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

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1 Progress in Lipid Research 38 (1999) 361±399 Phospholipid biosynthesis in the yeast Saccharomyces cerevisiae and interrelationship with other metabolic processes George M. Carman a, *, Susan A. Henry b a Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901, USA b Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA Contents 1. Introduction Phospholipid biosynthetic pathways Gene±enzyme relationships in phospholipid synthesis CDP-DAG pathway enzymes CDP-DAG synthase PS synthase PS decarboxylase Phospholipid N-methyltransferases CDP-choline pathway enzymes Choline kinase Phosphocholine cytidylyltransferase Choline phosphotransferase CDP-ethanolamine pathway enzymes Ethanolamine kinase Abbreviations: PA, phosphatidate; DAG, diacylglycerol; CDP-DAG, CDP-diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PG, phosphatidylglycerol; CL, cardiolipin; PLD, phospholipase D; PGP, phosphatidylglycerophosphate; DGPP, diacylglycerol pyrophosphate; TAG, triacylglycerol; UPR, unfolded protein response; TBP, TATA binding protein; TGN, trans-golgi network; M(IP) 2 C, mannosyl-diinositolphosphorylceramide; ARF, ADP ribosylation factor. * Corresponding author. Tel.: X-217; fax: address: carman@aesop.rutgers.edu (G.M. Carman) /99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S (99)

2 362 G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361± Phosphoethanolamine cytidylyltransferase Ethanolamine phosphotransferase Phosphatidylinositol pathway enzymes Inositol 1-phosphate synthase PI synthase Cardiolipin pathway enzymes PGP synthase CL synthase Other enzymes of phospholipid synthesis CTP synthetase PA phosphatase and DGPP phosphatase Regulation of phospholipid synthesis Genetic regulation Regulation of UAS INO -containing genes by phospholipid precursors Role of phospholipid metabolism in regulation of UAS INO -containing genes Regulation of UAS INO -containing genes in response to growth phase and nutrient starvation Biochemical regulation PS synthase Mg 2+ -dependent PA phosphatase Choline kinase CL synthase CTP synthetase Interrelationships of phospholipid synthesis with other metabolic pathways Relationships of the unfolded protein response and the glucose response signal transduction pathways to the expression and regulation of INO1 and other UAS INO - containing genes Connection to the unfolded protein response pathway Connection to the glucose response pathway Roles of DGPP phosphatase and Mg 2+ -independent PA phosphatase in lipid signaling pathways CTP synthetase regulation and phospholipid synthesis Sphingolipids and phospholipid synthesis Relationship of phospholipid metabolism to phospholipid transport and membrane tra cking Summary Acknowledgements References Introduction In this article, we will review recent progress in elucidating the mechanisms that regulate phospholipid biosynthesis in the yeast Saccharomyces cerevisiae and discuss advances in assigning gene±enzyme relationships in these pathways. Because these subjects have been covered in a number of comprehensive reviews [1±5], we will focus on aspects of this

3 G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361± metabolism that have not been covered in detail in previous reports. For example, we will not be covering the phosphatidylinositol (PI) kinases which have received much recent attention elsewhere [6±9]. Because a great deal of progress has been made recently in determining the gene±enzyme relationships in those parts of the phospholipid biosynthetic pathway that lead immediately to the phospholipid precursors diacylglycerol (DAG) and CDP-diacylglycerol (CDP-DAG), we will focus special attention on these reactions. We will also focus on recent progress in assigning the gene±enzyme relationships in cardiolipin (CL) synthesis, as well as the CDP-ethanolamine and CDP-choline pathways for phospholipid synthesis. This review will cover new information concerning mechanisms linking the control of phospholipid biosynthesis with other aspects of cellular metabolism, including nucleotide and sphingolipid metabolism and general nutrient control. We will also discuss emerging evidence that several major signal transduction pathways play a role in controlling the expression of genes that respond to the inositol-sensitive transcriptional regulation that coordinates major steps in phospholipid biosynthesis. 2. Phospholipid biosynthetic pathways The major phospholipids found in the membranes of S. cerevisiae include phosphatidylcholine (PC), phosphatidylethanolamine (PE), PI, and phosphatidylserine (PS) [1,2]. Mitochondrial membranes also contain phosphatidylglycerol (PG) and CL [1,2]. PC is the end product of phospholipid synthesis and the major membrane phospholipid found in S. cerevisiae [1,2]. In addition to serving as a major structural component of cellular membranes, PC serves as a reservoir for several second messengers [10]. Two alternative pathways, the CDP-DAG pathway and the CDP-choline (Kennedy) pathway (Fig. 1) synthesize PC. The Fig. 1. Pathways for the synthesis of the major phospholipids in S. cerevisiae. The pathways shown for synthesis of phospholipids include the relevant steps discussed in the text. The CDP-DAG, CDP-choline, and CDPethanolamine pathways are indicated. A more comprehensive description that includes additional steps in these pathways may be found in references [2,11]. The abbreviations used are: PA, phosphatidate; CDP-DAG, CDPdiacylglycerol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PME, phosphatidylmonomethylethanolamine; PDE, phosphatidyldimethylethanolamine; PC, phosphatidylcholine; DAG, diacylglycerol; TAG, triacylglycerol; DGPP, diacylglycerol pyrophosphate; PGP, phosphatidylglycerophosphate; PG, phosphatidylglycerol; CL, cardiolipin; SL, sphingolipids; PIPs, phosphoinositides.

4 364 G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361±399 pathways for the synthesis of PC in S. cerevisiae are carried out by enzymes common to mammalian cells, with the exception of the synthesis of PS. In yeast, PS is synthesized from CDP-DAG and serine [1,2,11] (Fig. 1), whereas in mammalian cells, PS is synthesized by an exchange reaction between PE or PC with serine [12]. PC is primarily synthesized in S. cerevisiae by the CDP-DAG pathway [1,2,11]. The CDPcholine pathway becomes essential for PC synthesis when the enzymes in the CDP-DAG pathway are defective. For example, mutants defective in the synthesis of PS, PE, or PC require choline for growth in order to synthesize PC via the CDP-choline pathway [1,2,11]. PS and PE synthesis mutants can also synthesize PC if they are supplemented with ethanolamine [1,2,11]. The ethanolamine is used for PE synthesis via the CDP-ethanolamine pathway [1,2,11] (Fig. 1). The PE is subsequently methylated to form PC in the CDP-DAG pathway (Fig. 1). The CDP-choline pathway was once viewed as an auxiliary or salvage pathway used by cells when the CDP-DAG pathway was compromised [1,2]. However, it is now known that the CDP-choline pathway contributes to PC synthesis even when wild-type cells are grown in the absence of exogenous choline [11,13]. The PC synthesized via the CDP-DAG pathway is constantly hydrolyzed back to free choline and phosphatidate (PA) by the reaction catalyzed by phospholipase D (PLD) [13] (Fig. 1). The free choline is incorporated back into PC via the CDP-choline pathway and the PA is recycled back into PC and other phospholipids via the CDP-DAG pathway [1,11,13]. CDP-DAG is also the direct precursor of PI, PG, and CL (Fig. 1). 3. Gene±enzyme relationships in phospholipid synthesis The availability of the Saccharomyces Genome Database has facilitated the identi cation and isolation of genes encoding enzymes of lipid metabolism. Although the deduced protein sequence of a gene can be useful in predicting gene function, the data derived is entirely predictive in nature. Sequence homology relationships without genetic and biochemical veri cation should not be used as the sole evidence in the assignment of a gene±enzyme relationship. Genetic analyses by themselves provide supportive, but not conclusive evidence of a gene± enzyme relationship. For example, a mutant defect leading to no activity or altered kinetic properties of an enzyme could be the result of mutation in another gene required for posttranslational modi cation. Likewise, a mutation in a regulatory gene required for transcription of another gene could also result in loss of activity. The overexpression of an enzyme activity from a plasmid bearing a cloned gene is suggestive of gene dosage e ects associated with structural genes. However, other regulatory enzymes can also be responsible for elevated levels of activity. The identi cation of a gene±enzyme relationship requires a conclusive level of evidence showing a direct and unambiguous connection between the enzyme in question and the gene purported to encode it. Unambiguous evidence that an enzyme is encoded by a particular gene is the demonstration that an amino acid sequence determined from a puri ed enzyme matches the deduced amino acid sequence of a cloned gene. Heterologous expression of a cloned gene and its encoded enzymatic activity and immunological experiments using antibodies directed

5 against an expressed gene or gene-fusion also provides convincing evidence of a gene±enzyme relationship. In this section of the review, we discuss established gene±enzyme relationships in phospholipid synthesis. A summary of genes whose product has been established is presented in Table CDP-DAG pathway enzymes G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361± CDP-DAG synthase The enzyme that catalyzes the formation of CDP-DAG and PP i from CTP and PA [14], the committed step in the CDP-DAG pathway, is CDP-DAG synthase. The enzyme is associated with the mitochondrial and microsomal fractions of the cell [15,16]. The structural gene encoding CDP-DAG synthase is the CDS1 gene [17]. CDS1, which is located on chromosome II, is an essential gene in S. cerevisiae [17]. CDP-DAG synthase has been puri ed to apparent homogeneity and characterized with respect to its enzymological and kinetic properties [15]. The subunit molecular mass of the puri ed enzyme (56 kda) is in reasonable agreement with the predicted size (51.8 kda) of the CDS1 gene product. The correlation between the CDS1 gene and the CDP-DAG synthase enzyme is supported by the heterologous expression of the CDS1 gene in Sf9 insect cells (W. Dowhan, unpublished data). Moreover, studies on the genetics and regulation of the enzyme [17±19] have provided evidence for the gene±enzyme relationship PS synthase The next step in the CDP-DAG pathway is catalyzed by PS synthase. The enzyme catalyzes the formation of PS by displacing CMP from CDP-DAG with serine [20]. PS synthase is associated with mitochondrial and microsomal membranes [16,21]. The structural gene encoding PS synthase is the CHO1/PSS gene [22±24]. The CHO1 gene is located on chromosome V. Mutants (cho1/pss ) defective in PS synthase are choline/ethanolamine auxotrophs [25±28]. These mutants require choline or ethanolamine for growth to synthesize PC or PE by the CDP-choline and CDP-ethanolamine pathways, respectively [1,2]. The CHO1 gene product has been puri ed and extensively characterized [21,24,29±34]. The subunit size of the puri ed enzyme (30 kda) is in good agreement with the size (30.8 kda) of the enzyme predicted from the open reading frame of CHO1. The gene±enzyme relationship of CHO1 and PS synthase has been shown conclusively by alignment of the N-terminal amino acid sequence of puri ed PS synthase with the deduced amino acid sequence of the CHO1 gene [24]. This relationship has also been demonstrated by immunological studies [29] PS decarboxylase PE is synthesized from PS by the reaction catalyzed by PS decarboxylase [20]. There are two genes encoding PS decarboxylase in S. cerevisiae, PSD1 [35,36] and PSD2 [37]. The PSD1- encoded PS decarboxylase is associated with the inner mitochondrial membrane [16] and the PSD2-encoded enzyme is associated with Golgi and vacuolar compartments [38]. The PSD1 and PSD2 genes are located on chromosomes XIV and VII, respectively. The predicted subunit molecular masses of the PSD1-encoded and PSD2-encoded enzymes are 56.6 and 130 kda, respectively. The gene±enzyme relationships of the PS decarboxylase enzymes have been

6 1 Table with established gene±enzyme relationships Enzymes Gene ORF Chromosome Enzyme Evidence of gene±enzyme relationship Reference YBR029C II CDP-DAG synthase Heterologous expression in Sf9 insect cells, CDS1 and regulation data genetic YER026C V PS synthase N-terminal amino acid sequence alignment CHO1/PSS deduced sequence with YNL169C VIV PS decarboxylase Heterologous expression in E. coli [35] PSD1 YGR170W VII PS decarboxylase Heterologous expression in Sf9 insect cells [37] PSD2 YGR157W VII PE methyltransferase Genetic and biochemical data [40±44] PEM1/CHO2 YJR073C X Phospholipid methyltransferase Genetic and biochemical data [40±44] PEM2/OPI3 YLR133W XII Choline kinase N-terminal amino acid sequence alignment CKI1 deduced sequence, heterologous with YDR212W VII Phosphocholine PCT1/CCT1 cytidylyltransferase [17±19] [24] in E. coli and Sf9 insect cells expression expression in E. coli [55] Heterologous YNL130C XIV Choline phosphotransferase Immunoblot analysis of HA-tagged CPT1- CPT1 enzyme encoded YDR147W IV Ethanolamine kinase Heterologous expression in Sf9 insect cells [66] EKI1 YGR007W VII Phosphoethanolamine ECT1 cytidylyltransferase [46,47] [61] Heterologous expression in E. coli [68] YHR123W VIII Ethanolamine phosphotransferase Genetic and biochemical data [69] EPT1 YJL153C X Inositol 1-phosphate synthase N-terminal amino acid sequence alignment INO1 with deduced sequence, immunological data YPR113W XVI PI synthase Heterologous expression in E. coli [78] PIS1 YCL004W III PGP synthase Heterologous expression in E. coli [64] PGS1/PEL1 YDL142C IV CL synthase Heterologous expression in Sf9 insect cells [65] CRD1/CLS1 YBL039C II CTP synthase N-terminal amino acid sequence alignment URA7 with deduced sequence, immunological data YJR103W X CTP synthetase Immunological data [101] URA8 YDR284C IV DGPP phosphatase N-terminal amino acid and internal sequence DPP1 with deduced sequence, alignment expression in Sf9 insect cells heterologous LPP1 YDR503C IV Mg 2+ -independent PA phosphatase Heterologous expression in Sf9 insect cells [106] [71,73] [100] [105] 366 G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361±399

7 G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361± documented by the heterologous expression of PSD1 in Escherichia coli [35] and the heterologous expression of PSD2 in Sf9 insect cells [37]. Although both enzymes catalyze the same reaction, the deduced protein sequences of the PSD1 and PSD2 genes show little overall identity (12.6%) and they have very di erent assay requirements [38]. The PSD1-encoded enzyme is responsible for >90% of the PS decarboxylase activity in the cell [35±37]. Neither one of the PS decarboxylase genes is essential nor do mutants defective in these genes exhibit any growth defects [35±38]. However, a psd1 psd2 double mutant is auxotrophic for choline or ethanolamine [37]. Like cho1 mutants, the psd1 psd2 double mutant requires these phospholipid precursors to synthesize PC or PE by the CDP-choline or CDP-ethanolamine pathways, respectively Phospholipid N-methyltransferases The phospholipid N-methyltransferases catalyze the three step AdoMet-dependent methylation of PE to PC [39]. Two separate enzymes catalyze these reactions [40,41]. The rst methylation reaction is catalyzed by the PEM1/CHO2-encoded PE methyltransferase [42,43] and the last two methylation reactions are catalyzed by the PEM2/OPI3-encoded phospholipid methyltransferase [42,44]. The later enzyme can also catalyze the rst methylation reaction [40,41]. The phospholipid N-methyltransferase enzymes are associated with the microsomal fraction of the cell and are likely to be ER proteins [16]. The PEM1/CHO2 and PEM2/OPI3 genes are located on chromosomes VII and X, respectively. The predicted subunit sizes of the PEM1/CHO2-encoded PE methyltransferase and the PEM2/OPI3-encoded phospholipid methyltransferase enzymes are and 23.2 kda, respectively, and show 9.3% amino acid sequence identity. There is no direct evidence linking the PEM1/CHO2 and PEM2/OPI3 gene products to the phospholipid N-methyltransferase enzymes. However, genetic and biochemical data provide strong support for the gene±enzyme relationships [40±44]. The PEM1/CHO2 and PEM2/OPI3 genes are not essential and mutants defective in either gene are not auxotrophic for choline [40,43,44]. However, a pem1/cho2 pem2/ opi3 double mutant is auxotrophic for choline to synthesize PC via the CDP-choline pathway [42±44] CDP-choline pathway enzymes Choline kinase The committed step in the synthesis of PC by the CDP-choline pathway is catalyzed by choline kinase. Choline kinase is a cytosolic enzyme that catalyzes the phosphorylation of choline with ATP to form phosphocholine and ADP [45]. The enzyme is encoded by the CKI1 gene [46], which is located on chromosome XII. The CKI1-encoded choline kinase has been puri ed to homogeneity and characterized [47]. The minimum subunit molecular mass (73 kda) of puri ed choline kinase is in good agreement with the predicted size (66.3 kda) of the CKI gene product. The N-terminal amino acid sequence of the puri ed choline kinase aligns perfectly with the amino acid sequence of the protein deduced from the open reading frame of the CKI1 gene [46,47]. This data unequivocally identi es the puri ed choline kinase as the product of the CKI1 gene. This relationship is also supported by the heterologous expression of the CKI1 gene in E. coli [46] and in Sf9 insect cells [47]. The CKI1 gene product also

8 368 G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361±399 exhibits ethanolamine kinase activity [46,47]. The preferred substrate is choline [47]. The C- terminal region of the choline kinase enzyme contains a phosphotransferase consensus sequence [48], presumed to be involved in catalytic function [49,50]. The CKI1 gene is not essential in S. cerevisiae [46] since this organism may synthesize PC by the CDP-DAG pathway Phosphocholine cytidylyltransferase The rate-limiting step in the CDP-choline pathway is catalyzed by phosphocholine cytidylyltransferase [51,52]. The enzyme catalyzes the formation of CDP-choline from CTP and phosphocholine [53]. Phosphocholine cytidylyltransferase activity is associated with the cytosolic and membranes fractions of the cell [51]. The PCT1/CCT1 gene in S. cerevisiae encodes the enzyme [54]. The PCT1 gene is located on chromosome VII. The predicted subunit molecular mass of the PCT1 gene product is 49.4 kda. The PCT1 gene and its encoded phosphocholine cytidylyltransferase activity has been expressed in E. coli [55] con rming the gene±enzyme relationship. The PCT1 gene in S. cerevisiae is not essential to cell viability [54] Choline phosphotransferase The nal step in the CDP-choline pathway is catalyzed by choline phosphotransferase [56]. This is a microsomal-associated enzyme that catalyzes the formation of PC from CDP-choline and DAG [57]. The CPT1 gene, located on chromosome XIV, encodes choline phosphotransferase activity [58,59]. The CPT1 gene is not essential for cell growth [58,60]. The predicted size of the subunit encoded by the CPT1 gene is 44.1 kda. Immunoblot analysis of cells carrying hemagglutinin (HA)-tagged CPT1-encoded protein has provided evidence for the gene±enzyme relationship [61]. The choline phosphotransferase enzyme [59] contains a conserved CDP-alcohol phosphotransferase motif found in several enzymes of phospholipid synthesis including PS synthase [23,24], PI synthase [62], ethanolamine phosphotransferase [63], PGP synthase [64], and CL synthase [65]. A mutational analysis of the choline phosphotransferase has con rmed that this motif plays a role in enzyme catalysis [61] CDP-ethanolamine pathway enzymes Ethanolamine kinase The committed step in the synthesis of PE by the CDP-ethanolamine pathway is catalyzed by ethanolamine kinase. Ethanolamine kinase is a cytosolic enzyme that catalyzes the phosphorylation of ethanolamine with ATP to form phosphoethanolamine and ADP [66]. The EKI1 gene [66], which is located on chromosome IV, encodes the enzyme. The predicted subunit size of ethanolamine kinase is 61.7 kda. That the product of the EKI1 gene is ethanolamine kinase is supported by the heterologous expression of the EKI1 gene in Sf9 insect cells [66]. The EKI1 gene product also exhibits choline kinase activity, however the preferred substrate is ethanolamine [66]. Like the CKI1-encoded choline kinase enzyme, the C-terminal region of the ethanolamine kinase contains a phosphotransferase consensus sequence [48]. The EKI1 gene is not essential in S. cerevisiae [66] since this organism may synthesize PE by the CDP-DAG pathway. Biochemical analyses of eki1d, cki1d, and eki1d cki1d mutants have shown that the EKI1

9 G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361± and CKI1 gene products encode all of the ethanolamine kinase and choline kinase activities in S. cerevisiae [66]. In vivo labeling experiments indicate that the EKI1 and CKI1 gene products have overlapping functions with respect to phospholipid synthesis. Whereas the EKI1 gene product is primarily responsible for PE synthesis via the CDP-ethanolamine pathway, the CKI1 gene product is primarily responsible for PC synthesis via the CDP-choline pathway [66] Phosphoethanolamine cytidylyltransferase The enzyme responsible for the second step in the CDP-ethanolamine pathway is phosphoethanolamine cytidylyltransferase [67]. This is a cytosolic-associated enzyme that catalyzes the formation of CDP-ethanolamine from CTP and phosphoethanolamine [68]. The ECT1 gene encodes the phosphoethanolamine cytidylyltransferase, which is located on chromosome VII [68]. The ECT1 gene is not essential for cell viability [68]. The predicted subunit size of the ECT1-encoded enzyme is 36.9 kda. Heterologous expression of the ECT1 gene fused to the glutathione S-transferase gene in E. coli has con rmed the gene±enzyme relationship [68] Ethanolamine phosphotransferase The last step in the CDP-ethanolamine pathway is catalyzed by ethanolamine phosphotransferase [67]. The enzyme is a microsomal-associated enzyme that is encoded by the EPT1 gene [60,63]. EPT1 is located on chromosome VIII. The deduced protein sequence (44.6 kda) of the enzyme contains the conserved CDP-alcohol phosphotransferase motif [61]. The EPT1 gene product shows 54.2% identity to the CPT1 gene product. In vitro, the EPT1 gene product exhibits choline phosphotransferase activity; however, in vivo, the enzyme only exhibits ethanolamine phosphotransferase activity [69]. The EPT1 gene is not essential for cell growth [58,60]. Although there is no direct evidence linking the EPT1 sequence to ethanolamine phosphotransferase, genetic and biochemical data support the gene±enzyme relationship [69] Phosphatidylinositol pathway enzymes Inositol 1-phosphate synthase Inositol 1-phosphate synthase is a cytosolic-associated enzyme that catalyzes the rate-limiting step in the synthesis of inositol, the water-soluble precursor of PI [70]. The enzyme catalyzes the formation of inositol 1-phosphate from glucose 6-phosphate [71]. The INO1 gene encodes inositol 1-phosphate synthase [72,73], which is located on chromosome X. The INO1-encoded inositol 1-phosphate synthase has been puri ed and characterized [71]. The open reading frame of the INO1 gene predicts a protein with a subunit molecular mass of 62.8 kda [73] which is in excellent agreement with the subunit molecular mass of 62 kda of the puri ed inositol 1- phosphate synthase [71]. Conclusive evidence for the gene±enzyme relationship has come from the alignment of the N-terminal amino acid sequence of the pure enzyme with the deduced sequence of the INO1 open reading frame [73]. The INO1 gene is not essential for growth, however, ino1 mutants are auxotrophic for inositol [70,71]. The INO1 gene is the most highly regulated gene in phospholipid synthesis [1,2,11]. Its regulation has a major impact on phospholipid metabolism (see below).

10 370 G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361± PI synthase The nal step in the synthesis of PI is catalyzed by PI synthase [74]. This microsomalassociated [16] enzyme catalyzes the formation of PI and CMP from CDP-DAG and inositol [75]. PI synthase is encoded by the PIS1 gene [62,76], which is located on chromosome XVI. The enzyme has been puri ed and characterized [77]. The subunit size (34 kda) of the puri ed PI synthase is somewhat larger than the size (24.8 kda) deduced from the open reading frame of the PIS1 gene [62,77]. This discrepancy has been attributed to anomalous behavior of the pure enzyme upon SDS-polyacrylamide gel electrophoresis or as due to posttranslational modi cation [74]. The heterologous expression of the PIS1 gene in E. coli has established that PIS1 is indeed the structural gene for PI synthase [78]. The PIS1 gene is essential for cell viability [62] because the product of the PI synthase reaction, PI, is essential to the growth and metabolism of S. cerevisiae [79±81]. Moreover, lipid derivatives of PI, such as the polyphosphoinositides and sphingolipids are also essential to cell viability [1,2,11,82] Cardiolipin pathway enzymes PGP synthase The committed and rate-limiting step in CL synthesis in S. cerevisiae is catalyzed by phosphatidylglycerophosphate (PGP) synthase [83]. The enzyme catalyzes the formation of PGP by displacing CMP from CDP-DAG with glycerol 3-phosphate [84]. PGP synthase activity is associated with the inner mitochondrial membrane [85]. The enzyme is encoded by the PGS1 gene [64], which is located on chromosome III. The deduced product derived from the open reading frame of PGS1 is a 59.3 kda protein [64]. Establishment of the gene±enzyme relationship of PGS1 and the PGP synthase enzyme has come from the heterologous expression of the gene in E. coli [64]. The PGS1 gene was rst identi ed as the PEL1 gene [86] encoding a second PS synthase enzyme [87]. This assignment was based solely on amino acid sequence homology with the E. coli pssa-encoded PS synthase [88,89]. The PGS1 gene is not essential to cell viability [64]. However, the deletion of the gene in a haploid strain results in a growth dependence on a fermentable carbon source, temperature sensitivity for growth, and a petite lethal phenotype [64,87]. These characteristics are consistent with the CL pathway being essential for mitochondrial function [64] CL synthase The nal step in the CL pathway is catalyzed by CL synthase [90]. CL synthase is an inner mitochondrial membrane protein that catalyzes the formation of CL and CMP from CDP- DAG and PG [90]. The structural gene encoding the enzyme is CRD1/CLS1, which is located on chromosome IV [65,91,92]. CL synthase has been puri ed from mitochondria membranes [93]. The subunit molecular mass of the puri ed enzyme (28 kda) is in reasonable agreement with the size of the product (31.9 kda) predicted from the CRD1 open reading frame [65,91,92]. The gene±enzyme relationship of CRD1 with CL synthase has been established by the heterologous expression of the CRD1 gene in Sf9 insect cells [65]. The CRD1 gene is not essential for growth on fermentable or non-fermentable carbon sources [65,91,92]. Therefore, CL, the major mitochondrial phospholipid previously thought to

11 G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361± be essential to mitochondrial function, is not required for aerobic growth at ambient temperatures [65,91,92]. However, at elevated temperatures, the crd1 (cls1) null mutant loses viability on both fermentable and non-fermentable carbon sources [94]. Accumulation of PG in crd1 mutants suggests that this anionic phospholipid may substitute for CL in some mitochondrial processes [65,92], but not adequately at 378C [94] Other enzymes of phospholipid synthesis CTP synthetase The enzyme responsible for the synthesis of the CTP that is required for the reactions catalyzed by CDP-DAG synthase, phosphocholine cytidylyltransferase, and phosphoethanolamine cytidylyltransferase is CTP synthetase. The enzyme catalyzes the nal step in the pyrimidine biosynthetic pathway [95]. CTP synthetase is a cytosolic-associated enzyme that catalyzes the ATP-dependent transfer of the amide nitrogen of glutamine to the C-4 position of UTP to form CTP [96,97]. GTP is an allosteric e ector that accelerates the formation of a covalent glutaminyl enzyme catalytic intermediate [97,98]. Duplicate genes named URA7 [95] and URA8 [99] encode CTP synthetase, and are located on chromosomes II and X, respectively. The deduced amino acid sequences of the open reading frames of the URA7 and URA8 genes show 78% identity [95,99]. Neither the URA7-nor the URA8-encoded CTP synthetase is essential provided that cells possess one functional CTP synthetase gene [95,99]. The URA7-encoded enzyme is more abundant than the URA8-encoded enzyme [52] and is responsible for the majority of the CTP synthesized in vivo [99]. The URA7- [100] and URA8-encoded [101] CTP synthetases have been puri ed to apparent homogeneity and characterized with respect to their enzymological and kinetic properties. The subunit molecular masses of the pure URA7-encoded (68 kda) and URA8-encoded (67 kda) CTP synthetases are in good agreement with the predicted molecular masses deduced from the open reading frames of the URA7 (64.7 kda) and URA8 (64.5 kda) genes, respectively. The gene±enzyme relationship of the URA7-encoded CTP synthetase has been documented by alignment of the N-terminal amino acid sequence of the pure protein to the deduced amino acid sequence of the open reading frame of URA7 [100]. Immunological experiments using antibodies directed against a fusion protein constructed from the coding sequences of the URA8 gene and expressed in E. coli have con rmed the gene±enzyme relationship of the enzyme [101]. The deduced protein products of the URA7 and URA8 genes contain a conserved glutamine amide transfer domain characteristic of glutamine amidotransferases [95,99] PA phosphatase and DGPP phosphatase PA phosphatase catalyzes the dephosphorylation of PA yielding DAG and Pi [102]. The DAG generated in this reaction is utilized for the synthesis of PC and PE via the CDP-choline and CDP-ethanolamine pathways, respectively, and for the synthesis of triacylglycerols (TAG) [1,2,103]. The PA phosphatase enzyme may also play a role in lipid signaling pathways [104]. Mg 2+ -dependent and Mg 2+ -independent PA phosphatase activities have been identi ed in S. cerevisiae [104]. Mg 2+ -dependent PA phosphatase is postulated to be responsible for the synthesis of phospholipids and TAG [104]. However, this hypothesis has not been established.

12 372 G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361±399 Two genes (DPP1 and LPP1 ) encoding Mg 2+ -independent PA phosphatase activity have been isolated and characterized [105,106]. However, genes encoding Mg 2+ -dependent PA phosphatases have not been isolated. The Mg 2+ -independent PA phosphatase has been identi ed as an activity of a membraneassociated DGPP phosphatase enzyme [107] encoded by the DPP1 gene [105]. Diacylglycerol pyrophosphate (DGPP) is a novel phospholipid recently identi ed in S. cerevisiae [107]. It is derived from PA via the reaction catalyzed by PA kinase [107]. Pure DGPP phosphatase catalyzes the removal of the b-phosphate of DGPP to yield PA, followed by removal of the phosphate from PA to yield DAG [107]. Although the DGPP phosphatase enzyme utilizes PA as a substrate in the absence of DGPP, the speci city constant for PA is 10-fold lower than that of DGPP [107]. This enzyme also utilizes lysopa, ceramide 1-phosphate, PGP, and isoprenoid phosphates as substrates [108±110]. The DPP1 gene is located on chromosome IV. The deduced subunit molecular mass (33.5 kda) derived from the DPP1 open reading frame [105] is in excellent agreement with the subunit molecular mass (34 kda) of the puri ed DGPP phosphatase enzyme [107]. The deduced amino acid sequence of the DPP1 gene matches the N-terminal amino acid and two internal amino acid sequences of the pure enzyme [105]. Moreover, the heterologous expression of the DPP1 gene in Sf-9 insect cells results in the expression of DGPP phosphatase and Mg 2+ -independent PA phosphatase activities [105]. These data provide conclusive evidence for the gene±enzyme relationship. The LPP1 gene has been identi ed [106] on the basis that its deduced protein product shows homology to the DPP1-encoded DGPP phosphatase [105] and to the mouse Mg 2+ - independent PA phosphatase [111]. The homologous regions of these proteins constitute a novel phosphatase sequence motif [112]. The LPP1 gene is also located on chromosome IV. The predicted protein product of the LPP1 gene has a subunit molecular mass of 31.6 kda. The LPP1-encoded protein is associated with the membrane fraction of the cell and possesses Mg 2+ -independent PA phosphatase activity [106]. The enzyme also utilizes lysopa and DGPP as substrates, however, PA appears to be the preferred substrate [106]. The LPP1-encoded and DPP1-encoded enzymes are similar insofar as they are Mg 2+ -independent phosphatases that utilize a variety of lipid phosphate substrates and contain a novel phosphatase motif [105,106]. However, other than the phosphatase motif, these enzymes show relatively little (23% identity) overall amino acid sequence homology. The LPP1 [106] and DPP1 [105] genes are not essential for cell viability. Moreover, an lpp1 dpp1 double mutant exhibits no obvious growth defects [106]. Analyses of lpp1 and dpp1 mutants show that the LPP1 and DPP1 gene products play a role in the regulation of the cellular levels of PI and PA [106]. 4. Regulation of phospholipid synthesis Because a number of comprehensive reviews of phospholipid biosynthesis in yeast have been published [1±3,11,103], this review will provide only a summary of key features of the regulatory mechanisms. We will discuss the genetic and biochemical regulation of phospholipid synthesis.

13 4.1. Genetic regulation G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361± Regulation of UAS INO -containing genes by phospholipid precursors The genes encoding many enzymes of phospholipid biosynthesis are subject to a complex pattern of coordinate transcriptional regulation [11]. These genes all contain variants of a repeated sequence, UAS INO, in their promoters [1,3]. A number of enzymes involved in phospholipid biosynthesis were initially observed to exhibit reduced activity levels in response to growth of cells in the presence of the phospholipid precursors, inositol and choline [1]. The activities of many of these same enzymes are reduced as cells entered stationary phase, even when inositol and choline are absent [113]. Following the cloning of the structural genes encoding many of these same enzymes, it was demonstrated that the regulation in response to both growth phase and phospholipid precursors occurs at the transcriptional level. This regulation requires the presence of the UAS INO cis-acting element in the promoters of the coregulated genes [1±3,11]. The basic pattern of regulation of UAS INO -containing genes is as follows: the transcripts of the co-regulated genes of phospholipid biosynthesis are maximally expressed during logarithmic growth in media lacking inositol and choline. If inositol is present in the growth medium, expression of these co-regulated genes is partially repressed; and the addition of choline to medium containing inositol leads to further repression. However, if choline is present in the absence of inositol, it has much less e ect than inositol has in the absence of choline. Other phospholipid precursors such as ethanolamine and serine have also been reported to have an e ect similar to choline when added to growth medium in the presence of inositol [113,114]. A large number of genes encoding enzymes of lipid metabolism show this general pattern of transcriptional regulation, but there is wide variation in the repression ratios exhibited by these genes. For example, expression of the INO1 gene, encoding inositol-1-phosphate synthase, shows at least 30-fold repression in response to inositol and choline [115] whereas CHO1, encoding PS synthase, exhibits a four- to ve-fold repression [116]. Other co-regulated genes such as those encoding CDP-DAG synthase, the phospholipid N-methyltransferases, and PGP synthase exhibit even lower repression ratios [117±120]. Curiously, the INO2 and OPI1 genes required for wild type expression of UAS INO -containing genes, also exhibit repression in response to inositol and choline [121,122]. Consistent with earlier studies of enzyme activities [113], the transcripts of genes exhibiting regulation by inositol and choline have been shown to be repressed as cells enter stationary phase even when inositol and choline are absent [122± 124]. Interestingly, there are a number of reactions that are regulated in an opposite fashion (i.e., they are up regulated by inositol and derepressed in stationary phase). These reactions include inositol-1-phosphate phosphatase [125], inositol phosphorylceramide synthase [126], and one form of Mg 2+ -dependent PA phosphatase [127,128]. Expression of CRD1, encoding CL synthase, is not regulated in response to inositol, but it is derepressed in stationary phase even when inositol and choline are absent [94]. PGP synthase does not t this pattern. Enzyme activity is decreased in cells grown in the presence of inositol. However, activity increases as cells enter stationary phase [129]. Aside from CL synthase, none of the activities that are derepressed in response to inositol and/or stationary phase have been studied at the level of transcription.

14 374 G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361±399 Based on the regulatory patterns described above, phospholipid biosynthesis in S. cerevisiae can be described as existing in two regulatory states, designated by `on' and `o ' as illustrated in Fig. 2. For purposes of this discussion, the system is de ned as being `on' when INO1 and other co-regulated UAS INO -containing genes are maximally expressed. In wild-type cells, the `on' regulatory state occurs only during the logarithmic phase of growth in the absence of inositol. The `o ' state, which occurs in wild-type cells in the presence of inositol and/or in stationary phase, is characterized by repression of INO1 and other UAS INO -containing, coregulated genes. Mutants such as ino2 and ino4, which are unable to derepress the UAS INO - containing genes including INO1, are inositol auxotrophs (Ino ÿ phenotype) and are permanently in the `o ' state. Mutants, which overexpress INO1, possess the Opi ÿ phenotype, are characterized by excretion of inositol into the growth medium. Some Opi ÿ mutants (such as opi1 ) fail to repress INO1 and other UAS INO -containing genes, either in stationary phase on in response to inositol, and are constitutively in the `on' state (Fig. 2). Sequencing and comparison of the promoters of INO1, CHO1, and other co-regulated genes resulted in the identi cation of the repeated element, consensus 5' CATGTGAAAT 3' [1,118,130]. This element, known as UAS INO (inositol-sensitive upstream activation sequence) or alternatively as ICRE (inositol choline responsive element) [1±3,11,130,131] is necessary and Fig. 2. Two regulatory states of phospholipid metabolism in yeast. Top Panel: `o ' corresponds to the regulatory state that occurs during stationary phase, regardless of the presence of phospholipid precursors. This regulatory state also occurs in logarithmic phase when inositol (I + ) is present in the growth medium. Bottom Panel: `on' corresponds to the regulatory state that occurs only in logarithmic phase in the absence of inositol (I ÿ ). Reactions that are derepressed, under each condition, are depicted with green arrows; repressed reactions are depicted in red. CL synthase (3) responds only to stationary phase regulation and is not repressed by inositol during logarithmic phase. Reactions depicted with black arrows are either not regulated by phospholipid precursors or growth phase (example: PI synthase), or have not been studied at the transcriptional level under these growth conditions (example: PLD). The SEC14 gene product (Sec14p) is shown exerting a negative regulatory e ect on choline phosphate cytidylyltransferase and PLD. The abbreviations used are: PL, phospholipid; PA, phosphatidate.

15 G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361± su cient for regulated expression in response to inositol and choline [115,118,130±132]. The UAS INO element contains the core consensus-binding site (CANNTG) for bhlh proteins and is the binding site for a heterodimer consisting of Ino2p and Ino4p, the products of the INO2 and INO4 regulatory genes [133±135]. Ino2p and Ino4p are DNA-binding proteins of the basic helix-loop-helix (bhlh) family of DNA-binding proteins [133,136,137]. All mutations in UAS INO that reduce the homology of its core sequence to the consensus bhlh binding site sequence (i.e., CATGTG) also reduce its ability to bind to the Ino2p/Ino4p heterodimer [132]. Such mutations also reduce the ability of the element to drive regulated expression of heterologous reporter genes [118,131,132]. The INO2 and INO4 regulatory genes also have UAS INO elements in their promoters and INO2 expression is repressed in response to inositol and choline [121,138,139]. However, the INO4 gene is constitutively expressed in response to inositol and choline [121,139]. Levels of Ino2p appear to be limiting for formation of the Ino2p/Ino4p heterodimer [133], while Ino4p levels do not appear to limit INO1 expression [121,140]. Ino2p contains an activation domain necessary for INO1 expression and Ino4p does not [135]. However, both Ino2p and Ino4p are required for INO1 expression [115] and exhibit other alterations in lipid composition and metabolism [141], consistent with the requirement of the Ino2p/Ino4p complex for derepression and maximal expression of all UAS INO -containing genes. Mutants that fail to repress INO1 and other UAS INO -containing genes were isolated on the basis of the Opi ÿ (overproduction of inositol) phenotype [142]. The opi1 mutants express the INO1 gene product, inositol-1-phosphate synthase, constitutively at a level about two-fold higher than the fully derepressed level observed in wild type cells growing in the absence of inositol [142,143]. Expression of INO1 and other UAS INO -containing genes in opi1 cells is not repressed in response to inositol and choline [115,116] and continues in stationary phase [122,123]. The OPI1 gene product (Opi1p) contains a leucine zipper and polyglutamine stretches, motifs that are often associated with DNA binding proteins [144]. However, there is no evidence that Opi1p binds directly to DNA [145] and its mechanism of action is unknown. As mentioned previously, the promoter of the OPI1 gene contains the UAS INO element and its transcription is repressed in response to inositol and choline and upon entry into stationary phase [122]. The OPI1 gene product appears to play some, as yet unde ned, negative regulatory function in the expression of UAS INO -containing genes, since opi1d (i.e., loss of function) mutations lead to high-level constitutive expression of INO1 and other UAS INO -containing genes [142,144]. Opi1p has been shown to function via UAS INO [132], but it does not appear to interact with Ino2p or Ino4p or to bind UAS INO directly (T. Graves, J. Yang, V. Bruno, S. Henry, unpublished data) Role of phospholipid metabolism in regulation of UAS INO -containing genes A unique feature of the regulation of UAS INO -containing genes is its responsiveness to the status of phospholipid biosynthesis and turnover. Mutations blocking any one of the reactions in the pathway that leads from PA to PC through the intermediates CDP-DAG, PS, PE, PME and PDE (Fig. 1) result in derepression of INO1 and other UAS INO -containing genes (`on' state, Fig. 2) even when inositol is present in the growth medium [18,43,44,119,146±148]. Such mutants have Opi ÿ phenotypes and some of them were isolated in screens for mutants

16 376 G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361±399 defective in INO1 regulation [11]. An Opi ÿ phenotype is also seen in a CTP synthetase (ura7 ) mutant (E161K) which is insensitive to CTP product inhibition [149]. An INO1 promoter lacz fusion reporter construct was found to exhibit more than two-fold elevated expression in this mutant compared to wild type [149]. Increased turnover of PC, via a PLD-mediated route, also results in derepression of the UAS INO -containing genes, even in the presence of inositol and/or during stationary phase and also produces an Opi ÿ phenotype [4,13]. Such regulatory phenotypes associated with mutants defective in structural genes suggest that an intermediate involved in phospholipid metabolism generates a signal that leads to derepression of UAS INO -containing genes. Henry and Patton-Vogt [11] presented a model of this regulatory network and hypothesized that PA, or a metabolite closely related to PA, is responsible for generating the signal for derepression of UAS INO -containing genes. Consistent with this hypothesis is the observation that INO1 is rapidly derepressed when PA is generated via a PLD-mediated turnover of PC [4,13]. The level of PA is also a ected by its rate of utilization as a precursor [18,32]. CDP-DAG synthase catalyzes the conversion of PA to CDP- DAG that then serves as the precursor to PI, PS, and PGP. Consistent with the hypothesis that elevated PA levels generate a signal for derepression of UAS INO -containing genes, both increased PLD-mediated turnover of PC and decreased expression of CDP-DAG synthase lead to rapid derepression of INO1 [13,119]. Addition of inositol to the growth medium of logarithmically growing cells results in a shift in the pattern of phospholipid biosynthesis, characterized by a higher rate of PI synthesis. The increase in PI synthesis occurs at the expense of cellular pools of CDP-DAG and its upstream precursor, PA [32], and consistent with the hypothesis described above, INO1 and other UAS INO -containing genes are repressed during logarithmic growth when inositol is present. Addition of inositol to the growth medium also causes an immediate change in the pattern of PS synthesis. Synthesis of PS, like PI, draws directly on the CDP-DAG pool (Fig. 1). The rate of PS synthesis is rapidly reduced when inositol is added to the growth medium because inositol is a non-competitive inhibitor of PS synthase [32]. In addition, expression of CHO1, the structural gene encoding PS synthase, is repressed by inositol via the coordinate transcriptional described above (Fig. 2). Thus, PS synthase activity is subject to allosteric inhibition by inositol and the rate of PS synthesis is rapidly reduced in the presence of inositol. Moreover, the activation of the RAS/cAMP pathway in S. cerevisiae results in a number of changes in overall lipid metabolism [150,151]. These changes include an increase in PI synthesis at the expense of PS synthesis [150]. The decrease in PS synthesis has been attributed to the phosphorylation and inhibition of PS synthase [33,150]. The increase in PI synthesis is not due to the phosphorylation of PI synthase [150]. Instead, PI synthesis increases due to a loss of competition between PI synthase and PS synthase for their common substrate CDP-DAG (Fig. 1) following down-regulation of PS synthase by phosphorylation [150]. The reduction in PS synthesis is subsequently reinforced by the slower mechanism of transcriptional regulation. Thus, when inositol is added to the growth medium, PI production is favored at the expense of the upstream precursors, CDP-DAG and PA, and competing reactions like PS synthesis. PI synthase activity, however, is constitutive, as is expression of its structural gene, PIS1 [114,152,153]. In addition to PI and PS synthesis, synthesis of PGP and DAG also compete for the available CDP-DAG and PA, respectively. All UAS INO -containing genes show an additional level of repression when choline is added

17 G.M. Carman, S.A. Henry / Progress in Lipid Research 38 (1999) 361± to medium that also contains inositol [113,117,124,154]. The synthesis of PC using choline, via the CDP-choline pathway, draws on DAG as its immediate lipid precursor, and DAG is derived from PA by dephosphorylation (Fig. 1). Thus, inositol and choline are used in reactions that draw upon the lipid precursors, CDP-DAG and DAG, respectively, which, in turn, draw on PA via separate and potentially additive pathways. Thus, if build-up of PA generates a signal for derepression of UAS INO -containing genes, the signal intensity should re ect the rate of PA production (from acylation of glycerol-3-phosphate and/or PLDmediated phospholipid turnover). This should be balanced against the rate of PA utilization as a precursor in reactions leading to CDP-DAG, DAG, DGPP, and all lipids downstream of these precursors. The model predicts that the system will be in the `on' state when PA is produced more rapidly than it is being consumed, and it will be in the `o ' state when PA is consumed more rapidly than it can be produced (Fig. 3). This additive feature of the model provides an explanation for the Opi ÿ regulatory phenotype of structural gene mutants (such as cds1, cho1, opi3, and cho2 ) blocked in reactions downstream of PA en route to PC [11]. The existing data are consistent with the identi cation of PA as the metabolite that generates the signal but at present, it is not possible to rule out other related upstream intermediates Fig. 3. Relationship of signal transduction pathways to regulation of UAS INO -containing genes. A red box marks genes if their gene products have a positive regulatory role, and a green box if their gene products have a negative role. Loss of function mutants for genes marked in green boxes lead to Opi ÿ phenotypes (`on' position, Fig. 2). Loss of function mutations of genes marked by red boxes have Ino ÿ phenotypes (`o ' state, Fig. 2). The protein kinases, Snf1p/Snf4p and Ire1p, which are associated with the glucose response and the unfolded protein response pathways, respectively, are required for derepression of UAS INO -containing genes, including INO1. Thus, ire1, snf1, and snf4 mutants are inositol auxotrophs. The product of the HAC1 gene (Hac1p) is required for expression of both genes under UPR control and UAS INO -containing genes, is expressed only after activation of Ire1p. hac1 mutants are also inositol auxotrophs. The GLC7 phosphatase (Glc7p), and its regulatory subunit Reg1p, are required to inactivate Snf1p/Snfp. reg1d mutants exhibit derepressed expression of catabolite repressed genes under control of the glucose response pathway. They also have an Opi ÿ phenotype and express UAS INO -containing genes constitutively.