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.     

The yeast phospholipid N-methyltransferases catalyzing the synthesis of phosphatidylcholine preferentially convert di-C16:1 substrates both in vivo and in vitro

Phosphatidylcholine (PC) is an important and abundant structural component of the membranes of eukaryotic cells. In the yeast Saccharomyces cerevisiae, the primary route for the biosynthesis of PC consists of three consecutive methylation steps of phosphatidylethanolamine (PE) catalyzed by the phospholipid N-methyltransferases Cho2p and Opi3p. To investigate how these biosynthetic enzymes contribute to the composition of the PC species profile, the precursor-product relationships between PE and newly synthesized PC were determined at the level of the molecular species by using electrospray ionization tandem mass spectrometry and stable isotope labeling. In vivo labeling of yeast cells for 10 min with [methyl-D3]methionine revealed the preferential methylation of di-C16:1 PE over a range of PE species compositions. A similar preferential conversion of di-C16:1 PE to PC was found in vitro upon incubating isolated microsomes with S-adenosyl[methyl-D3]methionine. Yeast opi3 and cho2 deletion strains were used to distinguish between the substrate selectivities of Cho2p and Opi3p, respectively. Both biosynthetic enzymes were found to participate in the speciesselective methylation with Cho2p contributing the most. The combined results indicate that the selective methylation of PE species by the methyltransferases plays an important role in shaping the steady-state profile of PC molecular species in yeast.     

Opi1p,Ume6p and Sin3p control expression from the promoter of the INO2 regulatory gene via a novel regulatory cascade

The INO2 gene of Saccharomyces cerevisiae is required for expression of most of the phospholipid biosynthetic genes. INO2 expression is regulated by a complex cascade that includes autoregulation, Opi1p-mediated repression and Ume6p-mediated activation. To screen for mutants with altered INO2 expression directly, we constructed an INO2-HIS3 reporter that provides a plate assay for INO2 promoter activity. This reporter was used to isolate mutants (dim1) that fail to repress expression of the INO2 gene in an otherwise wild-type strain. The dim1 mutants contain mutations in the OPI1 gene. To define further the mechanism for Ume6p regulation of INO2 expression, we isolated suppressors (rum1, 2, 3) of the ume6Delta mutation that overexpress the INO2-HIS3 gene. Two of the rum mutant groups contain mutations in the OPI1 and SIN3 genes showing that opi1 and sin3 mutations are epistatic to the ume6Delta mutation. These results are surprising given that Ume6p, Sin3p and Rpd3p are known to form a complex that represses the expression of a diverse set of yeast genes. This prompted us to examine the effect of sin3Delta and rpd3Delta mutants on INO2-cat expression. Surprisingly, the sin3Delta allele overexpressed INO2-cat, whereas the rpd3Delta mutant had no effect. We also show that the UME6 gene does not affect the expression of an OPI1-cat reporter. This suggests that Ume6p does not regulate INO2 expression indirectly by regulating OPI1 expression.     

Identification of novel suppressors for Mog1 implies its involvement in RNA metabolism, lipid metabolism and signal transduction

Mog1 is conserved from yeast to mammal, but its function is obscure. We isolated yeast genes that rescued a temperature-sensitive death of S. cerevisiae Scmog1Δ, and of S. pombe Spmog1^ts. Scmog1Δ was rescued by Opi3p, a phospholipid N-methyltransferase, in addition to S. cerevisiae Ran-homologue Gsp1p, and a RanGDP binding protein Ntf2p. On the other hand, Spmog1^ts was rescued by Cid13 that is a poly (A) polymerase specific for suc22⁺ mRNA encoding a subunit of ribonucleotide reductase, Ssp1 that is a protein kinase involved in stress response pathway, and Crp79 that is required for mRNA export, in addition to Spi1, S. pombe Ran-homologue, and Nxt2, S. pombe homologue of Ntf2p. Consistent with the identification of those suppressors, lack of ScMog1p dislocates Opi3p from the nuclear membrane and all of Spmog1^ts showed the nuclear accumulation of mRNA. Furthermore, SpMog1 was co-precipitated with Nxt2 and Cid13.     

Discrimination of yeast genes involved in methionine and phosphate metabolism on the basis of upstream motifs

MOTIVATION: In yeast, methionine and phosphate metabolism are regulated by the complexes Met4p/Met28p/Cbf1p and Pho4p, respectively. The binding sites for these factors share a common core CACGTG. We evaluate our capability to discriminate phosphate- and methionine-responding genes on the basis of putative regulatory elements, despite the similarity between Met4p/Met28p/Cbf1p and Pho4p consensus. RESULTS: We scanned upstream regions of methionine, phosphate and control genes with position-specific weight matrices for Pho4p, Met4p/Met28p/Cbf1p and Met31p/Met32p, and applied discriminant analysis to classify genes according to matrix matching scores. This analysis showed that matrix scores provided a good discrimination between phosphate, methionine and control genes. The optimal parameters have then been used to predict phosphate and methionine regulation at a genome scale. The genome-scale analysis predicts 37 genes as methionine-regulated and 40 as phosphate-regulated. We compare the predictive results with high throughput data and discuss the difference. AVAILABILITY: The programs for sequence retrieval and analysis, as well as the complete data and results, are available on the website on regulatory sequence analysis tools (http://rsat.scmbb.ulb.ac.be/rsat/). CONTACT: jvanheld@scmbb.ulb.ac.be SUPPLEMENTARY INFORMATION: The complete datasets and results are available at http://rsat.scmbb.ulb.ac.be/rsat/data/published_data/Gonze_MET_PHO/