Coordinate regulation of phospholipid biosynthesis in Saccharomyces cerevisiae: pleiotropically constitutive opi1 mutant.

Phospholipid metabolism in the Saccharomyces cerevisiae opi1 mutant, which excretes inositol and is constitutive for the biosynthetic enzyme inositol-1-phosphate synthase (M. Greenberg, P. Goldwasser, and S. Henry, Mol. Gen. Genet. 186:157-163, 1982), was examined and compared to that of a wild-type strain. In wild-type S. cerevisiae, the phospholipid composition and the relative rates of synthesis of individual phospholipids change in response to the availability of exogenous supplies of soluble phospholipid precursors, particularly inositol. The opi1 mutant, in contrast, displays a relatively invariant phospholipid composition, and its pattern of phospholipid synthesis does not change in response to exogenous phospholipid precursors. Phosphatidylinositol synthase was not found to be regulated in either wild-type or opi1 cells. In wild-type cells, phosphatidylserine synthase and the phospholipid N-methyltransferases are coordinately repressed in response to a combination of inositol and choline. However, in opi1 cells these activities are expressed constitutively. These results suggest that the gene product of the OPI1 locus participates in the coordinate regulation of phospholipid synthesis.     

Regulation of CDP-diacylglycerol synthase activity in Saccharomyces cerevisiae.

The addition of ethanolamine or choline to inositol-containing growth medium resulted in a reduction of CTP:phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) activity in Saccharomyces cerevisiae. The reduction of activity did not occur in the absence of inositol. CDP-diacylglycerol synthase activity was not regulated in a S. cerevisiae mutant strain (opi1; an inositol biosynthesis regulatory mutant) by the addition of phospholipid precursors to the growth medium.     

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.     

Coordinate regulation of phospholipid biosynthesis by serine in Saccharomyces cerevisiae.

The addition of L-serine to inositol-containing growth medium repressed membrane-associated CDPdiacylglycerol synthase (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41) and phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) activities and subunit levels in wild-type Saccharomyces cerevisiae. Enzyme activities and subunit levels were not repressed when inositol was absent from the growth medium. The addition of L-serine to the growth medium did not affect the phospholipid composition of wild-type cells. CDPdiacylglycerol synthase and phosphatidylserine synthase were not regulated in the S. cerevisiae inositol biosynthesis ino2, ino4, and opi1 regulatory mutants, suggesting that regulation by inositol plus L-serine is coupled to inositol synthesis. Inositol and L-serine did not affect the activities of purified CDPdiacylglycerol synthase and phosphatidylserine synthase. The addition of compounds structurally related to L-serine to the growth medium of wild-type cells also resulted in a repression of CDPdiacylglycerol synthase and phosphatidylserine synthase but only in the presence of inositol. Phosphatidylinositol synthase (CDPdiacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) was not regulated by inositol plus L-serine.     

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.     

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

A Saccharomyces cerevisiae mutant (cdg1 mutation) was isolated on the basis of an inositol excretion phenotype and exhibited pleiotropic deficiencies in phospholipid biosynthesis. Genetic analysis of the mutant confirmed that the cdg1 mutation represents a new genetic locus and that a defect in a single gene was responsible for the Cdg1 phenotype. CDP-diacylglycerol synthase activity in mutant haploid cells was 25% of the wild-type derepressed level. Biochemical and immunoblot analyses revealed that the defect in CDP-diacylglycerol synthase activity in the cdg1 mutant was due to a reduced level of the CDP-diacylglycerol synthase Mr-56,000 subunit rather than to an alteration in the enzymological properties of the enzyme. This defect resulted in a reduced rate of CDP-diacylglycerol synthesis, an elevated phosphatidate content, and alterations in overall phospholipid synthesis. Unlike wild-type cells, CDP-diacylglycerol synthase was not regulated in response to water-soluble phospholipid precursors. The cdg1 lesion also caused constitutive expression of inositol-1-phosphate synthase and elevated phosphatidylserine synthase. Phosphatidylinositol synthase was not affected in the cdg1 mutant.     

Effect of growth phase on phospholipid biosynthesis in Saccharomyces cerevisiae.

The effect of growth phase on the membrane-associated phospholipid biosynthetic enzymes CDP-diacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases in wild-type Saccharomyces cerevisiae was examined. Maximum activities were found in the exponential phase of cells grown in complete synthetic medium. As cells entered the stationary phase of growth, the activities of the CDP-diacylglycerol synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases decreased 2.5- to 5-fold. The subunit levels of phosphatidylserine synthase and the cytoplasmic-associated enzyme inositol-1-phosphate synthase were not significantly affected by the growth phase. When grown in medium supplemented with inositol-choline, cells in the exponential phase of growth had reduced CDP-diacylglycerol synthase, phosphatidylserine synthase, and phospholipid N-methyltransferase activities, with repressed subunit levels of phosphatidylserine synthase and inositol-1-phosphate synthase compared with cells grown without inositol-choline. Enzyme activity levels remained reduced in the stationary phase of growth of cells supplemented with inositol-choline. The phosphatidylserine synthase and inositol-1-phosphate synthase subunit levels, however, were depressed. Phosphatidylinositol synthase (activity and subunit) was not affected by growth in medium supplemented with or without inositol-choline or the growth phase of the culture. The phospholipid composition of cells in the exponential and stationary phase of growth was also examined. The phosphatidylinositol to phosphatidylserine ratio doubled in stationary-phase cells. The phosphatidylcholine to phosphatidylethanolamine ratio was not significantly affected by the growth phase of cells.     

Saccharomyces Cerevisiae Cho2 Mutants Are Deficient in Phospholipid Methylation and Cross-Pathway Regulation of Inositol Synthesis

Five allelic Saccharomyces cerevisiae mutants deficient in the methylation of phosphatidylethanolamine (PE) have been isolated, using two different screening techniques. Biochemical analysis suggested that these mutants define a locus, designated CHO2, that may encode a methyltransferase. Membranes of cho2 mutant cells grown in defined medium contain approximately 10% phosphatidylcholine (PC) and 40-50% PE as compared to wild-type levels of 40-45% PC and 15-20% PE. In spite of this greatly altered phospholipid composition, cho2 mutant cells are viable in defined medium and are not auxotrophic for choline or other phospholipid precursors such as monomethylethanolamine (MME). However, analysis of yeast strains carrying more than one mutation affecting phospholipid biosynthesis indicated that some level of methylated phospholipid is essential for viability. The cho2 locus was shown by tetrad analysis to be unlinked to other loci affecting phospholipid synthesis. Interestingly, cho2 mutants and other mutant strains that produce reduced levels of methylated phospholipids are unable to properly repress synthesis of the cytoplasmic enzyme inositol-1-phosphate synthase. This enzyme was previously shown to be regulated at the level of mRNA abundance in response to inositol and choline in the growth medium. We cloned the CHO2 gene on a 3.6-kb genomic DNA fragment and created a null allele of cho2 by disrupting the CHO2 gene in vivo. The cho2 disruptant, like all other cho2 mutants, is viable, exhibits altered regulation of inositol biosynthesis and is not auxotrophic for choline or MME.     

Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by inositol. Inositol is an inhibitor of phosphatidylserine synthase activity

The addition of inositol to the growth medium of Saccharomyces cerevisiae resulted in rapid changes in the rates of phospholipid biosynthesis. The partitioning of the phospholipid intermediate CDP-diacylglycerol was shifted to phosphatidylinositol at the expense of phosphatidylserine and its derivatives phosphatidylethanolamine and phosphatidylcholine. Serine at 133-fold greater concentrations than that of inositol shifted the partitioning of CDP-diacylglycerol to phosphatidylserine at the expense of phosphatidylinositol but to a much lesser degree. Kinetic experiments with pure phosphatidylserine synthase and phosphatidylinositol synthase indicated that the partitioning of CDP-diacylglycerol between phosphatidylserine and phosphatidylinositol was not governed by the affinities both enzymes have for their common substrate CDP-diacylglycerol. Instead, the main regulation of phosphatidylinositol and phosphatidylserine synthesis was through the exogenous supply of inositol. The Km of inositol (0.21 mM) for phosphatidylinositol synthase was 9-fold higher than cytosolic concentration of inositol (24 microM). The Km of serine (0.83 mM) for phosphatidylserine synthase was 3-fold below the cytosolic concentration of serine (2.6 mM). Therefore, inositol supplementation resulted in a dramatic increase in the rate of phosphatidylinositol synthesis, whereas serine supplementation resulted in little affect on the rate of phosphatidylserine synthesis. Inositol also contributed to the regulation of phosphatidylinositol and phosphatidylserine synthesis by having a direct affect on phosphatidylserine synthase activity. Kinetic experiments with pure phosphatidylserine synthase showed that inositol was a noncompetitive inhibitor of the enzyme with a Ki of 65 microM.     

Cardiolipin synthase expression is essential for growth at elevated temperature and is regulated by factors affecting mitochondrial development

Cardiolipin (CL) is a unique dimeric phospholipid localized primarily in the mitochondrial membrane. In eukaryotes, the enzyme CL synthase catalyses the synthesis of CL from two lipid substrates, CDP-diacylglycerol and phosphatidylglycerol. In earlier studies, we reported the purification of CL synthase from Saccharomyces cerevisiae and the cloning of the gene CRD1 (previously called CLS1 ) that encodes the enzyme. Because CL is an important component of the mitochondrial membrane, knowledge of its regulation will provide insight into the biogenesis of this organelle. To understand how CL synthesis is regulated, we analysed CRD1 expression by Northern blot analysis of RNA extracted from cells under a variety of growth conditions. CRD1 expression is regulated by mitochondrial development factors. CRD1 levels were 7- to 10-fold greater in stationary than in logarithmic growth phase, and threefold greater in wild-type than in ρ⁰ mutants. Expression was somewhat elevated during growth in glycerol/ethanol versus glucose media. In contrast, CRD1 expression was not regulated by the phospholipid precursors inositol and choline, and was not altered in the regulatory mutants ino2 , ino4 and opi1 . Mutations in cytochrome oxidase assembly, which led to reduced Crd1p enzyme activity, did not affect CRD1 expression. The crd1 null mutant makes a truncated CRD1 message. Although the null mutant can grow on both fermentable and non-fermentable carbon sources at lower temperatures, it cannot form colonies at 37°C. In conclusion, CRD1 expression is controlled by factors affecting mitochondrial development, but not by the phospholipid precursors inositol and choline. Expression of CRD1 is essential for growth at elevated temperatures, suggesting that either CL or Crd1p is required for an essential cellular function.     

Regulation of CDP-diacylglycerol synthesis and utilization by inositol and choline in Schizosaccharomyces pombe.

CDP-diacylglycerol (CDP-DG) is an important branchpoint intermediate in eucaryotic phospholipid biosynthesis and could be a key regulatory site in phospholipid metabolism. Therefore, we examined the effects of growth phase, phospholipid precursors, and the disruption of phosphatidylcholine (PC) synthesis on the membrane-associated phospholipid biosynthetic enzymes CDP-DG synthase, phosphatidylglycerolphosphate (PGP) synthase, phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase in cell extracts of the fission yeast Schizosaccharomyces pombe. In complete synthetic medium containing inositol, maximal expression of CDP-DG synthase, PGP synthase, PI synthase, and PS synthase in wild-type cells occurred in the exponential phase of growth and decreased two- to fourfold in the stationary phase of growth. In cells starved for inositol, this decrease in PGP synthase, PI synthase, and PS synthase expression was not observed. Starvation for inositol resulted in a twofold derepression of PGP synthase and PS synthase expression, while PI synthase expression decreased initially and then remained constant. Upon the addition of inositol to inositol-starved cells, there was a rapid and continued increase in PI synthase expression. We examined expression of these enzymes in cho2 and cho1 mutants, which are blocked in the methylation pathway for synthesis of PC. Choline starvation resulted in a decrease in PS synthase and CDP-DG synthase expression in cho1 but not cho2 cells. Expression of PGP synthase and PI synthase was not affected by choline starvation. Inositol starvation resulted in a 1.7-fold derepression of PGP synthase expression in cho2 but not cho1 cells when PC was synthesized. PS synthase expression was not depressed, while CDP-DG synthase and PI synthase expression decreased in cho2 and cho1 cells in the absence of inositol. These results demonstrate that (i) CDP-DG synthase, PGP synthase, PI synthase, and PS synthase are similarly regulated by growth phase; (ii) inositol affects the expression of PGP synthase, PI synthase, and PS synthase; (iii) disruption of the methylation pathway results in aberrant patterns of regulation of growth phase and phospholipid precursors. Important differences between S. pombe and Saccharomyces cerevisiae with regard to regulation of these enzymes are discussed.     

Inositol regulates phosphatidylglycerolphosphate synthase expression in Saccharomyces cerevisiae.

The enzyme phosphatidylglycerolphosphate synthase (PGPS; CDPdiacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase; EC 2.7.8.5) catalyzes the committed step in the synthesis of cardiolipin, a phospholipid found predominantly in the mitochondrial inner membrane. To determine whether PGPS is regulated by cross-pathway control, we analyzed PGPS expression under conditions in which the regulation of general phospholipid synthesis could be examined. The addition of inositol resulted in a three- to fivefold reduction in PGPS expression in wild-type cells in the presence or absence of exogenous choline. The reduction in enzyme activity in response to inositol was seen in minutes, suggesting that inactivation or degradation of the enzyme plays an important role in inositol-mediated repression of PGPS. In cho2 and opi3 mutants, which are blocked in phosphatidylcholine synthesis, inositol-mediated repression of PGPS did not occur unless choline was added to the media. Three previously identified genes that regulate general phospholipid synthesis, INO2, INO4, and OP11, did not affect PGPS expression. Thus, ino2 and ino4 mutants, which are unable to derepress biosynthetic enzymes involved in general phospholipid synthesis, expressed wild-type levels of PGPS activity under derepressing conditions. PGPS expression in the opi1 mutant, which exhibits constitutive synthesis of general phospholipid biosynthetic enzymes, was fully repressed in the presence of inositol and partially repressed even in the absence of inositol. These results demonstrate for the first time that an enzymatic step in cardiolipin synthesis is coordinately controlled with general phospholipid synthesis but that this control is not mediated by the same genetic regulatory circuit.     

Characterization of a yeast regulatory mutant constitutive for synthesis of inositol-1-phosphate synthase

Summary The enzyme inositol-1-phosphate synthase is repressed at least 50-fold in wild type yeast grown in inositol-supplemented media. Mutants which synthesize this enzyme constitutively have been isolated using a selection procedure based on excretion of inositol into the growth medium by putative mutants. Biochemical analysis of one of the mutants (opi1-1) confirmed that the nature of the mutations is regulatory, and not in the structural gene for the enzyme. Immunoprecipitation of crude extracts with antibody directed against purified inositol-1-phosphate synthase showed that a protein which reacts with the antibody is present in the mutant grown under both repressing and derepressing conditions, in contrast to the wild type which synthesizes the enzyme only when derepressed. Assay of inositol-1-phosphate synthase activity in crude extracts of the mutant verified synthase activity in cells grown under both repressing and drepressing conditions. Synthase purified from this mutant was characterized with respect to molecular weight, thermolability and affinity for substrates glucose-6-phosphate and NAD. These analyses indicated that purified mutant synthase was similar to the wild type enzyme.     

Regulation of phosphatidylserine decarboxylase in Saccharomyces cerevisiae by inositol and choline: kinetics of repression and derepression

The biosynthesis of phosphatidylserine (PS) and its conversion to phosphatidylcholine (PC) are regulated coordinately by inositol and choline in Saccharomyces cerevisiae (G. M. Carman and S. A. Henry, 1989, Annu. Rev. Biochem. 58, 635-669). In this study, PS decarboxylase activity is shown to be partially repressed when inositol is added to the medium of cells in the log phase of growth, and the extent of repression is augmented by the inclusion of choline, but not ethanolamine. The kinetics of repression and derepression of PS decarboxylase, PS synthase, and phospholipid N-methyltransferase (PNMT) activities, as regulatory responses to the availability of exogenous inositol and choline, have been characterized. When inositol was added to the medium of cell cultures growing exponentially, the three biosynthetic enzyme activities reached an intermediate level of repression (50-85% of control) within 60 min. After the addition of the combination of inositol and choline, PS decarboxylase, PS synthase, and PNMT activities decreased to the intermediate levels of repression in 60 min and were subsequently reduced to 15-40% of control values during a later stage of regulation (2-3 h). In a derepression study, the three enzyme activities remained relatively stable for approximately 60 min following the removal of choline and/or inositol from the growth medium, but the specific activities of PS decarboxylase, PS synthase, and PNMT increased to maximally derepressed levels within 2-3 h. The induction of the three biosynthetic activities was blocked by cycloheximide, but not by chloramphenicol. In summary, the level of PS decarboxylase activity in S. cerevisiae is partially and reversibly suppressed by inositol and further diminished by the combination of inositol and choline. The biphasic kinetics of repression by inositol and choline suggest that the effect of choline is dependent on earlier events mediated by inositol and possibly involves a separate regulatory factor(s).