Opi1 Mutant Research Articles

Efficient Production of Glucaric Acid by Engineered Saccharomyces cerevisiae.

Glucaric acid is a valuable chemical with applications in the detergent, polymer, pharmaceutical and food industries. In this study, two key enzymes for glucaric acid biosynthesis, MIOX4 (myo-inositol oxygenase) and Udh (uronate dehydrogenase), were fused and expressed with different peptide linkers. It was found that a strain harboring the fusion protein MIOX4-Udh linked by the peptide (EA3K)3 produced the highest glucaric acid titer and thereby resulted in glucaric acid production that was 5.7-fold higher than that of the free enzymes. Next, the fusion protein MIOX4-Udh linked by (EA3K)3 was integrated into delta sequence sites of the Saccharomyces cerevisiae opi1 mutant, and a strain, GA16, that produced a glucaric acid titer of 4.9 g/L in a shake flask fermentation was identified by a high-throughput screening method using an Escherichia coli glucaric acid biosensor. Strain improvement by further engineering was performed to regulate the metabolic flux of myo-inositol to increase the supply of glucaric acid precursors. The downregulation of ZWF1 and the overexpression of INM1 and ITR1 increased glucaric acid production significantly, and glucaric acid production was increased to 8.49 g/L in the final strain GA-ZII in a shake flask fermentation. Finally, in a 5-L bioreactor, GA-ZII produced a glucaric acid titer of 15.6 g/L through fed-batch fermentation. IMPORTANCE Glucaric acid is a value-added dicarboxylic acid that was synthesized mainly through the oxidation of glucose chemically. Due to the problems of the low selectivity, by-products, and highly polluting waste of this process, producing glucaric acid biologically has attracted great attention. The activity of key enzymes and the intracellular myo-inositol level were both rate-limiting factors for glucaric acid biosynthesis. To increase glucaric acid production, this work improved the activity of the key enzymes in the glucaric acid biosynthetic pathway through the expression of a fusion of Arabidopsis thaliana MIOX4 and Pseudomonas syringae Udh as well as a delta sequence-based integration. Furthermore, intracellular myo-inositol flux was optimized by a series of metabolic strategies to increase the myo-inositol supply, which improved glucaric acid production to a higher level. This study provided a way for constructing a glucaric acid-producing strain with good synthetic performance, making glucaric acid production biologically in yeast cells much more competitive.

Cross Regulation of Phospholipid and Unsaturated Fatty Acid Biosynthesis

As most cellular phospholipids require unsaturated fatty acids (UFAs), we investigated the potential cross regulation of phospholipid and UFA biosynthesis. The Δ‐9 desaturase, encoded by OLE1, is the sole source of endogenous UFAs in Saccharomyces cerevisiae. Inspection of the OLE1 promoter revealed several sequences that are close matches to the regulatory sequence UASINO found in many phospholipid biosynthetic genes, which is the target of regulation by Ino2p, Ino4p and Opi1p. We examined if these proteins also play a role in regulation of OLE1 expression. Reporter gene analysis showed that when uracil prototrophy was placed under control of the OLE1 promoter, poor expression was observed in the opi1 and ino4 mutants. When lacZ was placed under control of the OLE1 promoter, reporter assays confirmed that expression was poor in the tested mutants. qPCR analysis demonstrates that the down regulation of OLE1 occurs normally in opi1, ino2 and ino4 mutants, however, its expression under growth conditions without exogenous UFAs is poor compared to wild type cells. Fatty acid analysis revealed that normal regulation of OLE1 occurred in response to unsaturated fatty acid (UFA) supplement in the opi1 mutant, indicating that post‐transcriptional regulation is still functional. Finally, the 2‐hybrid assay was used to examine potential interactions between Ole1p and Opi1p, Ino2p, Ino4p and known OLE1 regulators Mga2p and Spt23p, however no unexpected interactions were detected. This preliminary work indicates that phospholipid regulators Opi1p, Ino2p, Ino4p play a role in OLE1 expression, but not through regulation in response to UFA supplements or through Mga2p and Spt23p.Support or Funding InformationResearch reported was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM132853

Cross Regulation of Phospholipid and Unsaturated Fatty Acid Biosynthesis

Considering the requirement for unsaturated fatty acids (UFAs) to produce phospholipids, we investigated the potential cross regulation of phospholipid and UFA biosynthesis. The Δ‐9 desaturase, encoded by OLE1, is the sole source of endogenous UFAs in Saccharomyces cerevisiae. Inspection of the OLE1 promoter revealed several sequences that are close matches to the regulatory sequence, UASINO, found in many phospholipid biosynthetic genes, and is the target of regulation by Ino2p, Ino4p and Opi1p. We then examined if these proteins also play a role in regulation of OLE1 expression. Reporter gene analysis showed that when uracil prototrophy was placed under control of the OLE1 promoter, poor expression was observed in an opi1 mutant. When lacZ was placed under control of the OLE1 promoter, reporter assays confirmed that expression was poor in the opi1 mutant. Fatty acid analysis revealed that normal regulation of OLE1 occurred in response to unsaturated fatty acid (UFA) supplement in the opi1 mutant, indicating that post‐transcriptional regulation is still functional. Finally, the 2 hybrid assay was used to examine potential interactions between Opi1p and Ole1p, Ino2p, Ino4p and known OLE1 regulators Mga2p and Spt23p. While no unexpected Opi1p interactions were detected, we observed that Ino4p interacts with Mga2p in this assay. This preliminary work indicates that phospholipid regulators also play a role in regulating OLE1 expression.Support or Funding InformationWe acknowledge the support of the Hope College Biology Department, and the Hope College Dean of Natural and Applied Sciences.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Opi1 mediates repression of phospholipid biosynthesis by phosphate limitation in the yeast Saccharomyces cerevisiae.

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are transcribed when precursor molecules inositol and choline (IC) are limiting. Gene expression is stimulated by the heterodimeric activator Ino2/Ino4, which binds to ICRE (inositol/choline-responsive element) promoter sequences. Activation is prevented by repressor Opi1, counteracting Ino2 when high concentrations of IC are available. Here we show that ICRE-dependent gene activation is repressed not only by an excess of IC but also under conditions of phosphate starvation. While PHO5 is activated by phosphate limitation, INO1 expression is repressed about 10-fold. Repression of ICRE-dependent genes by low phosphate is no longer observed in an opi1 mutant while repression is still effective in mutants of the PHO regulon (pho4, pho80, pho81 and pho85). In contrast, gene expression with high phosphate is reduced in the absence of pleiotropic sensor protein kinase Pho85. We could demonstrate that Pho85 binds to Opi1 in vitro and in vivo and that this interaction is increased in the presence of high concentrations of phosphate. Interestingly, Pho85 binds to two separate domains of Opi1 which have been previously shown to recruit pleiotropic corepressor Sin3 and activator Ino2, respectively. We postulate that Pho85 positively influences ICRE-dependent gene expression by phosphorylation-dependent weakening of Opi1 repressor, affecting its functional domains required for promoter recruitment and corepressor interaction. Copyright © 2016 John Wiley & Sons, Ltd.

Genomic Analysis of the Opi− Phenotype

Most of the phospholipid biosynthetic genes of Saccharomyces cerevisiae are coordinately regulated in response to inositol and choline. Inositol affects the intracellular levels of phosphatidic acid (PA). Opi1p is a repressor of the phospholipid biosynthetic genes and specifically binds PA in the endoplasmic reticulum. In the presence of inositol, PA levels decrease, releasing Opi1p into the nucleus where it represses transcription. The opi1 mutant overproduces and excretes inositol into the growth medium in the absence of inositol and choline (Opi(-) phenotype). To better understand the mechanism of Opi1p repression, the viable yeast deletion set was screened to identify Opi(-) mutants. In total, 89 Opi(-) mutants were identified, of which 7 were previously known to have the Opi(-) phenotype. The Opi(-) mutant collection included genes with roles in phospholipid biosynthesis, transcription, protein processing/synthesis, and protein trafficking. Included in this set were all nonessential components of the NuA4 HAT complex and six proteins in the Rpd3p-Sin3p HDAC complex. It has previously been shown that defects in phosphatidylcholine synthesis (cho2 and opi3) yield the Opi(-) phenotype because of a buildup of PA. However, in this case the Opi(-) phenotype is conditional because PA can be shuttled through a salvage pathway (Kennedy pathway) by adding choline to the growth medium. Seven new mutants present in the Opi(-) collection (fun26, kex1, nup84, tps1, mrpl38, mrpl49, and opi10/yol032w) were also suppressed by choline, suggesting that these affect PC synthesis. Regulation in response to inositol is also coordinated with the unfolded protein response (UPR). Consistent with this, several Opi(-) mutants were found to affect the UPR (yhi9, ede1, and vps74).

Negative regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by a Candida albicans orthologue of OPI1.

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are coordinately regulated by a UAS element, designated ICRE (inositol/choline-responsive element). Opi1 is a negative regulator responsible for repression of ICRE-dependent genes in the presence of an excess of inositol and choline. Gene regulation by phospholipid precursors has been also reported for the pathogenic yeast Candida albicans. Screening of a data base containing raw sequences of the C. albicans genome project allowed us to identify an open reading frame exhibiting weak similarity to Opi1. Expression of the putative CaOPI1 in an opi1 mutant of S. cerevisiae could restore repression of an ICRE-dependent reporter gene. Similar to OPI1, overexpression of CaOPI1 strongly inhibited derepression of ICRE-driven genes leading to inositol-requiring transformants. Previous work has shown that Opi1 mediates gene repression by interaction with the pleiotropic repressor Sin3. The genome of C. albicans also encodes a protein similar to Sin3 (CaSin3). By two-hybrid analyses and in vitro studies for protein-protein interaction we were able to show that CaOpi1 binds to ScSin3. ScOpi1 could also interact with CaSin3, while CaOpi1 failed to bind to CaSin3. Despite of some conservation of regulatory mechanisms between both yeasts, these results suggest that repression of phospholipid biosynthetic genes in C. albicans is mediated by a mechanism which does not involve recruitment of CaSin3 by CaOpi1.

Regulation of Phosphatidylglycerophosphate Synthase by Inositol in Saccharomyces cerevisiae Is Not at the Level of PGS1 mRNA Abundance

Phosphatidylglycerophosphate synthase catalyzes the committed step in the synthesis of the mitochondrial phospholipid cardiolipin. We showed previously that phosphatidylglycerophosphate synthase activity in Saccharomyces cerevisiae is increased in conditions favoring mitochondrial development and during growth in the absence of inositol. Interestingly, the regulatory effects of inositol were not altered in ino2, ino4, or opi1 mutants suggesting that regulation in response to inositol is not at the level of gene transcription. We report here that steady state mRNA levels of the PGS1 gene, which encodes phosphatidylglycerophosphate synthase, were not altered by inositol or choline. Growth in the presence of the inositol-depleting drug valproate led to an increase in phosphatidylglycerophosphate synthase activity unaccompanied by increased PGS1 mRNA. PGS1 mRNA abundance was not decreased in ino2 or ino4 mutants and was unaffected in an opi1 mutant. Therefore, regulation of phosphatidylglycerophosphate synthase by inositol is not mediated at the level of mRNA abundance and does not require the INO2-INO4-OPI1 regulatory circuit. PGS1 was increased in glycerol/ethanol compared with glucose media and was maximally expressed as cells entered the stationary phase. Deletion of the mitochondrial genome did not affect PGS1 expression. Thus, whereas inositol controls phosphatidylglycerophosphate synthase activity, regulation of PGS1 expression occurs primarily in response to mitochondrial development cues.

Lithium and Valproate Decrease Inositol Mass and Increase Expression of the Yeast INO1 and INO2Genes for Inositol Biosynthesis

Bipolar affective disorder (manic-depressive illness) is a chronic, severe, debilitating illness affecting 1-2% of the population. The Food and Drug Administration-approved drugs lithium and valproate are not completely effective in the treatment of this disorder, and the mechanisms underlying their therapeutic effects have not been established. We are employing genetic and molecular approaches to identify common targets of lithium and valproate in the yeast Saccharomyces cerevisiae. We show that both drugs affect molecular targets in the inositol metabolic pathway. Lithium and valproate cause a decrease in intracellular myo-inositol mass and an increase in expression of both a structural (INO1) and a regulatory (INO2) gene required for inositol biosynthesis. The opi1 mutant, which exhibits constitutive expression of INO1, is more resistant to inhibition of growth by lithium but not by valproate, suggesting that valproate may inhibit the Ino1p-catalyzed synthesis of inositol 1-phosphate. Consistent with this possibility, growth in valproate leads to decreased synthesis of inositol monophosphate. Thus, both lithium and valproate perturb regulation of the inositol biosynthetic pathway, albeit via different mechanisms. This is the first demonstration of increased expression of genes in the inositol biosynthetic pathway by both lithium and valproate. Because inositol is a key regulator of many cellular processes, the effects of lithium and valproate on inositol synthesis have far-reaching implications for predicting genetic determinants of responsiveness and resistance to these agents.

Overproduction of the Opi1 repressor inhibits transcriptional activation of structural genes required for phospholipid biosynthesis in the yeast Saccharomyces cerevisiae.

Transcription of structural genes required for phospholipid biosynthesis in the yeast Saccharomyces cerevisiae is repressed by high concentrations of inositol and choline. The ICRE (inositol/choline-responsive element), which is necessary and sufficient for regulation by phospholipid precursors, functions as a binding site for the heterodimeric Ino2/Ino4 activator. ICRE-dependent transcription becomes constitutive in the absence of the Opi1 repressor. Opi1 contains a leucine zipper motif and two glutamine-rich stretches. In this work we describe a molecular analysis of OPI1 function and expression. Opi1 mutant variants altered at the leucine zipper and a glutamine-rich region, respectively, were no longer functional repressors. In contrast, an Opi1 deletion variant lacking the N-terminal 106 amino acids still mediated negative regulation. Although the leucine zipper suggests that Opi1 may act as a DNA-binding protein, our data do not support a direct interaction with the ICRE. Despite its function as an antagonist of INO2 and INO4, expression of OPI1 is stimulated by an upstream ICRE. Overexpression of OPI1 under control of the GAL1 promoter severely inhibited activation of ICRE-dependent genes, leading to inositol-requiring cells. Growth inhibition of GAL1-OPI1 was observed with INO2 and INO4 alleles activated by either the natural promoter or a heterologous control region. Although induction of GAL1-OPI1 strongly repressed ICRE-dependent gene expression, the concentration of the Ino2/Ino4 activator remained unchanged. This finding suggests that differential expression of phospholipid biosynthetic genes may occur even in the presence of a constant amount of the specific activator.

Functional Characterization of an Inositol-sensitive Upstream Activation Sequence in Yeast: A cis-REGULATORY ELEMENT RESPONSIBLE FOR INOSITOL-CHOLINE MEDIATED REGULATION OF PHOSPHOLIPID BIOSYNTHESIS

A repeated element, the inositol-sensitive upstream activation sequence (UASINO), having the consensus sequence, 5'-CATGTGAAAT-3', is present in the promoters of genes encoding enzymes of phospholipid biosynthesis that are regulated in response to the phospholipid precursors, inositol and choline. None of the naturally occurring variants of the UASINO element exactly recapitulates the consensus (for review, see Carman, G. M., and Henry, S. A. (1989) Annu. Rev. Biochem. 58, 635-669 and Paltauf, F., Kolwhein, S., and Henry, S. A. (1992) in Molecular Biology of the Yeast Saccharomyces cerevisiae (Broach, J., Jones, E., and Pringle, J., eds) Vol. 2, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The first six bases of the UASINO element are homologous with canonical binding motif for proteins of the basic helix-loop-helix (bHLH) family. Two bHLH regulatory proteins, Ino2p and Ino4p from yeast, were previously shown to bind to promoter fragments containing this element. In the present study, an extensive analysis of UASINO function has been conducted. We report that any base substitution within the putative bHLH binding site resulted either in a dramatic reduction or in a complete obliteration of UASINO function as tested in an expression assay in vivo. Base substitutions in the 5' region that flanks the 10-base pair repeat, as well as sequences within the repeat itself at its 3' end outside the bHLH core, were also assessed. The two bases immediately flanking the 5' end of the element proved to be very important to its function as a UAS element as did the two bases immediately 3' of the bHLH core motif. Substitutions of the final two bases of the original ten base pair consensus (i.e. 5'-CATGTGAAAT-3') had less dramatic effects. We also tested a subset of the altered elements for their ability to serve as competitors in an assay of Ino2p x Ino4p binding. The strength of any given sequence as a UASINO element, as assayed in vivo, was strongly correlated with its strength as a competitor for Ino2p x Ino4p binding. We also tested a subset of the modified UASINO elements for their effects on expression in vivo in a strain carrying an opi1 mutation. The opi1 mutation renders the coregulated enzymes of phospholipid synthesis constitutive in the presence of phospholipid precursors. All elements that retained some residual UASINO activity when tested in the wild-type strain were constitutively expressed at a level comparable with the wild-type derepressed level when tested in the opi1 mutant.(ABSTRACT TRUNCATED AT 400 WORDS)

Production and reutilization of an extracellular phosphatidylinositol catabolite, glycerophosphoinositol, by Saccharomyces cerevisiae

Phosphatidylinositol catabolism in Saccharomyces cerevisiae is known to result in the formation of extracellular glycerophosphoinositol (GroPIns). We now report that S. cerevisiae not only produces but also reutilizes extracellular GroPIns and that these processes are regulated in response to inositol availability. A wild-type strain uniformly prelabeled with [3H] inositol displayed dramatically higher extracellular GroPIns levels when cultured in medium containing inositol than when cultured in medium lacking inositol. This difference in extracellular accumulation of GroPIns in response to inositol availability was shown to be a result of both regulated production and regulated reutilization. In a strain in which a negative regulator of phospholipid and inositol biosynthesis had been deleted (an opi1 mutant), this pattern of extracellular GroPIns accumulation in response to inositol availability was altered. An inositol permease mutant (itr1 itr2), which is unable to transport free inositol, was able to incorporate label from exogenous glycerophospho [3H]inositol, indicating that the inositol label did not enter the cell solely via the transporters encoded by itr1 and itr2. Kinetic studies of a wild-type strain and an itr1 itr2 mutant strain revealed that at least two mechanisms exist for the utilization of exogenous GroPIns: an inositol transporter-dependent mechanism and an inositol transporter-independent mechanism. The inositol transporter-independent pathway of exogenous GroPIns utilization displayed saturation kinetics and was energy dependent. Labeling studies employing [14C]glycerophospho[3H] inositol indicated that, while GroPIns enters the cell intact, the inositol moiety but not the glycerol moiety is incorporated into lipids.

Autoregulated expression of the yeast INO2 and INO4 helix-loop-helix activator genes effects cooperative regulation on their target genes.

In the yeast Saccharomyces cerevisiae, the phospholipid biosynthetic genes are highly regulated at the transcriptional level in response to the phospholipid precursors inositol and choline. In the absence of inositol and choline (derepressing), the products of the INO2 and INO4 genes form a heteromeric complex which binds to a 10-bp element, upstream activation sequence INO (UASINO), in the promoters of the phospholipid biosynthetic genes to activate their transcription. In the presence of inositol and choline (repressing), the product of the OPI1 gene represses transcription dictated by the UASINO element. Curiously, we identified a UASINO-like element in the promoters of both the INO2 and INO4 genes. The presence of the UASINO element in these two promoters suggested that the mechanism for the inositol-choline response would involved regulating expression of the two activator genes. Using a cat reporter gene, we find that INO2-cat expression was regulated 12-fold in response to inositol and choline but that INO4-cat was constitutively expressed. We further observed that INO2-cat was not expressed in either an ino2 or an ino4 mutant strain and was constitutively overexpressed in an opi1 mutant strain. Expression of the INO4-cat gene was affected only by mutation in the INO4 gene itself. Therefore, INO2-cat transcription is regulated by the products of both the INO2 and INO4 genes whereas INO4 must interact with another protein to activate its own transcription. Our data show that derepression of phospholipid biosynthetic gene expression involves two mechanisms: increasing the levels of the INO2 and INO4 gene products and inactivating the OPI1-mediated repression mechanism. We propose a model suggesting that this dual mechanism of regulation accounts for the observed cooperative stimulation of IN01 and CH01 gene expression (phospholipids biosynthetic genes).

The OPI1 gene of Saccharomyces cerevisiae, a negative regulator of phospholipid biosynthesis, encodes a protein containing polyglutamine tracts and a leucine zipper.

In Saccharomyces cerevisiae, recessive mutations at the OPI1 locus result in constitutively derepressed expression of inositol 1-phosphate synthase, the product of the INO1 gene. Many of the other enzymes involved in phospholipid biosynthesis are also expressed at high derepressed levels in opi1 mutants. Thus, the OPI1 gene is believed to encode a negative regulator that is required to repress a whole subset of structural genes encoding for phospholipid biosynthetic enzymes. In this study, the OPI1 gene was mapped to chromosome VIII and cloned. When transformed into an opi1 mutant, the cloned DNA was capable of complementing the mutant phenotype and restoring correct regulation to the INO1 structural gene. Construction of two opi1 disruption alleles and subsequent genetic analysis of strains bearing these alleles confirmed that the cloned DNA was homologous to the genomic OPI1 locus. Furthermore, the OPI1 gene was found to be nonessential to the organism since mutants bearing the null allele were viable and exhibited a phenotype similar to that of previously isolated opi1 mutants. Similar to other opi1 mutants, the opi1 disruption mutants accumulated INO1 mRNA constitutively to a level 2-3-fold higher than that observed in wild-type cells. The cloned OPI1 gene was sequenced, and translation of the open reading frame predicted a protein composed of 404 amino acid residues with a molecular weight of 40,036. The predicted Opi1 protein contained a well defined heptad repeat of leucine residues that has been observed in other regulatory proteins. In addition, the predicted protein contained polyglutamine residue stretches which have also been reported in yeast genes having regulatory functions. Sequencing of opi1 mutant alleles, isolated after chemical mutagenesis, revealed that several were the result of a chain termination mutation located within the largest polyglutamine residue stretch.

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.

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.

Regulation of phosphatidylserine synthase from Saccharomyces cerevisiae by phospholipid precursors.

The addition of ethanolamine or choline to inositol-containing growth medium of Saccharomyces cerevisiae wild-type cells resulted in a reduction of membrane-associated phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) activity in cell extracts. The reduction of activity did not occur when inositol was absent from the growth medium. Under the growth conditions where a reduction of enzyme activity occurred, there was a corresponding qualitative reduction of enzyme subunit as determined by immunoblotting with antiserum raised against purified phosphatidylserine synthase. Water-soluble phospholipid precursors did not effect purified phosphatidylserine synthase activity. Phosphatidylserine synthase (activity and enzyme subunit) was not regulated by the availability of water-soluble phospholipid precursors in S. cerevisiae VAL2C(YEp CHO1) and the opi1 mutant. VAL2C(YEp CHO1) is a plasmid-bearing strain that over produces phosphatidylserine synthase activity, and the opi1 mutant is an inositol biosynthesis regulatory mutant. The results of this study suggest that the regulation of phosphatidylserine synthase by the availability of phospholipid precursors occurs at the level of enzyme formation and not at the enzyme activity level. Furthermore, the regulation of phosphatidylserine synthase is coupled to inositol synthesis.

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.