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Measuring Phosphatidylinositol Generation on Biological Membranes

  • Mark G. WaughEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1376)

Abstract

Phosphatidylinositol (PI) is a phospholipid molecule required for the generation of seven different phosphoinositide lipids which have a diverse range of signaling and trafficking functions. The precise mechanism of phosphatidylinositol supply during receptor activated signaling and the cellular compartmentation of the synthetic process are still incompletely understood and remain controversial despite several decades of research in this area. The synthesis of phosphatidylinositol requires the activity of an enzyme called phosphatidylinositol synthase, also known as CDIPT, which catalyzes a reversible headgroup exchange reaction on its substrate liponucleotide CDP-diacylglycerol resulting in the incorporation of inositol to generate phosphatidylinositol and the release of CMP. This protocol describes a method for locating PI synthase activity in isolated, intact biological membranes and vesicles.

Key words

Phosphatidylinositol CDIPT Lipid Endoplasmic reticulum 

Notes

Acknowledgements

MGW acknowledges financial support from the Royal Free Charity.

References

  1. 1.
    Antonsson B (1997) Phosphatidylinositol synthase from mammalian tissues. Biochim Biophys Acta 1348:179–186PubMedCrossRefGoogle Scholar
  2. 2.
    Antonsson BE (1994) Purification and characterization of phosphatidylinositol synthase from human placenta. Biochem J 297(Pt 3):517–522PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Monaco ME, Feldman M, Kleinberg DL (1994) Identification of rat liver phosphatidylinositol synthase as a 21 kDa protein. Biochem J 304(Pt 1):301–305PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Cubitt AB, Gershengorn MC (1990) CMP activates reversal of phosphatidylinositol synthase and base exchange by distinct mechanisms in rat pituitary GH3 cells. Biochem J 272:813–816PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Lykidis A, Jackson PD, Rock CO, Jackowski S (1997) The role of CDP-diacylglycerol synthetase and phosphatidylinositol synthase activity levels in the regulation of cellular phosphatidylinositol content. J Biol Chem 272:33402–33409PubMedCrossRefGoogle Scholar
  6. 6.
    Waugh MG, Minogue S, Clayton EL, Hsuan JJ (2011) CDP-diacylglycerol phospholipid synthesis in detergent-soluble, non-raft, membrane microdomains of the endoplasmic reticulum. J Lipid Res 52:2148–2158PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Kim YJ, Guzman-Hernandez ML, Balla T (2011) A highly dynamic ER-derived phosphatidylinositol-synthesizing organelle supplies phosphoinositides to cellular membranes. Dev Cell 21:813–824PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Imai A, Gershengorn MC (1987) Regulation by phosphatidylinositol of rat pituitary plasma membrane and endoplasmic reticulum phosphatidylinositol synthase activities. A mechanism for activation of phosphoinositide resynthesis during cell stimulation. J Biol Chem 262:6457–6459PubMedGoogle Scholar
  9. 9.
    Imai A, Gershengorn MC (1987) Independent phosphatidylinositol synthesis in pituitary plasma membrane and endoplasmic reticulum. Nature 325:726–728PubMedCrossRefGoogle Scholar
  10. 10.
    Piatti E, Piacentini MP, Fraternale D, Bucchini A, Mangani F, Accorsi A (1999) myo-[3H]-inositol loaded erythrocytes and white ghosts: two models to investigate the phosphatidylinositol synthesis in human red cells. Biochimie 81:1011–1014PubMedCrossRefGoogle Scholar
  11. 11.
    Claro E, Wallace MA, Fain JN (1992) Concerted CMP-dependent [3H]inositol labeling of phosphoinositides and agonist activation of phospholipase C in rat brain cortical membranes. J Neurochem 58:2155–2161PubMedCrossRefGoogle Scholar
  12. 12.
    Sillence DJ, Downes CP (1993) Subcellular distribution of agonist-stimulated phosphatidylinositol synthesis in 1321 N1 astrocytoma cells. Biochem J 290(Pt 2):381–387PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Vaziri C, Downes CP, Macfarlane SC (1993) Direct labelling of hormone-sensitive phosphoinositides by a plasma-membrane-associated PtdIns synthase in turkey erythrocytes. Biochem J 294(Pt 3):793–799PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Kaur J, Sawhney M, Dattagupta S, Shukla NK, Srivastava A, Ralhan R (2010) Clinical significance of phosphatidyl inositol synthase overexpression in oral cancer. BMC Cancer 10:168PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Thakur PC, Davison JM, Stuckenholz C, Lu L, Bahary N (2014) Dysregulated phosphatidylinositol signaling promotes endoplasmic-reticulum-stress-mediated intestinal mucosal injury and inflammation in zebrafish. Dis Model Mech 7:93–106PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Murphy TR, Vihtelic TS, Ile KE, Watson CT, Willer GB, Gregg RG, Bankaitis VA, Hyde DR (2011) Phosphatidylinositol synthase is required for lens structural integrity and photoreceptor cell survival in the zebrafish eye. Exp Eye Res 93:460–474PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Waugh MG, Hsuan JJ (2009) Preparation of membrane rafts. Methods Mol Biol 462:403–414PubMedGoogle Scholar
  18. 18.
    Waugh MG, Lawson D, Tan SK, Hsuan JJ (1998) Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant density non-caveolar membranes. J Biol Chem 273:17115–17121PubMedCrossRefGoogle Scholar
  19. 19.
    Waugh MG, Chu KM, Clayton EL, Minogue S, Hsuan JJ (2011) Detergent-free isolation and characterization of cholesterol-rich membrane domains from trans-Golgi network vesicles. J Lipid Res 52:582–589PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Waugh MG, Minogue S, Blumenkrantz D, Anderson JS, Hsuan JJ (2003) Identification and characterization of differentially active pools of type IIalpha phosphatidylinositol 4-kinase activity in unstimulated A431 cells. Biochem J 376:497–503PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Minogue S, Chu KM, Westover EJ, Covey DF, Hsuan JJ, Waugh MG (2010) Relationship between phosphatidylinositol 4-phosphate synthesis, membrane organization, and lateral diffusion of PI4KIIalpha at the trans-Golgi network. J Lipid Res 51:2314–2324PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Carman GM, Fischl AS (1992) Phosphatidylinositol synthase from yeast. Methods Enzymol 209:305–312PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.School of Life and Medical SciencesUniversity College LondonLondonUK

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