Isolation of the yeast INO1 gene: located on an autonomously replicating plasmid, the gene is fully regulated.

Abstract

The Saccharomyces cerevisiae gene, INO), encoding the highly regulated enzyme, myo-inositol-l-phosphate synthase [1L-myo-inositol-l-phosphate Iyase (isomerizing), EC 5.5.1.4], was isolated by genetic complementation. The cloned sequence was shown to complement two independent INO) alleles (inol-5 and inol-13). One of these mutants (inol-5) fails to make any material that is crossreactive with antibody to the wild-type inositol-l-phosphate synthase. The cloned DNA restored not only inositol prototrophy to this mutant but also its ability to make material crossreactive with anti-inositol-l-phosphate synthase antibody. The sequence on an integrative plasmid was also shown to recombine with the INO) locus, thereby confirming its genetic identity. The DNA was subcloned and used for Southern blot analysis, revealing that the cloned DNA (5.4 kilobases long) represents a unique sequence in the yeast genome. Inositol-l-phosphate synthase was fully regulated when its gene was located extrachromosomally on the autonomously replicating plasmid. In cells (inol ) containing the cloned INOl gene on a high-copy-number plasmid, the enzyme was fully repressible. Furthermore, the gene product was not expressed when the plasmid was transferred into a strain containing an ino4 mutation, which also prevents expression of chromosomal copies of INOJ.These results establish that the intact structural gene and associated regulatory components have been isolated and that positioning of the gene in its normal chromosomal location is not required for full regulation of inositol-l-phosphate synthase.The synthesis of inositol, a critical phospholipid precursor, is a highly regulated process in yeast. myo-Inositol-1-phosphate synthase [Ins1P synthase; 1L-myo-inositol-1-phosphate lyase (isomerizing), EC 5.5.1.4], a repressible cytoplasmic enzyme (1), is the major biosynthetic enzyme in this pathway. This enzyme has been purified from several eukaryotic organisms, including mammals (2) and yeast (1). In yeast (1), the enzyme has a molecular size in excess of 200,000 daltons and is a multimer of identical subunits of 62,000 daltons. The reaction this enzyme catalyzes results in the conversion of glucose 6-phosphate to inositol 1-phosphate. The reaction includes a ring closure and a coupled oxidation and reduction in which NAD is a cofactor (1). Despite intensive biochemical analysis, the precise reaction mechanism remains to be proven, although it has been proposed to consist of three partial reactions involving the intermediates 5-ketoglucose 6-phosphate and inosose-2 1-phosphate (3).In yeast, InslP synthase is the gene product of the INOI gene (1), but a large number of other genes are known to affect its expression. For example, the ino2 and ino4 mutations, which are unlinked to INOI, prevent expression of InslP synthase, while the opil, opi2, and opi4 mutations render the enzyme constitutive (4). Several lines of evidence suggest that some of these unlinked regulatory genes are involved in the coordination of InslP synthase expression...

Regulation of phosphatidate phosphatase activity by inositol in Saccharomyces cerevisiae

“…The ino2 and ino4 mutants are inositol auxotrophs (11, 13) because they are unable to derepress inositol-1-phosphate synthase (10,12,29) and exhibit pleiotrophic effects on CDP-diacylglycerol synthase (23), phosphatidylserine synthase (3,23), and phospholipid N-methyltransferase (34) activities. PA phosphatase activity was reduced in ino2 mutant cells compared with wild-type cells grown with inositol (Fig.…”

Regulation of Phospholipid Synthesis in Saccharomyces cerevisiae by Zinc

“…Gene-The expression of the INO1 gene, which encodes inositol 3-phosphate synthase, 3 is regulated by inositol supplementation (105)(106)(107). This regulation is mediated by the transcription factors Ino2p, Ino4p, and Opi1p and occurs through the UA-S INO element in the INO1 promoter (67, 101, 102, 108 -115).…”

A Novel Salt-tolerant l-myo-Inositol-1-phosphate Synthase from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice

“…This mechanism is followed by all myo-inositolproducing organisms throughout the phylogenetic lines, and MIPS has been identified as an evolutionarily conserved protein (4). The structural gene coding for cytosolic MIPS, termed INO1, was first identified in yeast, Saccharomyces cerevisiae (5,6) and cloned by Klig and Henry (7). Subsequently, the complete nucleotide sequence of the full-length INO1 gene from S. cerevisiae was reported by Johnson and Henry (8).…”