BIOSYNTHESIS OF INOSITOL IN YEAST: STRUCTURAL STUDIES OF THE SACCHAROMYCES CEREVISIAE INO1 GENE AND ITS PRODUCT MYO-INOSITOL-1-PHOSPHATE SYNTHASE (EC 126.96.36.199)
JOHNSON, MARGARET DEAN
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A biochemical, molecular and genetic analysis of the S. cerevisiae INO1 gene and its product, MI-1-P synthase has provided, for the first time, a physical basis for the organization of the INO1 locus and regulation of its gene product, MI-1-P synthase. Establishment of the primary structure of this multifunctional enzyme makes its complex reaction mechanism, which has eluded classical biochemical analysis for over two decades, amenable to molecular dissection. Biosynthesis of the phospholipid precursor, inositol is one of the most highly regulated aspects of phospholipid metabolism in eukaryotic cells. In yeast, this regulation is centered on the cytoplasmic enzyme MI-1-P synthase which catalyzes the first committed step in the biosynthesis of inositol from glucose.;In the present study, the sequence of the entire INO1 gene and surrounding regions has been determined. Computer analysis of the DNA sequence predicts four peptides which are read in the same reading frame. The largest open reading frame of 553 amino acids corresponding to a 1.8 kb message has been shown to encode MI-1-P synthase. This open reading frame predicts a peptide with a molecular weight of 62,842. N-terminus analysis of MI-1-P synthase establishes the fact that this large open reading frame encodes MI-1-P synthase.;The largest of the three small ORFs adjacent to INO1 predicts a protein of 133 amino acids with a molecular weight of 15,182. The first 40 amino acids are similar in sequence to known signal sequences from a variety of sources. The remaining amino acid sequence has a long stretch of highly hydrophobic residues. These two features together suggest that the protein encoded by this small ORF is membrane associated. A null mutation in the chromosomal locus of this small ORF causes inositol to be excreted from the cell. A null mutation at INO1 is viable, producing an inositol auxotroph which fails to express any protein cross reactive to antibody directed against MI-1-P synthase.;MI-1-P synthase, like other enzymes involved in phospholipid metabolism is subject to coordinate regulation by the precursors inositol and choline. These studies will provide the physical foundation needed to dissect the complex regulatory network involved in the coordinate regulation of phospholipid biosynthesis in yeast.