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. 1993 Sep 15;294(Pt 3):793–799. doi: 10.1042/bj2940793

Direct labelling of hormone-sensitive phosphoinositides by a plasma-membrane-associated PtdIns synthase in turkey erythrocytes.

C Vaziri 1, C P Downes 1, S C Macfarlane 1
PMCID: PMC1134531  PMID: 8397510

Abstract

We have previously characterized phosphatidylinositol (PtdIns) synthase and PtdIns/myo-inositol-exchange enzyme activities in ghost membranes prepared by hypotonic lysis of turkey erythrocytes [McPhee, Lowe, Vaziri and Downes (1991) Biochem. J. 275, 187-192]. Here we show that PtdIns synthase activity is relatively enriched in plasma-membrane preparations of turkey erythrocytes and that inositol phospholipids labelled by both PtdIns synthase and PtdIns myo-inositol exchange enzymes are susceptible to hydrolysis by the receptor- and G-protein-regulated phospholipase C (PLC), which is present also in ghost preparations. Specific-radioactivity measurements of [3H]PtdIns from ghosts labelled to equilibrium under conditions favouring [3H]inositol incorporation by PtdIns synthase activity indicate that PtdIns synthase can directly access approx. 14% of the total erythrocyte ghost PtdIns. Approx. 16% of the [3H]PtdIns labelled by the PtdIns synthase reaction can be phosphorylated to polyphosphoinositides, which are then hydrolysed by the receptor- and G-protein-stimulated PLC. Since the mass of PtdIns declines to a similar extent as [3H]PtdIns during stimulation in the presence of guanine nucleotides and ATP, it is evident that both the labelled and unlabelled phosphoinositides are susceptible to hydrolysis by the relevant PLC. Phosphoinositides present in nuclei-free plasma membranes were also labelled by [3H]inositol under conditions favouring PtdIns synthase and PtdIns/myo-inositol-exchange enzyme activities respectively. These membranes lack PLC activity [Vaziri and Downes (1992) J. Biol. Chem. 267, 22973-22981], but the labelled lipids were sensitive to purinergic-receptor-stimulated hydrolysis in reconstitution assays using partially purified turkey erythrocyte PLC. The results strongly suggest that at least a portion of the PtdIns synthase in turkey erythrocytes is located in the plasma membrane and has direct access to an agonist-sensitive pool of inositol phospholipids.

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Selected References

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  1. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  2. Beam K. G., Alper S. L., Palade G. E., Greengard P. Hormonally regulated phosphoprotein of turkey erythrocytes: localization to plasma membrane. J Cell Biol. 1979 Oct;83(1):1–15. doi: 10.1083/jcb.83.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berridge M. J., Downes C. P., Hanley M. R. Neural and developmental actions of lithium: a unifying hypothesis. Cell. 1989 Nov 3;59(3):411–419. doi: 10.1016/0092-8674(89)90026-3. [DOI] [PubMed] [Google Scholar]
  4. Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature. 1989 Sep 21;341(6239):197–205. doi: 10.1038/341197a0. [DOI] [PubMed] [Google Scholar]
  5. Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
  6. Berry G., Yandrasitz J. R., Segal S. CMP-dependent phosphatidylinositol:myo-inositol exchange activity in isolated nerve-endings. Biochem Biophys Res Commun. 1983 May 16;112(3):817–821. doi: 10.1016/0006-291x(83)91690-x. [DOI] [PubMed] [Google Scholar]
  7. Bishop W. R., Bell R. M. Assembly of phospholipids into cellular membranes: biosynthesis, transmembrane movement and intracellular translocation. Annu Rev Cell Biol. 1988;4:579–610. doi: 10.1146/annurev.cb.04.110188.003051. [DOI] [PubMed] [Google Scholar]
  8. Boyer J. L., Waldo G. L., Evans T., Northup J. K., Downes C. P., Harden T. K. Modification of AlF-4- and receptor-stimulated phospholipase C activity by G-protein beta gamma subunits. J Biol Chem. 1989 Aug 15;264(23):13917–13922. [PubMed] [Google Scholar]
  9. Carpenter C. L., Cantley L. C. Phosphoinositide kinases. Biochemistry. 1990 Dec 25;29(51):11147–11156. doi: 10.1021/bi00503a001. [DOI] [PubMed] [Google Scholar]
  10. Cocco L., Gilmour R. S., Ognibene A., Letcher A. J., Manzoli F. A., Irvine R. F. Synthesis of polyphosphoinositides in nuclei of Friend cells. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation. Biochem J. 1987 Dec 15;248(3):765–770. doi: 10.1042/bj2480765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Divecha N., Banfić H., Irvine R. F. The polyphosphoinositide cycle exists in the nuclei of Swiss 3T3 cells under the control of a receptor (for IGF-I) in the plasma membrane, and stimulation of the cycle increases nuclear diacylglycerol and apparently induces translocation of protein kinase C to the nucleus. EMBO J. 1991 Nov;10(11):3207–3214. doi: 10.1002/j.1460-2075.1991.tb04883.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fischl A. S., Homann M. J., Poole M. A., Carman G. M. Phosphatidylinositol synthase from Saccharomyces cerevisiae. Reconstitution, characterization, and regulation of activity. J Biol Chem. 1986 Mar 5;261(7):3178–3183. [PubMed] [Google Scholar]
  13. Harden T. K., Hawkins P. T., Stephens L., Boyer J. L., Downes C. P. Phosphoinositide hydrolysis by guanosine 5'-[gamma-thio]triphosphate-activated phospholipase C of turkey erythrocyte membranes. Biochem J. 1988 Jun 1;252(2):583–593. doi: 10.1042/bj2520583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harden T. K., Stephens L., Hawkins P. T., Downes C. P. Turkey erythrocyte membranes as a model for regulation of phospholipase C by guanine nucleotides. J Biol Chem. 1987 Jul 5;262(19):9057–9061. [PubMed] [Google Scholar]
  15. Helms J. B., de Vries K. J., Wirtz K. W. Synthesis of phosphatidylinositol 4,5-bisphosphate in the endoplasmic reticulum of Chinese hamster ovary cells. J Biol Chem. 1991 Nov 15;266(32):21368–21374. [PubMed] [Google Scholar]
  16. Hokin L. E. Receptors and phosphoinositide-generated second messengers. Annu Rev Biochem. 1985;54:205–235. doi: 10.1146/annurev.bi.54.070185.001225. [DOI] [PubMed] [Google Scholar]
  17. Imai A., Gershengorn M. C. Independent phosphatidylinositol synthesis in pituitary plasma membrane and endoplasmic reticulum. Nature. 1987 Feb 19;325(6106):726–728. doi: 10.1038/325726a0. [DOI] [PubMed] [Google Scholar]
  18. Imai A., Gershengorn M. C. 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. 1987 May 15;262(14):6457–6459. [PubMed] [Google Scholar]
  19. Jolles J., Zwiers H., Dekker A., Wirtz K. W., Gispen W. H. Corticotropin-(1--24)-tetracosapeptide affects protein phosphorylation and polyphosphoinositide metabolism in rat brain. Biochem J. 1981 Jan 15;194(1):283–291. doi: 10.1042/bj1940283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. King C. E., Stephens L. R., Hawkins P. T., Guy G. R., Michell R. H. Multiple metabolic pools of phosphoinositides and phosphatidate in human erythrocytes incubated in a medium that permits rapid transmembrane exchange of phosphate. Biochem J. 1987 May 15;244(1):209–217. doi: 10.1042/bj2440209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kinney A. J., Carman G. M. Enzymes of phosphoinositide synthesis in secretory vesicles destined for the plasma membrane in Saccharomyces cerevisiae. J Bacteriol. 1990 Jul;172(7):4115–4117. doi: 10.1128/jb.172.7.4115-4117.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McPhee F., Lowe G., Vaziri C., Downes C. P. Phosphatidylinositol synthase and phosphatidylinositol/inositol exchange reactions in turkey erythrocyte membranes. Biochem J. 1991 Apr 1;275(Pt 1):187–192. doi: 10.1042/bj2750187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Michell R. H. Inositol phospholipids and cell surface receptor function. Biochim Biophys Acta. 1975 Mar 25;415(1):81–47. doi: 10.1016/0304-4157(75)90017-9. [DOI] [PubMed] [Google Scholar]
  24. Monaco M. E. Inositol metabolism in WRK-1 cells. Relationship of hormone-sensitive to -insensitive pools of phosphoinositides. J Biol Chem. 1987 Sep 25;262(27):13001–13006. [PubMed] [Google Scholar]
  25. Monaco M. E. The phosphatidylinositol cycle in WRK-1 cells. Evidence for a separate, hormone-sensitive phosphatidylinositol pool. J Biol Chem. 1982 Mar 10;257(5):2137–2139. [PubMed] [Google Scholar]
  26. Morris A. J., Waldo G. L., Downes C. P., Harden T. K. A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. I. Purification and properties. J Biol Chem. 1990 Aug 15;265(23):13501–13507. [PubMed] [Google Scholar]
  27. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 1988 Aug 25;334(6184):661–665. doi: 10.1038/334661a0. [DOI] [PubMed] [Google Scholar]
  28. Smith C. D., Wells W. W. Phosphorylation of rat liver nuclear envelopes. II. Characterization of in vitro lipid phosphorylation. J Biol Chem. 1983 Aug 10;258(15):9368–9373. [PubMed] [Google Scholar]
  29. Van Paridon P. A., Gadella T. W., Jr, Somerharju P. J., Wirtz K. W. On the relationship between the dual specificity of the bovine brain phosphatidylinositol transfer protein and membrane phosphatidylinositol levels. Biochim Biophys Acta. 1987 Sep 18;903(1):68–77. doi: 10.1016/0005-2736(87)90156-8. [DOI] [PubMed] [Google Scholar]
  30. Vaziri C., Downes C. P. Association of a receptor and G-protein-regulated phospholipase C with the cytoskeleton. J Biol Chem. 1992 Nov 15;267(32):22973–22981. [PubMed] [Google Scholar]
  31. Vaziri C., Downes C. P. G-protein-mediated activation of turkey erythrocyte phospholipase C by beta-adrenergic and P2y-purinergic receptors. Biochem J. 1992 Jun 15;284(Pt 3):917–922. doi: 10.1042/bj2840917. [DOI] [PMC free article] [PubMed] [Google Scholar]

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