Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1983 Apr;154(1):304–311. doi: 10.1128/jb.154.1.304-311.1983

Phosphatidylinositol biosynthesis in Saccharomyces cerevisiae: purification and properties of microsome-associated phosphatidylinositol synthase.

A S Fischl, G M Carman
PMCID: PMC217460  PMID: 6300035

Abstract

The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol synthase (cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified 1,000-fold from the microsomal fraction of Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of the microsomal membranes, CDPdiacylglycerol-Sepharose (Larson et al., Biochemistry 15:974-979, 1976) affinity chromatography, and chromatofocusing. The procedure resulted in the isolation of a nearly homogeneous protein preparation with an apparent minimum subunit molecular weight of 34,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Phosphatidylinositol synthase was dependent on manganese and Triton X-100 for maximum activity. The pH optimum was 8.0. Thioreactive agents inhibited enzyme activity. The energy of activation was found to be 35 kcal/mol (146,540 J/mol). The enzyme was reasonably stable at temperatures of up to 60 degrees C.

Full text

PDF
304

Images in this article

308Image
on p.308

Selected References

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

  1. Becker G. W., Lester R. L. Biosynthesis of phosphoinositol-containing sphingolipids from phosphatidylinositol by a membrane preparation from Saccharomyces cerevisiae. J Bacteriol. 1980 Jun;142(3):747–754. doi: 10.1128/jb.142.3.747-754.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Becker G. W., Lester R. L. Changes in phospholipids of Saccharomyces cerevisiae associated with inositol-less death. J Biol Chem. 1977 Dec 10;252(23):8684–8691. [PubMed] [Google Scholar]
  3. Belendiuk G., Mangnall D., Tung B., Westley J., Getz G. S. CTP-phosphatidic acid cytidyltransferase from Saccharomyces cerevisiae. Partial purification, characterization, and kinetic behavior. J Biol Chem. 1978 Jul 10;253(13):4555–4565. [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Carman G. M., Dowhan W. Phosphatidylserine synthase from Escherichia coli. The role of Triton X-100 in catalysis. J Biol Chem. 1979 Sep 10;254(17):8391–8397. [PubMed] [Google Scholar]
  6. Carman G. M., Matas J. Solubilization of microsomal-associated phosphatidylserine synthase and phosphatidylinositol synthase from Saccharomyces cerevisiae. Can J Microbiol. 1981 Nov;27(11):1140–1149. doi: 10.1139/m81-179. [DOI] [PubMed] [Google Scholar]
  7. Cobon G. S., Crowfoot P. D., Linnane A. W. Biogenesis of mitchondria. Phospholipid synthesis in vitro by yeast mitochondrial and microsomal fractions. Biochem J. 1974 Nov;144(2):265–275. doi: 10.1042/bj1440265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cousminer J. J., Carman G. M. Solubilization of membrane-associated phosphatidylserine synthase from Clostridium perfringens. Can J Microbiol. 1981 May;27(5):544–547. doi: 10.1139/m81-080. [DOI] [PubMed] [Google Scholar]
  9. Cousminer J. J., Fischl A. S., Carman G. M. Partial purification and properties of phosphatidylserine synthase from Clostridium perfringens. J Bacteriol. 1982 Sep;151(3):1372–1379. doi: 10.1128/jb.151.3.1372-1379.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DITTMER J. C., LESTER R. L. A SIMPLE, SPECIFIC SPRAY FOR THE DETECTION OF PHOSPHOLIPIDS ON THIN-LAYER CHROMATOGRAMS. J Lipid Res. 1964 Jan;5:126–127. [PubMed] [Google Scholar]
  11. Dowhan W., Wickner W. T., Kennedy E. P. Purification and properties of phosphatidylserine decarboxylase from Escherichia coli. J Biol Chem. 1974 May 25;249(10):3079–3084. [PubMed] [Google Scholar]
  12. Green P. R., Merrill A. H., Jr, Bell R. M. Membrane phospholipid synthesis in Escherichia coli. Purification, reconstitution, and characterization of sn-glycerol-3-phosphate acyltransferase. J Biol Chem. 1981 Nov 10;256(21):11151–11159. [PubMed] [Google Scholar]
  13. Hanson B. A., Lester R. L. Effects of inositol starvation on phospholipid and glycan syntheses in Saccharomyces cerevisiae. J Bacteriol. 1980 Apr;142(1):79–89. doi: 10.1128/jb.142.1.79-89.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Helenius A., Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta. 1975 Mar 25;415(1):29–79. doi: 10.1016/0304-4157(75)90016-7. [DOI] [PubMed] [Google Scholar]
  15. Hirabayashi T., Larson T. J., Dowhan W. Membrane-associated phosphatidylglycerophosphate synthetase from Escherichia coli: purification by substrate affinity chromatography on cytidine 5'-diphospho-1,2-diacyl-sn-glycerol sepharose. Biochemistry. 1976 Nov 30;15(24):5205–5211. doi: 10.1021/bi00669a002. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Larson T. J., Dowhan W. Ribosomal-associated phosphatidylserine synthetase from Escherichia coli: purification by substrate-specific elution from phosphocellulose using cytidine 5'-diphospho-1,2-diacyl-sn-glycerol. Biochemistry. 1976 Nov 30;15(24):5212–5218. doi: 10.1021/bi00669a003. [DOI] [PubMed] [Google Scholar]
  18. Larson T. J., Hirabayshi T., Dowhan W. Phosphatidylglycerol biosynthesis in Bacillus licheniformis Resolution of membrane-bound enzymes by affinity chromatography on cytidinediphospho-sn-1,2-diacylglycerol Sepharose. Biochemistry. 1976 Mar 9;15(5):974–979. doi: 10.1021/bi00650a005. [DOI] [PubMed] [Google Scholar]
  19. Lester R. L., Steiner M. R. The occurrence of diphosphoinositide and triphosphoinositide in Saccharomyces cerevisiae. J Biol Chem. 1968 Sep 25;243(18):4889–4893. [PubMed] [Google Scholar]
  20. March S. C., Parikh I., Cuatrecasas P. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal Biochem. 1974 Jul;60(1):149–152. doi: 10.1016/0003-2697(74)90139-0. [DOI] [PubMed] [Google Scholar]
  21. Ohta A., Waggoner K., Louie K., Dowhan W. Cloning of genes involved in membrane lipid synthesis. Effects of amplification of phosphatidylserine synthase in Escherichia coli. J Biol Chem. 1981 Mar 10;256(5):2219–2225. [PubMed] [Google Scholar]
  22. PAULUS H., KENNEDY E. P. The enzymatic synthesis of inositol monophosphatide. J Biol Chem. 1960 May;235:1303–1311. [PubMed] [Google Scholar]
  23. Raetz C. R., Dowhan W., Kennedy E. P. Partial purification and characterization of cytidine 5'-diphosphate-diglyceride hydrolase from membranes of Escherichia coli. J Bacteriol. 1976 Mar;125(3):855–863. doi: 10.1128/jb.125.3.855-863.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Raetz C. R., Larson T. J., Dowhan W. Gene cloning for the isolation of enzymes of membrane lipid synthesis: phosphatidylserine synthase overproduction in Escherichia coli. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1412–1416. doi: 10.1073/pnas.74.4.1412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Tanford C., Nozaki Y., Reynolds J. A., Makino S. Molecular characterization of proteins in detergent solutions. Biochemistry. 1974 May 21;13(11):2369–2376. doi: 10.1021/bi00708a021. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES