerevisiae. In the wild-type strains studied, deacylation of phosphatidylcholine to glycerophosphocholine is induced when choline is supplied to the culture medium and, also, when the culture temperature is raised from 30 to 37°C. In strains bearing mutations in any of the genes encoding enzymes of the CDP-choline pathway for phosphatidylcholine biosynthesis (CKI1, choline kinase; CPT1, 1, 2-diacylglycerol choline phosphotransferase; PCT1, CTP:phosphocholine cytidylyltransferase), no induction of phosphatidylcholine turnover and glycerophosphocholine production is seen in response to choline availability or elevated temperature. In contrast, the induction of phosphatidylcholine deacylation does occur in a strain bearing mutations in genes encoding enzymes of the methylation pathway for phosphatidylcholine biosynthesis (i.e. CHO2/PEM1 and OPI3/PEM2). Whereas the synthesis of PC via CDP-choline is accelerated when shifted from 30 to 37°C, synthesis of PC via the methylation pathway is largely unaffected by the temperature shift. These results suggest that the deacylation of PC to GroPC requires an active CDP-choline pathway for PC biosynthesis but not an active methylation pathway. Furthermore, the data indicate that the synthesis and turnover of CDP-choline-derived PC, but not methylation pathway-derived PC, are accelerated by the stress of elevated temperature.

In yeast, mutations in the CDP-choline pathway for phosphatidylcholine biosynthesis permit the cell to grow even when the SEC14 gene is completely deleted (Cleves, A., McGee, T., Whitters, E., Champion, K., Aitken, J., Dowhan, W., Goebl, M., and Bankaitis, V. (1991) Cell 64, 789 -800). We report that strains carrying mutations in the CDP-choline pathway, such as cki1, exhibit a choline excretion phenotype due to production of choline during normal turnover of phosphatidylcholine. Cells carrying cki1 in combination with sec14 ts , a temperature-sensitive allele in the gene encoding the phosphatidylinositol/phosphatidylcholine transporter, have a dramatically increased choline excretion phenotype when grown at the sec14 ts -restrictive temperature. We show that the increased choline excretion in sec14 ts cki1 cells is due to increased turnover of phosphatidylcholine via a mechanism consistent with phospholipase D-mediated turnover. We propose that the elevated rate of phosphatidylcholine turnover in sec14 ts cki1 cells provides the metabolic condition that permits the secretory pathway to function when Sec14p is inactivated.As phosphatidylcholine turnover increases in sec14 ts cki1 cells shifted to the restrictive temperature, the INO1 gene (encoding inositol-1-phosphate synthase) is also derepressed, leading to an inositol excretion phenotype (Opi ؊ ). Misregulation of the INO1 gene has been observed in many strains with altered phospholipid metabolism, and the relationship between phosphatidylcholine turnover and regulation of INO1 and other coregulated genes of phospholipid biosynthesis is discussed.

The SEC14 gene encodes a phosphatidylinositol/phosphatidylcholine transfer protein essential for secretion and growth in yeast (1). Mutations (cki1, cct1, and cpt1) in the CDP-choline pathway for phosphatidylcholine synthesis suppress the sec14 growth defect (2), permitting sec14 ts cki1, sec14 ts cct1, and sec14 ts cpt1 strains to grow at the sec14 ts restrictive temperature. Previously, we reported that these double mutant strains also excrete the phospholipid metabolites, choline and inositol (3). We now report that these choline and inositol excretion phenotypes are eliminated when the SPO14 (PLD1) gene encoding phospholipase D1 is deleted. In contrast to sec14 ts cki1 strains, sec14 ts cki1 pld1 strains are not viable at the sec14 ts restrictive temperature and exhibit a pattern of invertase secretion comparable with sec14 ts strains. Thus, the PLD1 gene product appears to play an essential role in the suppression of the sec14 ts defect by CDP-choline pathway mutations, indicating a role for phospholipase D1 in growth and secretion. Furthermore, sec14 ts strains exhibit elevated Ca 2؉ -independent, phophatidylinositol 4,5-bisphosphate-stimulated phospholipase D activity. We also propose that phospholipase D1-mediated phosphatidylcholine turnover generates a signal that activates transcription of INO1, the structural gene for inositol 1-phosphate synthase.

To study the consequences of depleting the major membrane phospholipid phosphatidylcholine (PC), exponentially growing cells of a yeast cho2opi3 double deletion mutant were transferred from medium containing choline to cholinefree medium. Cell growth did not cease until the PC level had dropped below 2% of total phospholipids after four to five generations. Increasing contents of phosphatidylethanolamine (PE) and phosphatidylinositol made up for the loss of PC. During PC depletion, the remaining PC was subject to acyl chain remodeling with monounsaturated species replacing diunsaturated species, as shown by mass spectrometry. The remodeling of PC did not require turnover by the SPO14-encoded phospholipase D. The changes in the PC species profile were found to reflect an overall shift in the cellular acyl chain composition that exhibited a 40% increase in the ratio of C16 over C18 acyl chains, and a 10% increase in the degree of saturation. The shift was stronger in the phospholipid than in the neutral lipid fraction and strongest in the species profile of PE. The shortening and increased saturation of the PE acyl chains were shown to decrease the nonbilayer propensity of PE. The results point to a regulatory mechanism in yeast that maintains intrinsic membrane curvature in an optimal range. INTRODUCTIONPhosphatidylcholine (PC) is an abundant glycerophospholipid present in the membranes of eukaryotic cells. Apart from being a major structural component of all organellar membranes, it serves as a reservoir of signaling molecules (Exton, 1994;Kent and Carman, 1999), and it has been implicated in apoptosis (Cui and Houweling, 2002). In the model eukaryote Saccharomyces cerevisiae, mutations in the genes encoding PC biosynthetic enzymes lead to respiratory deficiency (Griac et al., 1996), indicating that PC is important for mitochondrial function. PC was found to interact with Gut2p, the mitochondrial glycerol-3-phosphate dehydrogenase, in a photolabeling study (Janssen et al., 2002). Furthermore, the biosynthesis of PC is involved in the regulation of intracellular vesicle trafficking in yeast (reviewed in Howe and McMaster, 2001).The triple methylation of phosphatidylethanolamine (PE), catalyzed by the methyltransferases Cho2p (Pem1p) and Opi3p (Pem2p), is the primary route for the synthesis of PC in yeast in the absence of exogenous choline (Carman and Henry, 1999). When choline is supplied in the growth medium, the CDP-choline pathway contributes to the net synthesis of PC (Figure 1). However, also in the absence of choline, the CDP-choline pathway contributes to PC synthesis using (phospho)choline derived from the turnover of PC (McMaster and Bell, 1994). Electrospray ionization tandem mass spectrometry (ESI-MS/MS) in combination with stable isotope labeling revealed that the two biosynthetic routes produce the PC molecular species, i.e., PC molecules with specific acyl chains, in different ratios (Boumann et al., 2003).Whereas the biosynthesis of PC and its regulation have been extensively characterized (Carman...

Mutations in the Saccharomyces cerevisiae SNF1 gene affect a number of cellular processes, including the expression of genes involved in carbon source utilization and phospholipid biosynthesis. To identify targets of the Snf1 kinase that modulate expression of INO1, a gene required for an early, rate-limiting step in phospholipid biosynthesis, we performed a genetic selection for suppressors of the inositol auxotrophy of snf1⌬ strains. We identified mutations in ACC1 and FAS1, two genes important for fatty acid biosynthesis in yeast; ACC1 encodes acetyl coenzyme A carboxylase (Acc1), and FAS1 encodes the ␤ subunit of fatty acid synthase. Acc1 was shown previously to be phosphorylated and inactivated by Snf1. Here we show that snf1⌬ strains with increased Acc1 activity exhibit decreased INO1 transcription. Strains carrying the ACC1 suppressor mutation have reduced Acc1 activity in vitro and in vivo, as revealed by enzymatic assays and increased sensitivity to the Acc1-specific inhibitor soraphen A. Moreover, a reduction in Acc1 activity, caused by addition of soraphen A, provision of exogenous fatty acid, or conditional expression of ACC1, suppresses the inositol auxotrophy of snf1⌬ strains. Together, these findings indicate that the inositol auxotrophy of snf1⌬ strains arises in part from elevated Acc1 activity and that a reduction in this activity restores INO1 expression in these strains. These results reveal a Snf1-dependent connection between fatty acid production and phospholipid biosynthesis, identify Acc1 as a Snf1 target important for INO1 transcription, and suggest models in which metabolites that are generated or utilized during fatty acid biosynthesis can significantly influence gene expression in yeast.

No abstract

The highly conserved yeast cell wall integrity mitogen-activated protein kinase pathway regulates cellular responses to cell wall and membrane stress. We report that this pathway is activated and essential for viability under growth conditions that alter both the abundance and pattern of synthesis and turnover of membrane phospholipids, particularly phosphatidylinositol and phosphatidylcholine. Mutants defective in this pathway exhibit a choline-sensitive inositol auxotrophy, yet fully derepress INO1 and other Opi1p-regulated genes when grown in the absence of inositol. Under these growth conditions, Mpk1p is transiently activated by phosphorylation and stimulates the transcription of known targets of Mpk1p signaling, including genes regulated by the Rlm1p transcription factor. mpk1⌬ cells also exhibit severe defects in lipid metabolism, including an abnormal accumulation of phosphatidylcholine, diacylglycerol, triacylglycerol, and free sterols, as well as aberrant turnover of phosphatidylcholine. Overexpression of the NTE1 phospholipase B gene suppresses the choline-sensitive inositol auxotrophy of mpk1⌬ cells, whereas overexpression of other phospholipase genes has no effect on this phenotype. These results indicate that an intact cell wall integrity pathway is required for maintaining proper lipid homeostasis in yeast, especially when cells are grown in the absence of inositol.