Interactions among pathways for phosphatidylcholine metabolism, CTP synthesis and secretion through the Golgi apparatus.

Phosphatidylcholine is the major phospholipid in eukaryotic cells. It serves as a structural component of cell membranes and a reservoir of several lipid messengers. Recent studies in yeast and mammalian systems have revealed interrelationships between the two pathways of phosphatidylcholine metabolism, and between these pathways and those for CTP synthesis and secretion via the Golgi. These processes involve the regulation of the CDP-choline and phosphatidylethanolamine-methylation pathways of phosphatidylcholine synthesis, CTP synthetase, phospholipase D and the phospholipid-transfer protein Sec14p.

The membrane-associated enzyme phosphatidylserine synthase is regulated at the level of mRNA abundance.

To precisely define the functional sequence of the CHO1 gene from Saccharomyces cerevisiae, encoding the regulated membrane-associated enzyme phosphatidylserine synthase (PSS), we subcloned the original 4.5-kilobase (kb) CHO1 clone. In this report a 2.8-kb subclone was shown to complement the ethanolamine-choline auxotrophy and to repair the defect in the synthesis of phosphatidylserine, both of which are characteristic of cho1 mutants. When this subclone was used as a hybridization probe of Northern and slot blots of RNA from wild-type cells, the abundance of a 1.2-kb RNA changed in response to alterations in the levels of the soluble phospholipid precursors inositol and choline. The addition of inositol led to a 40% repression of the 1.2-kb RNA level, while the addition of choline and inositol led to an 85% repression. Choline alone had little repressive effect. The level of 1.2-kb RNA closely paralleled the level of PSS activity found in the same cells as determined by enzyme assays. Disruption of the CHO1 gene resulted in the simultaneous disappearance of 1.2-kb RNA and PSS activity. Cells bearing the ino2 or ino4 regulatory mutations, which exhibit constitutively repressed levels of a number of phospholipid biosynthetic enzymes, had constitutively repressed levels of 1.2-kb RNA and PSS activity. Another regulatory mutation, opi1, which causes the constitutive derepression of PSS and other phospholipid biosynthetic enzymes, caused the constitutive derepression of the 1.2-kb RNA. When cho1 mutant cells were transformed with the 2.8-kb subclone on a single-copy plasmid, the 1.2-kb RNA and PSS activity levels were regulated in a wild-type fashion. The presence of the 2.8-kb subclone on a multicopy plasmid, however, led to the constitutive overproduction of 1.2-kb RNA and PSS in cho1 cells.

Phospholipid biosynthesis in the yeast Saccharomyces cerevisiae and interrelationship with other metabolic processes.

In this review, we have discussed recent progress in the study of the regulation that controls phospholipid metabolism in S. cerevisiae. This regulation occurs on multiple levels and is tightly integrated with a large number of other cellular processes and related metabolic and signal transduction pathways. Progress in deciphering this complex regulation has been very rapid in the last few years, aided by the availability of the sequence of the entire Saccharomyces genome. The assignment of functions to the remaining unassigned open reading frames, as well as ascertainment of remaining gene-enzyme relationships in phospholipid biosynthesis in yeast, promises to provide detailed understanding of the genetic regulation of a crucial area of metabolism in a key eukaryotic model system. Since the processes of lipid metabolism, secretion, and signal transduction show fundamental similarities in all eukaryotes, the dissection of this regulation in yeast promises to have wide application to our understanding of metabolic control in all eukaryotes.

Phosphatidate phosphatases and diacylglycerol pyrophosphate phosphatases in Saccharomyces cerevisiae and Escherichia coli.

Phosphatidate phosphatase plays a major role in the synthesis of phospholipids and triacylglycerols in the yeast Saccharomyces cerevisiae. Membrane- and cytosolic-associated forms of the enzyme have been isolated and characterized. These enzymes are Mg2+-dependent and N-ethylmaleimide-sensitive. The expression of a membrane-associated form of phosphatidate phosphatase is regulated by growth phase and inositol supplementation, whereas enzyme activity is regulated by lipids, nucleotides, and by phosphorylation. Phosphatidate phosphatase is coordinately regulated with other phospholipid biosynthetic enzymes including phosphatidylserine synthase. Diacylglycerol pyrophosphate phosphatase is a novel enzyme of phospholipid metabolism which is present in S. cerevisiae, Escherichia coli, and mammalian cells. This enzyme possesses a phosphatidate phosphatase activity which is Mg2+-independent and N-ethylmaleimide-insensitive and is distinct from the Mg2+-dependent and N-ethylmaleimide-sensitive form of phosphatidate phosphatase. Genes encoding for diacylglycerol pyrophosphate phosphatase have been isolated from S. cerevisiae and E. coli. The deduced protein sequences of these genes show homology to the sequence of the mouse PAP2 (Mg2+-independent and N-ethylmaleimide-insensitive phosphatidate phosphatase) protein, especially in a novel phosphatase sequence motif. Rat liver PAP2 displays diacylglycerol pyrophosphate phosphatase activity.

Phosphatidylethanolamine methyltransferase and phospholipid methyltransferase activities from Saccharomyces cerevisiae. Enzymological and kinetic properties.

In the yeast Saccharomyces cerevisiae, two membrane-associated enzymes catalyze the three-step methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Phosphatidylethanolamine methyltransferase (PEMT) catalyzes the first methylation reactions (PE----phosphatidylmonomethylethanolamine (PMME] and phospholipid methyltransferase (PLMT) catalyzes the second two methylation reactions (PMME----phosphatidyldimethylethanolamine (PDME)----PC). Using gene disruption mutants of the S. cerevisiae OP13 and CHO2 genes, we independently studied the enzymological properties of microsome-associated PEMT and PLMT, respectively. The enzymological properties of the enzymes differed with respect to their pH optima, cofactor requirements and thermal lability. For the PEMT reactions, the apparent Km values for PE and S-Adenosylmethionine (AdoMet) were 57 microM and 110 microM, respectively. For the PLMT reactions, the apparent Km values for PMME and PDME were 380 microM and 180 microM, respectively. The apparent Km values for AdoMet were 54 microM and 59 microM with PMME and PDME as substrates, respectively. S-Adenosylhomocysteine (AdoHcy) was a competitive inhibitor of PEMT (Ki = 12 microM) and PLMT (Ki = 57 microM and Ki = 54 microM for PMME and PDME, respectively) with respect to AdoMet. AdoHcy was a noncompetitive inhibitor of PEMT (Ki = 160 microM) and PLMT (Ki = 120 microM) with respect to PE and PMME and PDME, respectively.

Enzymological properties of the LPP1-encoded lipid phosphatase from Saccharomyces cerevisiae.

The product of the LPP1 gene in Saccharomyces cerevisiae is a membrane-associated enzyme that catalyzes the Mg(2+)-independent dephosphorylation of phosphatidate (PA), diacylglycerol pyrophosphate (DGPP), and lysophosphatidate (LPA). The LPP1-encoded lipid phosphatase was overexpressed 681-fold in Sf-9 insect cells and used to examine the enzymological properties of the enzyme using PA, DGPP, and LPA as substrates. The optimum pH values for PA phosphatase, DGPP phosphatase, and LPA phosphatase activities were 7. 5, 7.0, and 7.0, respectively. Divalent cations (Mn(2+), Co(2+), and Ca(2+)), NaF, heavy metals, propranolol, phenylglyoxal, and N-ethylmaleimide inhibited the PA phosphatase, DGPP phosphatase, and LPA phosphatase activities of the enzyme. The inhibitory effects of N-ethylmaleimide and phenylglyoxal on the LPP1-encoded enzyme were novel properties when compared with other Mg(2+)-independent lipid phosphate phosphatases from S. cerevisiae and mammalian cells. The LPP1-encoded enzyme exhibited saturation kinetics with respect to the surface concentrations of PA (K(m)=0.05 mol%), DGPP (K(m)=0.07 mol%), and LPA (K(m)=0.08 mol%). Based on specificity constants (V(max)/K(m)LPA (1.3 units/mg/mol%). DGPP (K(i)=0.12 mol%) was a competitive inhibitor with respect to PA, and PA (K(i)=0.12 mol%) was a competitive inhibitor with respect to DGPP. This suggested that the binding sites for these substrates were the same. The enzymological properties of the LPP1-encoded enzyme differed significantly from those of the S. cerevisiae DPP1-encoded lipid phosphatase, a related enzyme that also utilizes PA, DGPP, and LPA as substrates.

Interactions of thiophosphatidic acid with enzymes which metabolize phosphatidic acid. Inhibition of phosphatidic acid phosphatase and utilization by CDP-diacylglycerol synthase.

Thiophosphatidic acid (1,2-diacyl-sn-glycero-3-phosphorothioate; thioPA) was chemically synthesized from egg phosphatidylcholine-derived 1,2-diacylglycerol and PSCl3 and tested for its effects on enzymes which utilize phosphatidic acid (PA) in phospholipid biosynthesis. The compound was not a substrate for rat liver cytosolic PA phosphatase and strongly inhibited this enzyme activity. ThioPA was also a potent inhibitor of purified membrane-associated PA phosphatase from Saccharomyces cerevisiae in a competitive manner and exhibited an apparent Ki = 60 microM. In contrast, purified CDPdiacylglycerol synthase (PA:CTP cytidylyltransferase) from this organism was able to convert thioPA to CDP-diacylglycerol. The apparent Vmax for thioPA was 7-fold lower than that for PA, whereas the apparent Km for thioPA (70 microM) was 4-fold lower than that for PA. Calculation of the specificity constant (Vmax/Km) demonstrated that PA was the preferred substrate. These properties of thioPA indicate that this substance may prove useful in studies of phospholipid metabolism and function.

Regulation of phosphatidylethanolamine methyltransferase and phospholipid methyltransferase by phospholipid precursors in Saccharomyces cerevisiae.

Phosphatidylethanolamine methyltransferase (PEMT) and phospholipid methyltransferase (PLMT), which are encoded by the CHO2 and OPI3 genes, respectively, catalyze the three-step methylation of phosphatidylethanolamine to phosphatidylcholine in Saccharomyces cerevisiae. Regulation of PEMT and PLMT as well as CHO2 mRNA and OPI3 mRNA abundance was examined in S. cerevisiae cells supplemented with phospholipid precursors. The addition of choline to inositol-containing growth medium repressed the levels of CHO2 mRNA and OPI3 mRNA abundance in wild-type cells. The major effect on the levels of the CHO2 mRNA and OPI3 mRNA occurred in response to inositol. Regulation was also examined in cho2 and opi3 mutants, which are defective in PEMT and PLMT activities, respectively. These mutants can synthesize phosphatidylcholine when they are supplemented with choline by the CDP-choline-based pathway but they are not auxotrophic for choline. CHO2 mRNA and OPI3 mRNA were regulated by inositol plus choline in opi3 and cho2 mutants, respectively. However, there was no regulation in response to inositol when the mutants were not supplemented with choline. This analysis showed that the regulation of CHO2 mRNA and OPI3 mRNA abundance by inositol required phosphatidylcholine synthesis by the CDP-choline-based pathway. The regulation of CHO2 mRNA and OPI3 mRNA abundance generally correlated with the activities of PEMT and PLMT, respectively. CDP-diacylglycerol synthase and phosphatidylserine synthase, which are regulated by inositol in wild-type cells, were examined in the cho2 and opi3 mutants. Phosphatidylcholine synthesis was not required for the regulation of CDP-diacylglycerol synthase and phosphatidylserine synthase by inositol.

Identification of a novel phosphatase sequence motif.

We have identified a novel, conserved phosphatase sequence motif, KXXXXXXRP-(X12-54)-PSGH-(X31-54)-SRXXXXX HXXXD, that is shared among several lipid phosphatases, the mammalian glucose-6-phosphatases, and a collection of bacterial nonspecific acid phosphatases. This sequence was also found in the vanadium-containing chloroperoxidase of Curvularia inaequalis. Several lines of evidence support this phosphatase motif identification. Crystal structure data on chloroperoxidase revealed that all three domains are in close proximity and several of the conserved residues are involved in the binding of the cofactor, vanadate, a compound structurally similar to phosphate. Structure-function analysis of the human glucose-6-phosphatase has shown that two of the conserved residues (the first domain arginine and the central domain histidine) are essential for enzyme activity. This conserved sequence motif was used to identify nine additional putative phosphatases from sequence databases, one of which has been determined to be a lipid phosphatase in yeast.

Regulation of phospholipid synthesis in yeast by zinc.

The yeast Saccharomyces cerevisiae has the ability to cope with a variety of stress conditions (e.g. zinc deficiency) by regulating the expression of enzyme activities including those involved with phospholipid synthesis. Zinc is an essential mineral required for the growth and metabolism of S. cerevisiae. Depletion of zinc from the growth medium of wild-type cells results in alterations in phospholipid composition including an increase in PI (phosphatidylinositol) and a decrease in phosphatidylethanolamine. These changes can be attributed to an increase in PIS1-encoded PI synthase activity and a decrease in the activities of several CDP-diacylglycerol pathway enzymes including the CHO1-encoded PS (phosphatidylserine) synthase. The reduction in PS synthase in response to zinc depletion is due to a repression mechanism that involves the UAS(INO) (inositol upstream activating sequence) element in the CHO1 promoter and the negative transcription factor Opi1p. These factors are also responsible for the inositol-mediated repression of CHO1. This regulation may play an important role in allowing cells to adapt to zinc deficiency given the essential roles that phospholipids play in the structure and function of cellular membranes.