The INO2 and INO4 loci of Saccharomyces cerevisiae are pleiotropic regulatory genes.

We isolated a mutant of Saccharomyces cerevisiae defective in the formation of phosphatidylcholine via methylation of phosphatidylethanolamine. The mutant synthesized phosphatidylcholine at a reduced rate and accumulated increased amounts of methylated phospholipid intermediates. It was also found to be auxotrophic for inositol and allelic to an existing series of ino4 mutants. The ino2 and ino4 mutants, originally isolated on the basis of an inositol requirement, are unable to derepress the cytoplasmic enzyme inositol-1-phosphate synthase (myo-inositol-1-phosphate synthase; EC 5.5.1.4). The INO4 and INO2 genes were, thus, previously identified as regulatory genes whose wild-type product is required for expression of the INO1 gene product inositol-1-phosphate synthase (T. Donahue and S. Henry, J. Biol. Chem. 256:7077-7085, 1981). In addition to the identification of a new ino4-allele, further characterization of the existing series of ino4 and ino2 mutants, reported here, demonstrated that they all have a reduced capacity to convert phosphatidylethanolamine to phosphatidylcholine. The pleiotropic phenotype of the ino2 and ino4 mutants described in this paper suggests that the INO2 and INO4 loci are involved in the regulation of phospholipid methylation in the membrane as well as inositol biosynthesis in the cytoplasm.     

Regulatory Mutations of Inositol Biosynthesis in Yeast: Isolation of Inositol-Excreting Mutants

The enzyme inositol-1-phosphate synthase (I-1-P synthase), product of the INO1 locus, catalyzes the synthesis of inositol-1-phosphate from the substrate glucose-6-phosphate. The activity of this enzyme is dramatically repressed in the presence of inositol. By selecting for mutants which overproduce and excrete inositol, we have identified mutants constitutive for inositol-1-phosphate synthase as well as a mutation in phospholipid biosynthesis. Genetic analysis of the mutants indicates that at least three loci (designated OPI1, OPI2 and OPI4) direct inositol-mediated repression of I-1-P synthase. Mutants of these loci synthesize I-1-P synthase constitutively. Three loci are unlinked to each other and to INO1, the structural gene for the enzyme. A mutant of a fourth locus, OPI3, does not synthesize I-1-P synthase constitutively, despite its inositol excretion phenotype. This mutant is preliminarily identified as having a defect in phospholipid synthesis.     

Regulation of the yeast INO1 gene. The products of the INO2, INO4 and OPI1 regulatory genes are not required for repression in response to inositol

The ino2Delta, ino4Delta, opi1Delta, and sin3Delta mutations all affect expression of INO1, a structural gene for inositol-1-phosphate synthase. These same mutations affect other genes of phospholipid biosynthesis that, like INO1, contain the repeated element UAS(INO) (consensus 5' CATGTGAAAT 3'). In this study, we evaluated the effects of these four mutations, singly and in all possible combinations, on growth and expression of INO1. All strains carrying an ino2Delta or ino4Delta mutation, or both, failed to grow in medium lacking inositol. However, when grown in liquid culture in medium containing limiting amounts of inositol, the opi1Delta ino4Delta strain exhibited a level of INO1 expression comparable to, or higher than, the wild-type strain growing under the same conditions. Furthermore, INO1 expression in the opi1Delta ino4Delta strain was repressed in cells grown in medium fully supplemented with both inositol and choline. Similar results were obtained using the opi1Delta ino2Delta ino4Delta strain. Regulation of INO1 was also observed in the absence of the SIN3 gene product. Therefore, while Opi1p, Sin3p, and the Ino2p/Ino4p complex all affect the overall level of INO1 expression in an antagonistic manner, they do not appear to be responsible for transmitting the signal that leads to repression of INO1 in response to inositol. Various models for Opi1p function were tested and no evidence for binding of Opi1p to UAS(INO), or to Ino2p or Ino4p, was obtained.     

Biosynthesis of inositol in yeast. Primary structure of myo-inositol-1-phosphate synthase (EC 5.5.1.4) and functional analysis of its structural gene, the INO1 locus

A biochemical, molecular, and genetic analysis of the Saccharomyces cerevisiae INO1 gene and its product, L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) has been carried out. The sequence of the entire INO1 gene and surrounding regions has been determined. Computer analysis of the DNA sequence revealed four potential peptides. The largest open reading frame of 553 amino acids predicted a peptide with a molecular weight of 62,842. The amino acid composition and amino terminus of purified L-myo-inositol-1-phosphate synthase were chemically determined and compared to the amino acid composition and amino terminus of the protein predicted from the DNA sequence of the large open reading frame. This analysis established that the large open reading frame encodes L-myo-inositol-1-phosphate synthase. The largest of several small open reading frames adjacent to INO1 predicted a protein of 133 amino acids with a molecular weight of 15,182 and features which suggested that the encoded protein may be membrane-associated. A gene disruption was constructed at INO1 by eliminating a portion of the coding sequence and replacing it with another sequence. Strains carrying the gene disruption failed to express any protein cross-reactive to antibody directed against L-myo-inositol-1-phosphate synthase. Although auxotrophic for inositol, strains carrying the gene disruption were completely viable when supplemented with inositol. In a similar fashion, a gene disruption was constructed in the chromosomal locus of the 133-amino acid open reading frame. This mutation did not affect viability but did cause inositol to be excreted from the cell.     

The Mycobacterium tuberculosis ino1 gene is essential for growth and virulence

Inositol is utilized by Mycobacterium tuberculosis in the production of its major thiol and of essential cell wall lipoglycans. We have constructed a mutant lacking the gene encoding inositol-1-phosphate synthase (ino1), which catalyses the first committed step in inositol synthesis. This mutant is only viable in the presence of extremely high levels of inositol. Mutant bacteria cultured in inositol-free medium for four weeks showed a reduction in levels of mycothiol, but phosphatidylinositol mannoside, lipomannan and lipoarabinomannan levels were not altered. The ino1 mutant was attenuated in resting macrophages and in SCID mice. We used site-directed mutagenesis to alter four putative active site residues; all four alterations resulted in a loss of activity, and we demonstrated that a D310N mutation caused loss of the active site Zn2+ ion and a conformational change in the NAD+ cofactor.     

Regulatory gene INO4 of yeast phospholipid biosynthesis is positively autoregulated and functions as a transactivator of fatty acid synthase genes FAS1 and FAS2 from Saccharomyces cerevisiae.

The sequence motif 5' TYTTCACATGY 3' functions as an upstream activation site common to both yeast fatty acid synthase genes, FAS1 and FAS2. In addition, this UASFAS element is shared by all so far characterized genes of yeast phospholipid biosynthesis. We have investigated the influence of a functional INO4 gene previously described as a regulator of inositol biosynthesis on the expression of FAS1 and FAS2. In a delta ino4 null allele strain, both genes are expressed at only 50% of wild type level. Using individual UASFAS sequence motifs inserted into a heterologous test system, a drastic decrease of reporter gene expression to 2-10% of the wild type reference was observed in the delta ino4 mutant. In gel retardation assays, the protein-DNA complex involving the previously described FAS binding factor 1, Fbf1, was absent when using a protein extract from the delta ino4 mutant. On the other hand, this signal was enhanced with an extract from cells grown under conditions of inositol/choline derepression. Subsequent experiments demonstrated that INO4 expression is itself affected by phospholipid precursors, mediated by an UASFAS element in the INO4 upstream region. Thus, in addition of being an activator of phospholipid biosynthetic genes, INO4 is also subject to a positive autoregulatory loop in its own biosynthesis.     

A dominant mutation that alters the regulation of INO1 expression in Saccharomyces cerevisiae.

A dominant, single nuclear gene mutation, CSE1, caused inositol auxotrophy in yeast cells. The inositol requirement was marked when choline was present in the medium. Inositol-1-phosphate synthase, the regulatory enzyme of inositol synthesis, is repressed by inositol, or more profoundly by a combination of inositol and choline in the wild type. In CSE1, the level of inositol-1-phosphate synthase was low and was greatly repressed on the addition of choline alone. In accordance with this, INO1 mRNA encoding the enzyme was low even under the depressed conditions and was profoundly decreased by choline in CSE1. But in the wild type, the addition of choline alone had little effect. An INO1-lacZ fusion was constructed and the control of the INO1 promoter in CSE1 was studied. lacZ expression was repressed not only by inositol, but also by choline in CSE1, whereas it was repressed by inositol, but only slightly by choline in the wild type. CSE1 was unlinked to the INO1 structural gene. Thus CSE1 was thought to be a regulatory mutation. Furthermore, when the CDP-choline pathway was mutationally blocked, choline did not affect INO1 expression, indicating that the metabolism of choline via the CDP-choline pathway is required for INO1 repression.     

Inositol Mutants of SACCHAROMYCES CEREVISIAE: Mapping the ino1 Locus and Characterizing Alleles of the ino1, ino2 and ino4 Loci

An extensive genetic analysis of inositol auxotrophic mutants of yeast is reported. The analysis includes newly isolated mutants, as well as those previously reported (Culbertson and Henry 1975). Approximately 70% of all inositol auxotrophs isolated are shown to be alleles of the ino1 locus, the structural gene for inositol-1-phosphate synthase, the major enzyme involved in inositol biosynthesis. Alleles of two other loci, ino2 and ino4, comprise 9% of total mutants, with the remainder representing unique loci or complementation groups. The ino1 locus was mapped by trisomic analysis with an n + 1 disomic strain constructed with complementing alleles at this locus. The ino1 locus is shown to be located between ura2 (11.1 cm) and cdc6 (21.8 cm) on chromosome X. An extended map of chromosome X of yeast is presented. Unlike most yeast loci, but similar to the his1 locus, the ino1 locus lacks allelic representatives that are suppressible by known suppressors. This finding suggests that premature termination of translation of the ino1 gene product may be incompatible with cell viability.     

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.     

Mutations in the Saccharomyces Cerevisiae Opi3 Gene: Effects on Phospholipid Methylation, Growth and Cross-Pathway Regulation of Inositol Synthesis

We report the isolation of two new opi3 mutants by EMS mutagenesis, and construction of an insertion allele in vitro using the cloned gene. We have demonstrated that the opi3 mutations cause a deficiency in the two terminal phospholipid N-methyltransferase (PLMT) activities required for the de novo synthesis of PC (phosphatidylcholine). The opi3 mutants, under certain growth conditions, produce membrane virtually devoid of PC although, surprisingly, none of the mutants displays a strict auxotrophic requirement for choline. Although the opi3 mutants grow without supplements, we have shown that the atypical membrane affects the ability of the mutant strains to initiate log phase growth and to sustain viability at stationary phase. The commencement of log phase growth is enhanced by addition of choline or to a lesser extent DME (dimethylethanolamine), and retarded by addition of MME (monomethylethanolamine). The mutant cells lose viability at the stationary phase of the cell cycle in the absence of DME or choline, and are also temperature sensitive for growth at 37 degrees especially in media containing MME. These growth defects have been correlated to the presence of specific phospholipids in the membrane. The opi3 growth defects are suppressed by an unusual mutation in the phospholipid methylation pathway that perturbs the N-methyltransferase (PEMT) activity immediately preceding the reactions affected by the opi3 lesion. We believe this mutation, cho2-S, alters the substrate specificity of the PEMT. A secondary effect of opi3 mutations is disruption of the cross pathway regulation of the synthesis of the PI (phosphatidylinositol) precursor inositol. Synthesis of inositol is controlled through regulation of the INO1 gene which encodes inositol-1-phosphate synthase. This highly regulated gene is expressed constitutively in opi3 mutants. We have used the opi3 strains to demonstrate that synthesis of either PC or PD (phosphatidyldimethylethanolamine) will restore normal regulation of the INO1 gene.     

A Pleiotropic Phospholipid Biosynthetic Regulatory Mutation in Saccharomyces Cerevisiae Is Allelic to Sin3 (Sdi1, Ume4, Rpd1)

Three mutants were identified in a genetic screen using an INO1-lacZ fusion to detect altered INO1 regulation in Saccharomyces cerevisiae. These strains harbor mutations that render the cell unable to fully repress expression of INO1, the structural gene for inositol-1-phosphate synthase. The Cpe(-) (constitutive phospholipid gene expression) phenotype associated with these mutations segregated 2:2, indicating that it was the result of a single gene mutation. The mutations were shown to be recessive and allelic. A strain carrying the tightest of the three alleles was examined in detail and was found to express the set of co-regulated phospholipid structural genes (INO1, CHO1, CHO2 and OPI3) constitutively. The Cpe(-) mutants also exhibited a pleiotropic defect in sporulation. The mutations were mapped to the right arm of chromosome XV, close to the centromere, where it was discovered that they were allelic to the previously identified regulatory mutation sin3 (sdi1, ume4, rpd1, gam2). A sin3 null mutation failed to complement the mutation conferring the Cpe(-) phenotype. A mutant harboring a sin3 null allele exhibited the same altered INO1 expression pattern observed in strains carrying the Cpe(-) mutations isolated in this study.     

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.     

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.