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. 2001 Aug;81(2):994–1005. doi: 10.1016/S0006-3495(01)75757-8

ADP ribosylation factor 6 binding to phosphatidylinositol 4,5-bisphosphate-containing vesicles creates defects in the bilayer structure: an electron spin resonance study.

M Ge 1, J S Cohen 1, H A Brown 1, J H Freed 1
PMCID: PMC1301569  PMID: 11463641

Abstract

The effects of binding of myristoylated ADP ribosylation factor 6 (myr-ARF6), an activator of phospholipase D (PLD), to a model membrane were investigated using an electron spin resonance (ESR) labeling technique. Initial studies were conducted in vesicles composed of 1-palmitoyl-2-oleoyl phosphatidylethanolamine, dipalmitoylphosphatidylcholine, phosphatidylinositol 4,5-biphosphate (PIP(2)), and cholesterol. Recombinant ARF6 binding significantly enhances defects in both the headgroup and acyl-chain regions of the membrane, which are revealed by the emergence of sharp components in the spectra from a headgroup label, 1,2-dipalmitoylphosphatidyl-2,2,6,6-tetramethyl-1-piperidinyloxy-choline (DPPTC), and a chain label, 10PC, after myr-ARF6 binding. Binding of non-myristoylated ARF6 (non-ARF6) shows markedly reduced effects. Interestingly, no change in spectra from DPPTC was observed upon myr-ARF6 binding when PIP(2) in the vesicles was replaced by other negatively charged lipids, including phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol, even when normalized for charge. The production of the sharp peak appears to be a specific event, because another GTP binding protein, CDC42, which binds PIP(2) and activates PLD, fails to induce changes in vesicle structure. These results suggest a previously unappreciated role for ARF in mediating a protein/lipid interaction that produces defects in lipid bilayers. This function may serve as an initial event in destabilizing membrane structure for subsequent membrane fusion or biogenesis of vesicles.

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Selected References

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  1. Antonny B., Beraud-Dufour S., Chardin P., Chabre M. N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange. Biochemistry. 1997 Apr 15;36(15):4675–4684. doi: 10.1021/bi962252b. [DOI] [PubMed] [Google Scholar]
  2. Batenburg A. M., Hibbeln J. C., Verkleij A. J., de Kruijff B. Melittin induces HII phase formation in cardiolipin model membranes. Biochim Biophys Acta. 1987 Sep 18;903(1):142–154. doi: 10.1016/0005-2736(87)90164-7. [DOI] [PubMed] [Google Scholar]
  3. Bittman R., Blau L. The phospholipid-cholesterol interaction. Kinetics of water permeability in liposomes. Biochemistry. 1972 Dec 5;11(25):4831–4839. doi: 10.1021/bi00775a029. [DOI] [PubMed] [Google Scholar]
  4. Bloom M., Evans E., Mouritsen O. G. Physical properties of the fluid lipid-bilayer component of cell membranes: a perspective. Q Rev Biophys. 1991 Aug;24(3):293–397. doi: 10.1017/s0033583500003735. [DOI] [PubMed] [Google Scholar]
  5. Brown H. A., Gutowski S., Moomaw C. R., Slaughter C., Sternweis P. C. ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity. Cell. 1993 Dec 17;75(6):1137–1144. doi: 10.1016/0092-8674(93)90323-i. [DOI] [PubMed] [Google Scholar]
  6. Brown H. A., Sternweis P. C. Stimulation of phospholipase D by ADP-ribosylation factor. Methods Enzymol. 1995;257:313–324. doi: 10.1016/s0076-6879(95)57035-7. [DOI] [PubMed] [Google Scholar]
  7. Burack W. R., Dibble A. R., Allietta M. M., Biltonen R. L. Changes in vesicle morphology induced by lateral phase separation modulate phospholipase A2 activity. Biochemistry. 1997 Aug 26;36(34):10551–10557. doi: 10.1021/bi970509f. [DOI] [PubMed] [Google Scholar]
  8. Chen Y. G., Siddhanta A., Austin C. D., Hammond S. M., Sung T. C., Frohman M. A., Morris A. J., Shields D. Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network. J Cell Biol. 1997 Aug 11;138(3):495–504. doi: 10.1083/jcb.138.3.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cockcroft S., Thomas G. M., Fensome A., Geny B., Cunningham E., Gout I., Hiles I., Totty N. F., Truong O., Hsuan J. J. Phospholipase D: a downstream effector of ARF in granulocytes. Science. 1994 Jan 28;263(5146):523–526. doi: 10.1126/science.8290961. [DOI] [PubMed] [Google Scholar]
  10. Cohen J. S., Brown H. A. Phospholipases stimulate secretion in RBL mast cells. Biochemistry. 2001 Jun 5;40(22):6589–6597. doi: 10.1021/bi0103011. [DOI] [PubMed] [Google Scholar]
  11. Cullis P. R., De Kruyff B., Richards R. E. Factors affecting the motion of the polar headgroup in phospholipid bilayers. A 31P NMR study of unsonicated phosphatidylcholine liposomes. Biochim Biophys Acta. 1976 Mar 19;426(3):433–446. doi: 10.1016/0005-2736(76)90388-6. [DOI] [PubMed] [Google Scholar]
  12. Cullis P. R., Hope M. J. The bilayer stabilizing role of sphingomyelin in the presence of cholesterol: a 31P NMR study. Biochim Biophys Acta. 1980 Apr 24;597(3):533–542. doi: 10.1016/0005-2736(80)90225-4. [DOI] [PubMed] [Google Scholar]
  13. Dufourcq J., Faucon J. F., Fourche G., Dasseux J. L., Le Maire M., Gulik-Krzywicki T. Morphological changes of phosphatidylcholine bilayers induced by melittin: vesicularization, fusion, discoidal particles. Biochim Biophys Acta. 1986 Jul 10;859(1):33–48. doi: 10.1016/0005-2736(86)90315-9. [DOI] [PubMed] [Google Scholar]
  14. Düzgüneş N., Wilschut J., Hong K., Fraley R., Perry C., Friend D. S., James T. L., Papahadjopoulos D. Physicochemical characterization of large unilamellar phospholipid vesicles prepared by reverse-phase evaporation. Biochim Biophys Acta. 1983 Jul 13;732(1):289–299. doi: 10.1016/0005-2736(83)90214-6. [DOI] [PubMed] [Google Scholar]
  15. Earle K. A., Moscicki J. K., Ge M., Budil D. E., Freed J. H. 250-GHz electron spin resonance studies of polarity gradients along the aliphatic chains in phospholipid membranes. Biophys J. 1994 Apr;66(4):1213–1221. doi: 10.1016/S0006-3495(94)80905-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Exton J. H. Regulation of phospholipase D. Biochim Biophys Acta. 1999 Jul 30;1439(2):121–133. doi: 10.1016/s1388-1981(99)00089-x. [DOI] [PubMed] [Google Scholar]
  17. Fettiplace R., Haydon D. A. Water permeability of lipid membranes. Physiol Rev. 1980 Apr;60(2):510–550. doi: 10.1152/physrev.1980.60.2.510. [DOI] [PubMed] [Google Scholar]
  18. Field K. A., Holowka D., Baird B. Fc epsilon RI-mediated recruitment of p53/56lyn to detergent-resistant membrane domains accompanies cellular signaling. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9201–9205. doi: 10.1073/pnas.92.20.9201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Franco M., Chardin P., Chabre M., Paris S. Myristoylation of ADP-ribosylation factor 1 facilitates nucleotide exchange at physiological Mg2+ levels. J Biol Chem. 1995 Jan 20;270(3):1337–1341. doi: 10.1074/jbc.270.3.1337. [DOI] [PubMed] [Google Scholar]
  20. Freed J. H. New technologies in electron spin resonance. Annu Rev Phys Chem. 2000;51:655–689. doi: 10.1146/annurev.physchem.51.1.655. [DOI] [PubMed] [Google Scholar]
  21. Freyberg Z., Sweeney D., Siddhanta A., Bourgoin S., Frohman M., Shields D. Intracellular localization of phospholipase D1 in mammalian cells. Mol Biol Cell. 2001 Apr;12(4):943–955. doi: 10.1091/mbc.12.4.943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Frohman M. A., Sung T. C., Morris A. J. Mammalian phospholipase D structure and regulation. Biochim Biophys Acta. 1999 Jul 30;1439(2):175–186. doi: 10.1016/s1388-1981(99)00093-1. [DOI] [PubMed] [Google Scholar]
  23. Ge M., Budil D. E., Freed J. H. An electron spin resonance study of interactions between phosphatidylcholine and phosphatidylserine in oriented membranes. Biophys J. 1994 May;66(5):1515–1521. doi: 10.1016/S0006-3495(94)80942-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ge M., Freed J. H. Electron-spin resonance study of aggregation of gramicidin in dipalmitoylphosphatidylcholine bilayers and hydrophobic mismatch. Biophys J. 1999 Jan;76(1 Pt 1):264–280. doi: 10.1016/S0006-3495(99)77195-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gordeliy V. I., Ivkov V. G., Ostanevich YuM, Yaguzhinskij L. S. Detection of structural defects in phosphatidylcholine membranes by small-angle neutron scattering. The cluster model of a lipid bilayer. Biochim Biophys Acta. 1991 Jan 9;1061(1):39–48. doi: 10.1016/0005-2736(91)90266-b. [DOI] [PubMed] [Google Scholar]
  26. Haun R. S., Tsai S. C., Adamik R., Moss J., Vaughan M. Effect of myristoylation on GTP-dependent binding of ADP-ribosylation factor to Golgi. J Biol Chem. 1993 Apr 5;268(10):7064–7068. [PubMed] [Google Scholar]
  27. Hong J. X., Haun R. S., Tsai S. C., Moss J., Vaughan M. Effect of ADP-ribosylation factor amino-terminal deletions on its GTP-dependent stimulation of cholera toxin activity. J Biol Chem. 1994 Apr 1;269(13):9743–9745. [PubMed] [Google Scholar]
  28. Hui S. W., Stewart T. P., Boni L. T., Yeagle P. L. Membrane fusion through point defects in bilayers. Science. 1981 May 22;212(4497):921–923. doi: 10.1126/science.7233185. [DOI] [PubMed] [Google Scholar]
  29. Ivanova P. T., Cerda B. A., Horn D. M., Cohen J. S., McLafferty F. W., Brown H. A. Electrospray ionization mass spectrometry analysis of changes in phospholipids in RBL-2H3 mastocytoma cells during degranulation. Proc Natl Acad Sci U S A. 2001 Jun 19;98(13):7152–7157. doi: 10.1073/pnas.131195098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jones D., Morgan C., Cockcroft S. Phospholipase D and membrane traffic. Potential roles in regulated exocytosis, membrane delivery and vesicle budding. Biochim Biophys Acta. 1999 Jul 30;1439(2):229–244. doi: 10.1016/s1388-1981(99)00097-9. [DOI] [PubMed] [Google Scholar]
  31. Kleinschmidt J. H., Mahaney J. E., Thomas D. D., Marsh D. Interaction of bee venom melittin with zwitterionic and negatively charged phospholipid bilayers: a spin-label electron spin resonance study. Biophys J. 1997 Feb;72(2 Pt 1):767–778. doi: 10.1016/s0006-3495(97)78711-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ktistakis N. T., Brown H. A., Waters M. G., Sternweis P. C., Roth M. G. Evidence that phospholipase D mediates ADP ribosylation factor-dependent formation of Golgi coated vesicles. J Cell Biol. 1996 Jul;134(2):295–306. doi: 10.1083/jcb.134.2.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lodge J. K., Jackson-Machelski E., Devadas B., Zupec M. E., Getman D. P., Kishore N., Freeman S. K., McWherter C. A., Sikorski J. A., Gordon J. I. N-myristoylation of Arf proteins in Candida albicans: an in vivo assay for evaluating antifungal inhibitors of myristoyl-CoA: protein N-myristoyltransferase. Microbiology. 1997 Feb;143(Pt 2):357–366. doi: 10.1099/00221287-143-2-357. [DOI] [PubMed] [Google Scholar]
  34. McLaughlin S., Aderem A. The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions. Trends Biochem Sci. 1995 Jul;20(7):272–276. doi: 10.1016/s0968-0004(00)89042-8. [DOI] [PubMed] [Google Scholar]
  35. Patyal B. R., Crepeau R. H., Freed J. H. Lipid-gramicidin interactions using two-dimensional Fourier-transform electron spin resonance. Biophys J. 1997 Oct;73(4):2201–2220. doi: 10.1016/S0006-3495(97)78252-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Randazzo P. A., Weiss O., Kahn R. A. Preparation of recombinant ADP-ribosylation factor. Methods Enzymol. 1995;257:128–135. doi: 10.1016/s0076-6879(95)57018-7. [DOI] [PubMed] [Google Scholar]
  37. Roth M. G., Sternweis P. C. The role of lipid signaling in constitutive membrane traffic. Curr Opin Cell Biol. 1997 Aug;9(4):519–526. doi: 10.1016/s0955-0674(97)80028-2. [DOI] [PubMed] [Google Scholar]
  38. Rothman J. E. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. doi: 10.1038/372055a0. [DOI] [PubMed] [Google Scholar]
  39. Sen A., Isac T. V., Hui S. W. Bilayer packing stress and defects in mixed dilinoleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine and their susceptibility to phospholipase A2. Biochemistry. 1991 May 7;30(18):4516–4521. doi: 10.1021/bi00232a021. [DOI] [PubMed] [Google Scholar]
  40. Shiffer K. A., Rood L., Emerson R. K., Kuypers F. A. Effects of phosphatidylinositol diphosphate on phospholipid asymmetry in the human erythrocyte membrane. Biochemistry. 1998 Mar 10;37(10):3449–3458. doi: 10.1021/bi972218c. [DOI] [PubMed] [Google Scholar]
  41. Shin Y. K., Freed J. H. Thermodynamics of phosphatidylcholine-cholesterol mixed model membranes in the liquid crystalline state studied by the orientational order parameter. Biophys J. 1989 Dec;56(6):1093–1100. doi: 10.1016/S0006-3495(89)82757-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Singer W. D., Brown H. A., Bokoch G. M., Sternweis P. C. Resolved phospholipase D activity is modulated by cytosolic factors other than Arf. J Biol Chem. 1995 Jun 23;270(25):14944–14950. doi: 10.1074/jbc.270.25.14944. [DOI] [PubMed] [Google Scholar]
  43. Singer W. D., Brown H. A., Jiang X., Sternweis P. C. Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase activity. J Biol Chem. 1996 Feb 23;271(8):4504–4510. doi: 10.1074/jbc.271.8.4504. [DOI] [PubMed] [Google Scholar]
  44. Spink C. H., Yeager M. D., Feigenson G. W. Partitioning behavior of indocarbocyanine probes between coexisting gel and fluid phases in model membranes. Biochim Biophys Acta. 1990 Mar 30;1023(1):25–33. doi: 10.1016/0005-2736(90)90005-9. [DOI] [PubMed] [Google Scholar]
  45. Straume M., Litman B. J. Equilibrium and dynamic structure of large, unilamellar, unsaturated acyl chain phosphatidylcholine vesicles. Higher order analysis of 1,6-diphenyl-1,3,5-hexatriene and 1-[4-(trimethylammonio)phenyl]- 6-phenyl-1,3,5-hexatriene anisotropy decay. Biochemistry. 1987 Aug 11;26(16):5113–5120. doi: 10.1021/bi00390a033. [DOI] [PubMed] [Google Scholar]
  46. Südhof T. C. The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature. 1995 Jun 22;375(6533):645–653. doi: 10.1038/375645a0. [DOI] [PubMed] [Google Scholar]
  47. Terui T., Kahn R. A., Randazzo P. A. Effects of acid phospholipids on nucleotide exchange properties of ADP-ribosylation factor 1. Evidence for specific interaction with phosphatidylinositol 4,5-bisphosphate. J Biol Chem. 1994 Nov 11;269(45):28130–28135. [PubMed] [Google Scholar]
  48. Walker S. J., Wu W. J., Cerione R. A., Brown H. A. Activation of phospholipase D1 by Cdc42 requires the Rho insert region. J Biol Chem. 2000 May 26;275(21):15665–15668. doi: 10.1074/jbc.M000076200. [DOI] [PubMed] [Google Scholar]
  49. Yan E. C., Eisenthal K. B. Effect of cholesterol on molecular transport of organic cations across liposome bilayers probed by second harmonic generation. Biophys J. 2000 Aug;79(2):898–903. doi: 10.1016/S0006-3495(00)76345-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zheng Y., Glaven J. A., Wu W. J., Cerione R. A. Phosphatidylinositol 4,5-bisphosphate provides an alternative to guanine nucleotide exchange factors by stimulating the dissociation of GDP from Cdc42Hs. J Biol Chem. 1996 Sep 27;271(39):23815–23819. doi: 10.1074/jbc.271.39.23815. [DOI] [PubMed] [Google Scholar]

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