Regulation of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae.

Phospholipids are key molecules that contribute to the structural definition of cells and that participate in the regulation of cellular processes. Phospholipid metabolism is a major activity that cells engage in throughout their growth. The yeast, Saccharomyces cerevisiae, serves as a model system in which to study the regulation of phospholipid synthesis and its regulation in eucaryotes. Its membranous organelles, the lipids that comprise these membranes, and the phospholipid biosynthetic pathways that generate these membranes typify eucaryotic cells (1, 2). Many of the structural genes encoding for the phospholipid biosynthetic enzymes have been cloned and characterized (Table I) (3–25), and a number of mutations in these genes have been isolated (3, 7–9, 11–13, 17, 23, 26–33). In addition, a number of the phospholipid biosynthetic enzymes have been purified and studied (Table I) (34–44). The characterization of the wild-type and mutant genes, as well as the gene products encoded by these alleles, has significantly advanced our understanding both of phospholipid biosynthesis and of its regulation. Results from these genetic, molecular, and biochemical studies have shown that the regulation of phospholipid synthesis is a complex, highly coordinated process. The mechanisms that govern this regulation mediate the mRNA and protein levels of the biosynthetic enzymes as well as their activity and localization (1, 2, 45). This review summarizes our current understanding of the regulation of phospholipid metabolism in S. cerevisiae with a particular focus on the regulation of the activity of the biosynthetic enzymes. For more comprehensive reviews, the reader is directed to recent articles by Paltauf et al. (2) and Greenberg and Lopes (45).

Phospholipids are key molecules that contribute to the structural definition of cells and that participate in the regulation of cellular processes. Phospholipid metabolism is a major activity that cells engage in throughout their growth. The yeast, Saccharomyces cerevisiae, serves as a model system in which to study the regulation of phospholipid synthesis and its regulation in eucaryotes. Its membranous organelles, the lipids that comprise these membranes, and the phospholipid biosynthetic pathways that generate these membranes typify eucaryotic cells (1,2). Many of the structural genes encoding for the phospholipid biosynthetic enzymes have been cloned and characterized (Table I)  , and a number of mutations in these genes have been isolated (3, 7-9, 11-13, 17, 23, 26 -33). In addition, a number of the phospholipid biosynthetic enzymes have been purified and studied (Table I) (34 -44). The characterization of the wild-type and mutant genes, as well as the gene products encoded by these alleles, has significantly advanced our understanding both of phospholipid biosynthesis and of its regulation. Results from these genetic, molecular, and biochemical studies have shown that the regulation of phospholipid synthesis is a complex, highly coordinated process. The mechanisms that govern this regulation mediate the mRNA and protein levels of the biosynthetic enzymes as well as their activity and localization (1,2,45). This review summarizes our current understanding of the regulation of phospholipid metabolism in S. cerevisiae with a particular focus on the regulation of the activity of the biosynthetic enzymes. For more comprehensive reviews, the reader is directed to recent articles by Paltauf et al. (2) and Greenberg and Lopes (45).

Phospholipid Composition of S. cerevisiae
The major phospholipids found in mitotically growing cells are PC, 1 PE, PI, and PS (2). Phospholipid composition can vary dramatically when culture conditions are altered (2). Examples of this include: inositol supplementation of wild-type cells (46,47); inositol starvation of ino1 mutant cells (48 -50); choline/ethanolamine starvation of cho1 mutant cells (51); fumonisin B 1 supplementation of wild-type cells (52); and glucose starvation of wild-type and respiratory deficient cells (53,54). Although the proportions of the individual phospholipids change with these growth conditions, the average charge of the membrane phospholipids remains relatively constant (2,48). Therefore, mechanisms exist in S. cerevisiae that compensate for changes in the levels of phospholipids of one charge by orchestrating parallel changes in the levels of phospholipids of the opposite charge. The mechanisms that mediate these processes and other aspects of phospholipid metabolism include genetic regulation and biochemical regulation of the phospholipid biosynthetic enzymes.

Phospholipid Biosynthetic Pathways
Phospholipid biosynthesis is a complex process that contains a number of branch points (Fig. 1). PS, PE, and PC are synthesized from PA by the CDP-DG pathway (indicated in Fig. 1 by the color blue), while PE and PC are also synthesized by the Kennedy (CDPcholine and CDP-ethanolamine) pathway (indicated in Fig. 1 by the color red) (1,2,55,56). CDP-DG is also used for the synthesis of other phospholipids, including inositol-containing lipids (phosphoinositides and sphingolipids) and CL. The CDP-DG pathway is used by wild-type cells for the synthesis of PE and PC when they are grown in the absence of ethanolamine or choline (1,2,57,58). The Kennedy pathway assumes a critical role in PC synthesis when the enzymes in the CDP-DG pathway are defective or repressed (1,2,45). Mutants defective in the CDP-DG pathway require choline for growth and synthesize PC via CDP-choline (9 -12, 26, 29, 59, 60). Mutants defective in PS synthase (26,59) and PS decarboxylase (9,60) can also synthesize PC if they are supplemented with ethanolamine. Under these conditions, PE is synthesized from CDP-ethanolamine. The PE may be subsequently methylated by the phospholipid N-methyltransferases to form PC (Fig. 1). It is not clear what the relative contributions of the CDP-DG and Kennedy pathways are to PE and PC synthesis when ethanolamine and/or choline is present in the growth media.
The utilization of the CDP-DG and Kennedy pathways is also regulated by the cellular levels of CTP (61). The elevation of cellular levels of CTP results in a 2-fold increase in the utilization of the Kennedy pathway for PC synthesis. This has been attributed to an increase in substrate availability for the choline-P cytidylyltransferase reaction in the Kennedy pathway and the inhibition of PS synthase activity by CTP in the CDP-DG pathway (61).

Regulation of Phospholipid Biosynthesis
A number of factors regulate phospholipid biosynthesis including inositol, choline, ethanolamine, lipids (e.g. PA and CDP-DG), nucleotides (e.g. ATP and CTP), and growth phase. The regulation of phospholipid biosynthetic enzymes by inositol has been the most extensively characterized (2,45).
Inositol Effects on the CDP-DG and Kennedy Pathways-The addition of inositol to the growth medium of wild-type cells alters phospholipid composition. The level of PI increases while the levels of PS, PE, and PC decrease (46,47). These changes are due in part to repression mechanisms. These mechanisms regulate mRNA and protein levels and/or the activity of the phospholipid biosynthetic enzymes. For example, the activity and/or levels of the CDP-DG pathway enzymes (i.e. CDP-DG synthase (62,63), PS synthase (46, 64 -66), PS decarboxylase (67)(68)(69), and the two phospholipid Nmethyltransferases (46, 67, 70 -73)) are reduced when wild-type cells are supplemented with inositol. In many instances, the repressive effects of inositol are enhanced by the inclusion of ethanolamine or choline in the growth medium. This regulation is absolutely dependent on inositol (1,2,45). Under these growth conditions, the exogenous ethanolamine and choline is used to synthesize PE and PC via the Kennedy pathway (1, 2). The coordinate regulation of the CDP-DG pathway enzymes by inositol requires ongoing PC synthesis (70,74). Data from recent studies have shown that, even in the absence of exogenous ethanolamine and choline, the Kennedy pathway contributes to the synthesis of PC (61,75,76). Data suggest that the choline required is derived from the turnover of PC synthesized by the CDP-DG pathway (75,76). This may indicate that the PC generated by each pathway has distinct as well as overlapping functions in cell physiology. The relative contributions of the Kennedy and CDP-DG pathways to phospholipid synthesis in the absence of exogenous ethanolamine or choline are not known. An apparent paradox in the regulation of phospholipid synthesis is the repression by inositol of the mRNA abundance of the Kennedy pathway enzymes choline kinase (77), cholinephosphotransferase (78), and ethanolaminephosphotransferase (74). In addition, the inositol (79,80) and choline (81) transporters are repressed by inositol. If the Kennedy pathway is needed for PE and PC synthesis when the CDP-DG pathway enzymes are repressed, then why are these enzymes repressed?
Cross-regulation of the Pathways for the Synthesis of PI and PC-The level of inositol 1-phosphate synthase (encoded by the INO1 gene) is reduced in cells supplemented with inositol, and this effect is enhanced by the addition of choline (19,20,32,38,82). Thus, inositol regulates enzymes in the pathways leading to the synthesis of PI and PC suggesting that these pathways are coordinately regulated (1,2). In fact, data indicate that at least one level of coordinate regulation exists that involves the transcriptional regulators Ino2p, Ino4p, and Opi1p (1,2,45). For example, Ino2p and Ino4p activate the expression of the genes encoding for inositol 1-phosphate synthase and PS synthase, while Opi1p represses the expression of these genes (32,38,46,64,65,(83)(84)(85)(86)(87)(88)(89). In contrast, PI synthase, which utilizes inositol for the synthesis of PI, is not regulated by inositol alone or in combination with ethanolamine or choline (46,63,90). However, IPC synthase, which utilizes PI for the synthesis of sphingolipids, is regulated by inositol (91). IPC synthase activity is elevated in wild-type cells supplemented with inositol, and this effect is dependent on the INO4 regulatory gene (91).
Inositol effects are also observed with enzymes that function at earlier steps in the biosynthetic pathway. One example of this is PA phosphatase. PA phosphatase catalyzes the formation of DG. DG is used for the CDP-ethanolamine-and CDP-choline-based reactions in the Kennedy pathway ( Fig. 1) (1, 2). Two membrane-associated forms of PA phosphatase (45 and 104 kDa) have been identified in S. cerevisiae (36,37). The addition of inositol to the growth medium of wild-type cells results in the elevation of the levels of the 45-kDa PA phosphatase, while the levels of the 104-kDa PA phosphatase are not altered (37). Choline, in the absence or presence of inositol, has no effect on the PA phosphatases (37). Mutations in genes (OPI1, INO2) that alter the expression of INO1 also influence the levels of the 45-kDa PA phosphatase (37,92). These observations are consistent with a model that predicts that the expression of the gene that encodes this PA phosphatase is regulated in response to inositol.

Biochemical Regulation of Phospholipid
Biosynthetic Enzymes The rapid changes in the rates of phospholipid synthesis in response to inositol supplementation (47), choline/ethanolamine starvation of cho1 mutant cells (51), fumonisin B 1 supplementation of wild-type cells (52), and glucose starvation of wild-type and respiratory deficient cells (53,54) cannot be simply ascribed to genetic mechanisms. It is likely that the direct regulation of enzyme activities also mediates phospholipid synthesis. A number of the biosynthetic enzymes (e.g. CDP-DG synthase (34), PS synthase (35), PA phosphatase (36,37), PI synthase (39), and PI 4-kinase) have been purified to near homogeneity, and defined studies of their biochemical regulation have been conducted (Table II). This regulation will be discussed in the context of phospholipid synthesis.
Regulation of DG/CDP-DG Synthesis-A major branch point in phospholipid synthesis involves the enzymes PA phosphatase and CDP-DG synthase. These enzymes utilize PA as a substrate (Fig.  1). The partitioning of PA at this step in the pathway would influence the levels of individual phospholipids and would also alter the proportions of the phospholipids and the neutral lipids, DG and TG. Based on the relative K m values for PA, the 45-and 104-kDa forms of PA phosphatase have a greater affinity for PA than does CDP-DG synthase (34,36,37). This suggests that the partitioning of PA between CDP-DG and DG may be primarily governed by the regulation of PA phosphatase activity. The 45-and 104-kDa PA phosphatase activities are each inhibited by sphingoid bases (i.e. sphinganine and phytosphingosine) (93) and ATP (54). However, they are regulated differentially by phosphorylation (94). cAMP-dependent protein kinase phosphorylates and activates the 45-kDa enzyme but has no effect on the 104-kDa PA phosphatase (94). The regulation of PA phosphatase activity by sphingoid bases, ATP, and phosphorylation correlates with observed changes in the synthesis of phospholipids and TG (52, 54, 92, 94 -96). Both PA phosphatase activities are activated by CL, CDP-DG, and PI (97). Since the activation constants for these phospholipids are within the range of their cellular concentrations (97), this activation may be physiologically relevant. In contrast to the PA phosphatases, CDP-DG synthase activity is not regulated by phosphorylation, nucleotides, sphingoid bases, or phospholipids (52,98,99).
Regulation of PS/PI Synthesis-A second branch point in phos- pholipid biosynthesis involves the enzymes PS synthase and PI synthase. These enzymes both utilize CDP-DG as a substrate (Fig.  1). Data suggest that the partitioning of CDP-DG between PS and PI is primarily determined by the level of PS synthase activity. Inositol regulates the expression of PS synthase (64,65) and regulates the activity of the enzyme by acting as a noncompetitive inhibitor (47). PS synthase activity is also inhibited by sphingoid bases (52) and by cAMP-dependent protein kinase phosphorylation (100). The inhibition of PS synthase activity by inositol (47), sphingoid bases (52), and phosphorylation (99) results in an increase in PI synthesis and a concomitant reduction in PS synthesis in vivo. PA, CL, and DG also regulate PS synthase activity (101). PA activates PS synthase activity while CL and DG inhibit its activity. The activation constants for these lipids are within the range of their cellular concentrations (101), which suggests that the regulation of PS synthase activity by these lipids may be physiologically relevant.
In contrast to PS synthase, PI synthase activity is not regulated by phospholipid precursors (47,90), phospholipids (101), sphingoid bases (52), or phosphorylation (99). Data indicate that the partitioning of CDP-DG between PS and PI is not governed by the affinities that PI synthase and PS synthase have for CDP-DG (47). Given the low intracellular levels of inositol and the relative high K m value for inositol, the synthesis of PI by PI synthase in vivo is likely to be regulated primarily by the availability of this substrate (47).
Regulation of Phosphoinositide/Sphingolipid Synthesis-PI is a branch point intermediate for the synthesis of the phosphoinositides (PIP, PIP 2 , and PI 3-P) and sphingolipids (IPC, MIPC, and M(IP) 2 C) (Fig. 1). Given this, the regulation of PI 4-kinase and IPC synthase activities could play a pivotal role in the partitioning of PI between these lipids. Two membrane-associated forms of PI 4-kinase (45 and 55 kDa) have been identified and characterized (40 -42). Regulation of the 45-and 55-kDa PI 4-kinase activities by ATP and ADP plays a major role in the synthesis of PIP and PIP 2 in vivo (102). The activities of these PI 4-kinases are not regulated by cAMP-dependent protein kinase phosphorylation (102), a mechanism previously thought to regulate the membrane-associated forms of the enzyme (103,104). The 45-kDa PI 4-kinase is inhibited by CDP-DG, and the inhibitor constant for the enzyme is within its plasma membrane concentration (50). Moreover, regulation of the 45-kDa PI 4-kinase activity by CDP-DG is coordinated with the regulation of enzymes in the CDP-DG pathway (50). IPC synthase has not been purified, and little is known about its biochemical regulation. However, studies with a solubilized preparation of the enzyme have shown that IPC synthase activity is inhibited by sphingoid bases and that this inhibition correlates with a decrease in sphingolipid synthesis (52).
Regulation of DG/PS Synthesis-The responses of PA phosphatase and PS synthase to various modulators further illustrate the reciprocal nature of the regulation of phospholipid synthesis. The DG generated from PA by the PA phosphatase can be used to synthesize TG and phospholipids by the Kennedy pathway, while PS synthase can use the CDP-DG derived from PA by the action of CDP-DG synthase (Fig. 1). Inositol supplementation elevates levels of the 45-kDa PA phosphatase (37) but reduces levels of the PS synthase (64,65). Phosphorylation of the 45-kDa PA phosphatase by cAMP-dependent protein kinase stimulates its activity (94), while phosphorylation of PS synthase inhibits its activity (100). Both enzymes are regulated by phospholipids but in a complementary manner. PS synthase activity is activated by PA (101) while PA phosphatase activity is activated by CDP-DG (97). Thus, the phospholipid substrate for PA phosphatase activates PS synthase, while the phospholipid substrate for PS synthase activates PA phosphatase. In addition, DG (the product of the PA phosphatase reaction) inhibits PS synthase activity (101). Finally, CL activates PA phosphatase activity (97) but inhibits PS synthase activity (101). These results suggest that the differential regulation of PA phosphatase and PS synthase plays a central role in controlling the pathways by which phospholipids and neutral lipids are synthesized.

Novel Enzymes of Phospholipid Metabolism
DGPP phosphatase is a membrane-associated enzyme recently identified in S. cerevisiae (44). This enzyme catalyzes the dephosphorylation of DGPP to generate PA. DGPP is a novel metabolite that contains a pyrophosphate group attached to DG (105). This phospholipid accounts for 0.18 mol % of the total phospholipid content in S. cerevisiae (44). When DGPP is supplied as a substrate in vitro, the enzyme removes the ␤-phosphate of DGPP to generate PA and then removes the ␣-phosphate to generate DG (44). In fact, DGPP phosphatase can utilize PA as a substrate in the absence of DGPP, although the enzyme has a 10-fold higher specificity constant for DGPP (44). In vitro, the DGPP phosphatase activity of the enzyme is not significantly altered by PC, PE, PI, PS, or DG (44). In addition, PA does not alter DGPP phosphatase activity (44). However, DGPP does competitively inhibit the PA phosphatase activity of the DGPP phosphatase enzyme (44). In contrast, DGPP stimulates the activities of the 45-and 104-kDa PA phosphatases (44). Moreover, these PA phosphatase enzymes do not utilize DGPP as a substrate (44). These data indicate that the activities of the DGPP phosphatase enzyme and of the DGPP phospholipid may influence PA levels in vivo. Since PA plays a major role in phospholipid metabolism, it is likely that the activities of the DGPP phosphatase enzyme will influence these processes.
Another enzyme activity that has been recently identified in S. cerevisiae is PA kinase (44). This enzyme catalyzes the phosphorylation of PA to generate DGPP. The PA kinase from yeast has not been purified, and its response to various lipid regulators is not known. Since the activity of PA kinase and DGPP phosphatase will contribute to the levels of DGPP, PA, and DG, the enzymes likely participate in a novel cycle for the regulation of the levels of these lipids.

Concluding Comments
Research on phospholipid synthesis in S. cerevisiae has significantly advanced our understanding of this process. It is clear from studies of phospholipid enzymes and their genes that the mechanisms that govern this metabolism are intricate and are integrated with other aspects of cell physiology. Investigators are using a combination of approaches (genetic, molecular, and biochemical) to help resolve this complexity. This effort requires the cloning of those genes that encode enzymes that have been identified as well as the purification and characterization of the products of these genes. In addition, as the recent discovery of DGPP phosphatase and PA kinase illustrates, there may be other components of phospholipid metabolism yet to be identified.