Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 1989 Jun;122(2):317–330. doi: 10.1093/genetics/122.2.317

Mutations in the Saccharomyces Cerevisiae Opi3 Gene: Effects on Phospholipid Methylation, Growth and Cross-Pathway Regulation of Inositol Synthesis

P McGraw 1, S A Henry 1
PMCID: PMC1203704  PMID: 2670666

Abstract

We report the isolation of two new opi3 mutants by EMS mutagenesis, and construction of an insertion allele in vitro using the cloned gene. We have demonstrated that the opi3 mutations cause a deficiency in the two terminal phospholipid N-methyltransferase (PLMT) activities required for the de novo synthesis of PC (phosphatidylcholine). The opi3 mutants, under certain growth conditions, produce membrane virtually devoid of PC although, surprisingly, none of the mutants displays a strict auxotrophic requirement for choline. Although the opi3 mutants grow without supplements, we have shown that the atypical membrane affects the ability of the mutant strains to initiate log phase growth and to sustain viability at stationary phase. The commencement of log phase growth is enhanced by addition of choline or to a lesser extent DME (dimethylethanolamine), and retarded by addition of MME (monomethylethanolamine). The mutant cells lose viability at the stationary phase of the cell cycle in the absence of DME or choline, and are also temperature sensitive for growth at 37° especially in media containing MME. These growth defects have been correlated to the presence of specific phospholipids in the membrane. The opi3 growth defects are suppressed by an unusual mutation in the phospholipid methylation pathway that perturbs the N-methyltransferase (PEMT) activity immediately preceding the reactions affected by the opi3 lesion. We believe this mutation, cho2-S, alters the substrate specificity of the PEMT. A secondary effect of opi3 mutations is disruption of the cross pathway regulation of the synthesis of the PI (phosphatidylinositol) precursor inositol. Synthesis of inositol is controlled through regulation of the INO1 gene which encodes inositol-1-phosphate synthase. This highly regulated gene is expressed constitutively in opi3 mutants. We have used the opi3 strains to demonstrate that synthesis of either PC or PD (phosphatidyldimethylethanolamine) will restore normal regulation of the INO1 gene.

Full Text

The Full Text of this article is available as a PDF (6.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Atkinson K. D., Jensen B., Kolat A. I., Storm E. M., Henry S. A., Fogel S. Yeast mutants auxotrophic for choline or ethanolamine. J Bacteriol. 1980 Feb;141(2):558–564. doi: 10.1128/jb.141.2.558-564.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Audubert F., Vance D. E. Pitfalls and problems in studies on the methylation of phosphatidylethanolamine. J Biol Chem. 1983 Sep 10;258(17):10695–10701. [PubMed] [Google Scholar]
  3. Bailis A. M., Poole M. A., Carman G. M., Henry S. A. The membrane-associated enzyme phosphatidylserine synthase is regulated at the level of mRNA abundance. Mol Cell Biol. 1987 Jan;7(1):167–176. doi: 10.1128/mcb.7.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
  5. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. CROCKEN B. J., NYC J. F. PHOSPHOLIPID VARIATIONS IN MUTANT STRAINS OF NEUROSPORA CRASSA. J Biol Chem. 1964 Jun;239:1727–1730. [PubMed] [Google Scholar]
  7. Dean-Johnson M., Henry S. A. Biosynthesis of inositol in yeast. Primary structure of myo-inositol-1-phosphate synthase (EC 5.5.1.4) and functional analysis of its structural gene, the INO1 locus. J Biol Chem. 1989 Jan 15;264(2):1274–1283. [PubMed] [Google Scholar]
  8. Donahue T. F., Henry S. A. myo-Inositol-1-phosphate synthase. Characteristics of the enzyme and identification of its structural gene in yeast. J Biol Chem. 1981 Jul 10;256(13):7077–7085. [PubMed] [Google Scholar]
  9. Elion E. A., Warner J. R. The major promoter element of rRNA transcription in yeast lies 2 kb upstream. Cell. 1984 Dec;39(3 Pt 2):663–673. doi: 10.1016/0092-8674(84)90473-2. [DOI] [PubMed] [Google Scholar]
  10. Fried H. M., Warner J. R. Cloning of yeast gene for trichodermin resistance and ribosomal protein L3. Proc Natl Acad Sci U S A. 1981 Jan;78(1):238–242. doi: 10.1073/pnas.78.1.238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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. 1982;186(2):157–163. doi: 10.1007/BF00331845. [DOI] [PubMed] [Google Scholar]
  12. Greenberg M. L., Klig L. S., Letts V. A., Loewy B. S., Henry S. A. Yeast mutant defective in phosphatidylcholine synthesis. J Bacteriol. 1983 Feb;153(2):791–799. doi: 10.1128/jb.153.2.791-799.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Greenberg M. L., Reiner B., Henry S. A. Regulatory mutations of inositol biosynthesis in yeast: isolation of inositol-excreting mutants. Genetics. 1982 Jan;100(1):19–33. doi: 10.1093/genetics/100.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  15. Hirsch J. P., Henry S. A. Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis. Mol Cell Biol. 1986 Oct;6(10):3320–3328. doi: 10.1128/mcb.6.10.3320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. KENNEDY E. P., WEISS S. B. The function of cytidine coenzymes in the biosynthesis of phospholipides. J Biol Chem. 1956 Sep;222(1):193–214. [PubMed] [Google Scholar]
  18. Kelley M. J., Bailis A. M., Henry S. A., Carman G. M. Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by inositol. Inositol is an inhibitor of phosphatidylserine synthase activity. J Biol Chem. 1988 Dec 5;263(34):18078–18085. [PubMed] [Google Scholar]
  19. Klig L. S., Homann M. J., Carman G. M., Henry S. A. Coordinate regulation of phospholipid biosynthesis in Saccharomyces cerevisiae: pleiotropically constitutive opi1 mutant. J Bacteriol. 1985 Jun;162(3):1135–1141. doi: 10.1128/jb.162.3.1135-1141.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kodaki T., Yamashita S. Yeast phosphatidylethanolamine methylation pathway. Cloning and characterization of two distinct methyltransferase genes. J Biol Chem. 1987 Nov 15;262(32):15428–15435. [PubMed] [Google Scholar]
  21. Kovác L., Gbelská I., Poliachová V., Subík J., Kovácová V. Membrane mutants: a yeast mutant with a lesion in phosphatidylserine biosynthesis. Eur J Biochem. 1980 Oct;111(2):491–501. doi: 10.1111/j.1432-1033.1980.tb04965.x. [DOI] [PubMed] [Google Scholar]
  22. LINDEGREN C. C., LINDEGREN G., SHULT E., HWANG Y. L. Centromeres, sites of affinity and gene loci on the chromosomes of Saccharomyces. Nature. 1962 Apr 21;194:260–265. doi: 10.1038/194260a0. [DOI] [PubMed] [Google Scholar]
  23. Letts V. A., Henry S. A. Regulation of phospholipid synthesis in phosphatidylserine synthase-deficient (chol) mutants of Saccharomyces cerevisiae. J Bacteriol. 1985 Aug;163(2):560–567. doi: 10.1128/jb.163.2.560-567.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lindegren G., Hwang Y. L., Oshima Y., Lindegren C. C. Genetical mutants induced by ethyl methanesulfonate in Saccharomyces. Can J Genet Cytol. 1965 Sep;7(3):491–499. doi: 10.1139/g65-064. [DOI] [PubMed] [Google Scholar]
  25. Loewy B. S., Henry S. A. The INO2 and INO4 loci of Saccharomyces cerevisiae are pleiotropic regulatory genes. Mol Cell Biol. 1984 Nov;4(11):2479–2485. doi: 10.1128/mcb.4.11.2479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  27. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ridgway N. D., Vance D. E. Purification of phosphatidylethanolamine N-methyltransferase from rat liver. J Biol Chem. 1987 Dec 15;262(35):17231–17239. [PubMed] [Google Scholar]
  29. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  30. Rozek C. E., Davidson N. Drosophila has one myosin heavy-chain gene with three developmentally regulated transcripts. Cell. 1983 Jan;32(1):23–34. doi: 10.1016/0092-8674(83)90493-2. [DOI] [PubMed] [Google Scholar]
  31. Scarborough G. A., Nyc J. F. Methylation of ethanolamine phosphatides by microsomes from normal and mutant strains of Neurospora crassa. J Biol Chem. 1967 Jan 25;242(2):238–242. [PubMed] [Google Scholar]
  32. Steiner M. R., Lester R. L. In vitro studies of phospholipid biosynthesis in Saccharomyces cerevisiae. Biochim Biophys Acta. 1972 Feb 21;260(2):222–243. doi: 10.1016/0005-2760(72)90035-5. [DOI] [PubMed] [Google Scholar]
  33. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Summers E. F., Letts V. A., McGraw P., Henry S. A. Saccharomyces cerevisiae cho2 mutants are deficient in phospholipid methylation and cross-pathway regulation of inositol synthesis. Genetics. 1988 Dec;120(4):909–922. doi: 10.1093/genetics/120.4.909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Waechter C. J., Lester R. L. Differential regulation of the N-methyl transferases responsible for phosphatidylcholine synthesis in Saccharomyces cerevisiae. Arch Biochem Biophys. 1973 Sep;158(1):401–410. doi: 10.1016/0003-9861(73)90637-1. [DOI] [PubMed] [Google Scholar]
  37. Waechter C. J., Lester R. L. Regulation of phosphatidylcholine biosynthesis in Saccharomyces cerevisiae. J Bacteriol. 1971 Mar;105(3):837–843. doi: 10.1128/jb.105.3.837-843.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yamashita S., Oshima A., Nikawa J., Hosaka K. Regulation of the phosphatidylethanolamine methylation pathway in Saccharomyces cerevisiae. Eur J Biochem. 1982 Nov 15;128(2-3):589–595. doi: 10.1111/j.1432-1033.1982.tb07005.x. [DOI] [PubMed] [Google Scholar]
  39. Yamashita S., Oshima A. Regulation of phosphatidylethanolamine methyltransferase level by myo-inositol in Saccaromyces cerevisiae. Eur J Biochem. 1980 Mar;104(2):611–616. doi: 10.1111/j.1432-1033.1980.tb04465.x. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES