Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis.

Abstract

The INO1 gene of Saccharomyces cerevisiae encodes the regulated enzyme inositol-1-phosphate synthase, which catalyzes the first committed step in the synthesis of inositol-containing phospholipids. The expression of this gene was analyzed under conditions known to regulate phospholipid synthesis. RNA blot hybridization with a genomic clone for INO1 detected two RNA species of 1.8 and 0.6 kb. The abundance of the 1.8-kb RNA was greatly decreased when the cells were grown in the presence of the phospholipid precursor inositol, as was the enzyme activity of the synthase. Complementation analysis showed that this transcript encoded the INO1 gene product. The level of INO1 RNA was repressed 12-fold when the cells were grown in medium containing inositol, and it was repressed 33-fold when the cells were grown in the presence of inositol and choline together. The INO1 transcript was present at a very low level in cells containing mutations (ino2 and ino4) in regulatory genes unlinked to INO1 that result in inositol auxotrophy. The transcript was constitutively overproduced in cells containing a mutation (opi1) that causes constitutive expression of inositol-1-phosphate synthase and results in excretion of inositol. The expression of INO1 RNA was also examined in cells containing a mutation (cho2) affecting the synthesis of phosphatidylcholine. In contrast to what was observed in wild-type cells, growth of cho2 cells in medium containing inositol did not result in a significant decrease in INO1 RNA abundance. Inositol and choline together were required for repression of the INO1 transcript in these cells, providing evidence for a regulatory link between the synthesis of inositol- and choline-containing lipids. The level of the 0.6-kb RNA was affected, although to a lesser degree, by many of the same factors that influence INO1 expression.

Genome-wide Analysis Reveals Inositol, Not Choline, as the Major Effector of Ino2p-Ino4p and Unfolded Protein Response Target Gene Expression in Yeast

“…For example, it was originally reported that INO1 expression is repressed by an additional 2-3-fold in cells grown in inositol-containing medium when choline is present compared with cells grown in inositol-containing medium when choline is absent (16). Similar simultaneous effects of inositol and choline have also been observed for CHO1 (17,20), OP13 (21), CHO2 (21), CKI1 (52), and CPT1 (53).…”

“…Strains were maintained on yeast extract/peptone/dextrose (YPD) plates (1% yeast extract, 2% bactopeptone, 2% glucose, and 2% agar). All experiments were performed using chemically defined synthetic complete media (16,40), containing (per liter): 20 g of glucose, 5 g of ammonium sulfate, 1 g of potassium phosphate, 0.5 of g magnesium sulfate, 1 g of sodium chloride, 0.1 g of calcium chloride, 0.5 mg of boric acid, 0.04 mg of cupric sulfate, 0.1 mg of potassium iodide, 0.2 mg of ferric chloride, 0.4 mg of manganese sulfate, 0.2 mg of sodium molybdate, 0.4 mg of zinc sulfate, 2 g of biotin, 400 g of calcium pantothenate, 2 g of folic acid, 400 g of niacin, 200 g of p-aminobenzoic acid, 400 g of pyridoxine hydrochloride, 200 g of riboflavin, 400 g of thiamine, 20 mg of adenine sulfate, 20 mg of arginine, 20 mg of histidine, 60 mg of leucine, 230 mg of lysine, 20 mg of methionine, 300 mg of threonine, 20 mg of tryptophan, and 40 mg of uracil. Where indicated, media were supplemented with 75 M myo-inositol and/or 1 mM choline.…”

“…pSJ32-HO was constructed by PCR-amplifying the HO ORF and inserting the 365-bp PstI-BamHI fragment into pGEM1. Membranes were hybridized with either INO1, KAR2, PDI1, VTC3, HO, ACT1, Ty1, or Ty2 probes in formamide hybridization buffer and washed to a final stringency of 0.2ϫ SSC/0.1% SDS at 60°C as described previously (16,41). Quantitation was performed by analysis on a STORM 860 PhosphorImager (Amersham Biosciences) and analyzed with ImageQuant software.…”

Dimethyl Sulfoxide Exposure Facilitates Phospholipid Biosynthesis and Cellular Membrane Proliferation in Yeast Cells

“…The expression of INO1 and OIP3 had been shown previously to be regulated at the level of transcript abundance. The rate-limiting step in inositol phospholipid biosynthesis is mediated by the INO1 gene product (30,31). Therefore, we propose that PC synthesis in yeast cells is promoted by Me 2 SO exposure, because there was significant enhancement in the levels of mRNA for INO1 and OPI3 as shown in Table I and Fig.…”

Epi-inositol regulates expression of the yeast INO1 gene encoding inositol-1-P synthase

“…RNA probes for northern analysis were synthesized using the Gemini II core system from plasmids linearized with a restriction enzyme as follows (plasmid, restriction enzyme, RNA polymerase): pJH310, HindIII, T7 (INO1); pAB309, EcoRI, SP6 (TCM1). 30 Hybridization with the pJH310-derived probe 31 detects a single 1.8-kb INO1 transcript, which is not detected in RNA from strains containing a deletion of the INO1 gene. The results of northern blot hybridization were visualized by autoradiography, and relative levels of INO1 and TCM1 were quantitated by phosphorimaging.…”