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.
Similar content being viewed by others
References
Barnett JEG, Brice, RE, Corina DL (1970) A colorimetric determination of inositol monophosphates as an assay for D-glucose-6-phosphate-L-myo-inositol-1-phosphate cyclase. Biochem J 119:183
Chen I, Charalamopus FC (1966) Biochemical studies on inositol. IX. Inositol-1-phosphate as intermediate in the biosynthesis of inositol from glucose-6-phosphate and characteristics of two reactions in this biosynthesis. J Biol Chem 241:2194
Culbertson MR, Donahue, TF, Henry SA (1976a) Control of inositol biosynthesis in Saccharomyces cerevisiae: I. Properties of a repressible enzyme system in extracts of wild type (ino+) cells. J Bacteriol 126:232
Culbertson MR, Donahue TF, Henry SA (1976) Control of inositol biosynthesis in Saccharomyces cerevisiae: II. Inositol-1-phosphate synthetase mutants. J Bacteriol 126:243
Donahue TF (1979) Biochemical and genetic analysis of inositol-1-phosphate mutants of yeast. Ph.D. dissertation, Albert Einstein College of Medicine, New York
Donahue TF, Atkinson A, Kolat A, Henry S (1978) Inositol-1-phosphate synthase mutants of yeast Saccharomyces cerevisiae. In: Eisenberg F, Wells W (eds) Cyclitols and phosphoinositides. Academic Press, New York, pp 311–316
Donahue TF, Henry SA (1981) L-myo-inositol-1-phosphate synthase: characteristics of the enzyme and identification of its structural gene in yeast. J Biol Chem 256:7077–7085
Greenberg ML, Reiner B, Henry SA (1982) Regulatory mutations of inositol biosynthesis in yeast: Isolation of inositol-excreting mutants. Genetics, (in press)
Jackson M, Shin S (1980) Analysis of inositol starvation in an inositol requiring mutant of CHO-K1. J Cell Biol 87:293a
Keesey J, Bigelis R, Fink GR (1979) The product of the his4 gene cluster in Saccharomyces cerevisiae: A trifunctional polypeptide. J Biol Chem 254:7427
Laemmli UK (1978) Cleavage of structural proteins during the assembly of the head of bacteriphage T4. Nature 227:680
Majumder A, Duttagupta S, Goldwasser P, Donahue T, Henry S (1981) Mechanism of interallelic complementation at the Ino1 locus in yeast: Immunological analysis of mutant. Mol Gen Genet 184:347–354
Oshima Y (1982) Regulatory circuts for gene expression: The metabolism of galactose and of phosphate. In: Strathern J Jones E, Broach JR (eds) The molecular biology of the yeast Saccharomyces. Cold Springer Harbor Press, New York, in press
Paukert JL, Williams GR, Robinowitz JC (1977) Formylmethenyl-methylene-tetrahydrofolate synthetase (combined): Correlation of enzymic activities with limited proteolytic degradation of the protein from yeast. Biochem Biophys Res Commun 77:147
Schweizer E, Werkmeister K, Jain M (1978) Fatty acid biosynthesis in yeast. Mol Cell Biochem 21:95
Stewart PR (1975) Analytical methods for yeast. In: Methods in cell biology: 12:111–147
Strauss BS (1958) Cell death and unbalanced growth in Neurospora. J Gen Microbiol 18:658–669
Thomas PL (1972) Increased frequency of auxotrophic mutants of Ustilago hordei after combined UV irradiation and inositol starvation. Can J Genet Cytol 14:785–788
Author information
Authors and Affiliations
Additional information
Communicated by G.R. Fink
Rights and permissions
About this article
Cite this article
Greenberg, M.L., Goldwasser, P. & Henry, S.A. Characterization of a yeast regulatory mutant constitutive for synthesis of inositol-1-phosphate synthase. Mol Gen Genet 186, 157–163 (1982). https://doi.org/10.1007/BF00331845
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00331845