Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Dec 1;109(6):2801–2808. doi: 10.1083/jcb.109.6.2801

A specific interaction in vitro between pancreatic zymogen granules and plasma membranes: stimulation by G-protein activators but not by Ca2+

PMCID: PMC2115933  PMID: 2512300

Abstract

The molecular details of the final step in the process of regulated exocytosis, the fusion of the membrane of the secretory granule with the plasma membrane, are at present obscure. As a first step in an investigation of this membrane fusion event, we have developed a cell- free assay for the interaction between pancreatic zymogen granules and plasma membranes. We show here that plasma membranes are able to trigger the release of the granule contents, and that this effect is specific to pancreatic membranes, involves membrane fusion, requires membrane proteins, and is stimulated by activators of G-proteins but not by Ca2+. The assay is simple, reliable, and rapid, and should permit the identification of proteins that are involved in the exocytotic fusion event.

Full Text

The Full Text of this article is available as a PDF (953.9 KB).

Selected References

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

  1. BAGINSKI E., ZAK B. Micro-determination of serum phosphate and phospholipids. Clin Chim Acta. 1960 Nov;5:834–838. doi: 10.1016/0009-8981(60)90117-0. [DOI] [PubMed] [Google Scholar]
  2. Baker P. F., Knight D. E. Calcium-dependent exocytosis in bovine adrenal medullary cells with leaky plasma membranes. Nature. 1978 Dec 7;276(5688):620–622. doi: 10.1038/276620a0. [DOI] [PubMed] [Google Scholar]
  3. Balch W. E., Dunphy W. G., Braell W. A., Rothman J. E. Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell. 1984 Dec;39(2 Pt 1):405–416. doi: 10.1016/0092-8674(84)90019-9. [DOI] [PubMed] [Google Scholar]
  4. Balch W. E., Wagner K. R., Keller D. S. Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system. J Cell Biol. 1987 Mar;104(3):749–760. doi: 10.1083/jcb.104.3.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barrowman M. M., Cockcroft S., Gomperts B. D. Two roles for guanine nucleotides in the stimulus-secretion sequence of neutrophils. Nature. 1986 Feb 6;319(6053):504–507. doi: 10.1038/319504a0. [DOI] [PubMed] [Google Scholar]
  6. Bittner M. A., Holz R. W., Neubig R. R. Guanine nucleotide effects on catecholamine secretion from digitonin-permeabilized adrenal chromaffin cells. J Biol Chem. 1986 Aug 5;261(22):10182–10188. [PubMed] [Google Scholar]
  7. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  8. Braell W. A. Fusion between endocytic vesicles in a cell-free system. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1137–1141. doi: 10.1073/pnas.84.5.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burgoyne R. D. G proteins: control of exocytosis. Nature. 1987 Jul 9;328(6126):112–113. doi: 10.1038/328112a0. [DOI] [PubMed] [Google Scholar]
  10. Cockcroft S., Howell T. W., Gomperts B. D. Two G-proteins act in series to control stimulus-secretion coupling in mast cells: use of neomycin to distinguish between G-proteins controlling polyphosphoinositide phosphodiesterase and exocytosis. J Cell Biol. 1987 Dec;105(6 Pt 1):2745–2750. doi: 10.1083/jcb.105.6.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Crabb J. H., Jackson R. C. In vitro reconstitution of exocytosis from plasma membrane and isolated secretory vesicles. J Cell Biol. 1985 Dec;101(6):2263–2273. doi: 10.1083/jcb.101.6.2263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Davis B., Lazarus N. R. An in Vitro system for studying insulin release caused by secretory granules-plasma membrane interaction: definition of the system. J Physiol. 1976 Apr;256(3):709–729. doi: 10.1113/jphysiol.1976.sp011347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Emmelot P., Bos C. J., van Hoeven R. P., van Blitterswijk W. J. Isolation of plasma membranes from rat and mouse livers and hepatomas. Methods Enzymol. 1974;31:75–90. doi: 10.1016/0076-6879(74)31008-7. [DOI] [PubMed] [Google Scholar]
  14. Fabiato A., Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 1979;75(5):463–505. [PubMed] [Google Scholar]
  15. Fernandez J. M., Neher E., Gomperts B. D. Capacitance measurements reveal stepwise fusion events in degranulating mast cells. 1984 Nov 29-Dec 5Nature. 312(5993):453–455. doi: 10.1038/312453a0. [DOI] [PubMed] [Google Scholar]
  16. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  17. Griffiths G., Simons K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science. 1986 Oct 24;234(4775):438–443. doi: 10.1126/science.2945253. [DOI] [PubMed] [Google Scholar]
  18. Gunther G. R. Effect of 12-O-tetradecanoyl-phorbol-13-acetate on Ca2+ efflux and protein discharge in pancreatic acini. J Biol Chem. 1981 Dec 10;256(23):12040–12045. [PubMed] [Google Scholar]
  19. Haggerty J. G., Jackson R. C. Release of granule contents from sea urchin egg cortices. New assay procedures and inhibition by sulfhydryl-modifying reagents. J Biol Chem. 1983 Feb 10;258(3):1819–1825. [PubMed] [Google Scholar]
  20. Hinton R. H., Dobrota M., Fitzsimons J. T., Reid E. Preparation of a plasma membrane fraction from rat liver by zonal centrifugation. Eur J Biochem. 1970 Feb;12(2):349–359. doi: 10.1111/j.1432-1033.1970.tb00857.x. [DOI] [PubMed] [Google Scholar]
  21. Hodges T. K., Leonard R. T. Purification of a plasma membrane-bound adenosine triphosphatase from plant roots. Methods Enzymol. 1974;32:392–406. doi: 10.1016/0076-6879(74)32039-3. [DOI] [PubMed] [Google Scholar]
  22. Hoekstra D., de Boer T., Klappe K., Wilschut J. Fluorescence method for measuring the kinetics of fusion between biological membranes. Biochemistry. 1984 Nov 20;23(24):5675–5681. doi: 10.1021/bi00319a002. [DOI] [PubMed] [Google Scholar]
  23. Howell T. W., Cockcroft S., Gomperts B. D. Essential synergy between Ca2+ and guanine nucleotides in exocytotic secretion from permeabilized rat mast cells. J Cell Biol. 1987 Jul;105(1):191–197. doi: 10.1083/jcb.105.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Howell T. W., Gomperts B. D. Rat mast cells permeabilised with streptolysin O secrete histamine in response to Ca2+ at concentrations buffered in the micromolar range. Biochim Biophys Acta. 1987 Feb 18;927(2):177–183. doi: 10.1016/0167-4889(87)90132-7. [DOI] [PubMed] [Google Scholar]
  25. Jackson R. C., Ward K. K., Haggerty J. G. Mild proteolytic digestion restores exocytotic activity to N-ethylmaleimide-inactivated cell surface complex from sea urchin eggs. J Cell Biol. 1985 Jul;101(1):6–11. doi: 10.1083/jcb.101.1.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Knight D. E., Baker P. F. Guanine nucleotides and Ca-dependent exocytosis. Studies on two adrenal cell preparations. FEBS Lett. 1985 Sep 23;189(2):345–349. doi: 10.1016/0014-5793(85)81053-x. [DOI] [PubMed] [Google Scholar]
  27. Knight D. E., Koh E. Ca2+ and cyclic nucleotide dependence of amylase release from isolated rat pancreatic acinar cells rendered permeable by intense electric fields. Cell Calcium. 1984 Aug;5(4):401–418. doi: 10.1016/0143-4160(84)90007-1. [DOI] [PubMed] [Google Scholar]
  28. Konings F., De Potter W. Calcium-dependent in vitro interaction between bovine adrenal medullary cell membranes and chromaffin granules as a model for exocytosis. FEBS Lett. 1981 Apr 6;126(1):103–106. doi: 10.1016/0014-5793(81)81043-5. [DOI] [PubMed] [Google Scholar]
  29. Meldolesi J., Jamieson J. D., Palade G. E. Composition of cellular membranes in the pancreas of the guinea pig. I. Isolation of membrane fractions. J Cell Biol. 1971 Apr;49(1):109–129. doi: 10.1083/jcb.49.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Merritt J. E., Rubin R. P. Pancreatic amylase secretion and cytoplasmic free calcium. Effects of ionomycin, phorbol dibutyrate and diacylglycerols alone and in combination. Biochem J. 1985 Aug 15;230(1):151–159. doi: 10.1042/bj2300151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Milutinović S., Argent B. E., Schulz U., Sachs G. Studies on isolated subcellular components of cat pancreas. II. A Ca++-dependent interaction between membranes and zymogen granules of cat pancreas. J Membr Biol. 1977 Sep 14;36(2-3):281–295. doi: 10.1007/BF01868155. [DOI] [PubMed] [Google Scholar]
  32. Mullock B. M., Hinton R. H., Dobrota M., Peppard J., Orlans E. Distribution of secretory component in hepatocytes and its mode of transfer into bile. Biochem J. 1980 Sep 15;190(3):819–826. doi: 10.1042/bj1900819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Omura T., Takesue S. A new method for simultaneous purification of cytochrome b5 and NADPH-cytochrome c reductase from rat liver microsomes. J Biochem. 1970 Feb;67(2):249–257. doi: 10.1093/oxfordjournals.jbchem.a129248. [DOI] [PubMed] [Google Scholar]
  34. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  35. Pandol S. J., Schoeffield M. S., Sachs G., Muallem S. Role of free cytosolic calcium in secretagogue-stimulated amylase release from dispersed acini from guinea pig pancreas. J Biol Chem. 1985 Aug 25;260(18):10081–10086. [PubMed] [Google Scholar]
  36. Pâquet M. R., St-Jean P., Roberge M., Beaudoin A. R. Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins. Eur J Cell Biol. 1982 Aug;28(1):20–26. [PubMed] [Google Scholar]
  37. Rinderknecht H., Wilding P., Haverback B. J. A new method for the determination of alpha-amylase. Experientia. 1967 Oct 15;23(10):805–805. doi: 10.1007/BF02146851. [DOI] [PubMed] [Google Scholar]
  38. Rogers J., Hughes R. G., Matthews E. K. Cyclic GMP inhibits protein kinase C-mediated secretion in rat pancreatic acini. J Biol Chem. 1988 Mar 15;263(8):3713–3719. [PubMed] [Google Scholar]
  39. Rogers J., Matthews E. K., McKay D. B. Effects of a cytosolic protein on the interaction of rat pancreatic zymogen granules in vitro. Biochim Biophys Acta. 1987 Feb 26;897(2):217–228. doi: 10.1016/0005-2736(87)90418-4. [DOI] [PubMed] [Google Scholar]
  40. Rosenzweig S. A., Miller L. J., Jamieson J. D. Identification and localization of cholecystokinin-binding sites on rat pancreatic plasma membranes and acinar cells: a biochemical and autoradiographic study. J Cell Biol. 1983 May;96(5):1288–1297. doi: 10.1083/jcb.96.5.1288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rothman S. S., Burwen S., Liebow C. The zymogen granule: intragranular organization and its functional significance. Adv Cytopharmacol. 1974;2:341–348. [PubMed] [Google Scholar]
  42. STEIN E. A., FISCHER E. H. The resistance of alpha-amylases towards proteolytic attack. J Biol Chem. 1958 Jun;232(2):867–879. [PubMed] [Google Scholar]
  43. Sips H. J., Brown D., Oonk R., Orci L. Orientation of rat-liver plasma membrane vesicles. A biochemical and ultrastructural study. Biochim Biophys Acta. 1982 Nov 22;692(3):447–454. doi: 10.1016/0005-2736(82)90396-0. [DOI] [PubMed] [Google Scholar]
  44. Tartakoff A., Vassalli P., Détraz M. Comparative studies of intracellular transport of secretory proteins. J Cell Biol. 1978 Dec;79(3):694–707. doi: 10.1083/jcb.79.3.694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Toutant M., Aunis D., Bockaert J., Homburger V., Rouot B. Presence of three pertussis toxin substrates and Go alpha immunoreactivity in both plasma and granule membranes of chromaffin cells. FEBS Lett. 1987 May 11;215(2):339–344. doi: 10.1016/0014-5793(87)80174-6. [DOI] [PubMed] [Google Scholar]
  46. VALLEE B. L., STEIN E. A., SUMERWELL W. N., FISCHER E. H. Metal content of alpha-amylases of various origins. J Biol Chem. 1959 Nov;234:2901–2905. [PubMed] [Google Scholar]
  47. Whitaker M. J., Baker P. F. Calcium-dependent exocytosis in an in vitro secretory granule plasma membrane preparation from sea urchin eggs and the effects of some inhibitors of cytoskeletal function. Proc R Soc Lond B Biol Sci. 1983 Jul 22;218(1213):397–413. doi: 10.1098/rspb.1983.0047. [DOI] [PubMed] [Google Scholar]
  48. Woodman P. G., Edwardson J. M. A cell-free assay for the insertion of a viral glycoprotein into the plasma membrane. J Cell Biol. 1986 Nov;103(5):1829–1835. doi: 10.1083/jcb.103.5.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES