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. 1994 Jan 1;124(1):43–53. doi: 10.1083/jcb.124.1.43

Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis

PMCID: PMC2119894  PMID: 8294505

Abstract

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3- like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.

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Selected References

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  1. Amsterdam A., Solomon T. E., Jamieson J. D. Sequential dissociation of the exocrine pancreas into lobules, acini, and individual cells. Methods Cell Biol. 1978;20:361–378. doi: 10.1016/s0091-679x(08)62028-2. [DOI] [PubMed] [Google Scholar]
  2. Araki S., Kikuchi A., Hata Y., Isomura M., Takai Y. Regulation of reversible binding of smg p25A, a ras p21-like GTP-binding protein, to synaptic plasma membranes and vesicles by its specific regulatory protein, GDP dissociation inhibitor. J Biol Chem. 1990 Aug 5;265(22):13007–13015. [PubMed] [Google Scholar]
  3. Arvan P., Castle J. D. Phasic release of newly synthesized secretory proteins in the unstimulated rat exocrine pancreas. J Cell Biol. 1987 Feb;104(2):243–252. doi: 10.1083/jcb.104.2.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Balch W. E. Small GTP-binding proteins in vesicular transport. Trends Biochem Sci. 1990 Dec;15(12):473–477. doi: 10.1016/0968-0004(90)90301-q. [DOI] [PubMed] [Google Scholar]
  5. Baldini G., Hohl T., Lin H. Y., Lodish H. F. Cloning of a Rab3 isotype predominantly expressed in adipocytes. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5049–5052. doi: 10.1073/pnas.89.11.5049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burgess T. L., Kelly R. B. Constitutive and regulated secretion of proteins. Annu Rev Cell Biol. 1987;3:243–293. doi: 10.1146/annurev.cb.03.110187.001331. [DOI] [PubMed] [Google Scholar]
  7. Cameron R. S., Cameron P. L., Castle J. D. A common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands. J Cell Biol. 1986 Oct;103(4):1299–1313. doi: 10.1083/jcb.103.4.1299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chavrier P., Parton R. G., Hauri H. P., Simons K., Zerial M. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell. 1990 Jul 27;62(2):317–329. doi: 10.1016/0092-8674(90)90369-p. [DOI] [PubMed] [Google Scholar]
  9. Chavrier P., Vingron M., Sander C., Simons K., Zerial M. Molecular cloning of YPT1/SEC4-related cDNAs from an epithelial cell line. Mol Cell Biol. 1990 Dec;10(12):6578–6585. doi: 10.1128/mcb.10.12.6578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cornell-Bell A. H., Otake L. R., Sadler K., Thomas P. G., Lawrence S., Olsen K., Gumkowski F., Peterson J. R., Jamieson J. D. Membrane glycolipid trafficking in living, polarized pancreatic acinar cells: assessment by confocal microscopy. Methods Cell Biol. 1993;38:221–240. doi: 10.1016/s0091-679x(08)61005-5. [DOI] [PubMed] [Google Scholar]
  11. De Camilli P., Harris S. M., Jr, Huttner W. B., Greengard P. Synapsin I (Protein I), a nerve terminal-specific phosphoprotein. II. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes. J Cell Biol. 1983 May;96(5):1355–1373. doi: 10.1083/jcb.96.5.1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fischer von Mollard G., Mignery G. A., Baumert M., Perin M. S., Hanson T. J., Burger P. M., Jahn R., Südhof T. C. rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1988–1992. doi: 10.1073/pnas.87.5.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fischer von Mollard G., Südhof T. C., Jahn R. A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature. 1991 Jan 3;349(6304):79–81. doi: 10.1038/349079a0. [DOI] [PubMed] [Google Scholar]
  14. Gomperts B. D. GE: a GTP-binding protein mediating exocytosis. Annu Rev Physiol. 1990;52:591–606. doi: 10.1146/annurev.ph.52.030190.003111. [DOI] [PubMed] [Google Scholar]
  15. Hall A. The cellular functions of small GTP-binding proteins. Science. 1990 Aug 10;249(4969):635–640. doi: 10.1126/science.2116664. [DOI] [PubMed] [Google Scholar]
  16. Jamieson J. D., Palade G. E. Synthesis, intracellular transport, and discharge of secretory proteins in stimulated pancreatic exocrine cells. J Cell Biol. 1971 Jul;50(1):135–158. doi: 10.1083/jcb.50.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jena B. P., Padfield P. J., Ingebritsen T. S., Jamieson J. D. Protein tyrosine phosphatase stimulates Ca(2+)-dependent amylase secretion from pancreatic acini. J Biol Chem. 1991 Sep 25;266(27):17744–17746. [PubMed] [Google Scholar]
  18. 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]
  19. Lambert M., Bui N. D., Christophe J. Novel GTP-binding proteins in plasma membranes and zymogen granule membranes from rat pancreas and in pancreatic AR 4-2J cell membranes. FEBS Lett. 1990 Oct 1;271(1-2):19–22. doi: 10.1016/0014-5793(90)80362-m. [DOI] [PubMed] [Google Scholar]
  20. Lapetina E. G., Reep B. R. Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2261–2265. doi: 10.1073/pnas.84.8.2261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Matteoli M., Takei K., Cameron R., Hurlbut P., Johnston P. A., Südhof T. C., Jahn R., De Camilli P. Association of Rab3A with synaptic vesicles at late stages of the secretory pathway. J Cell Biol. 1991 Nov;115(3):625–633. doi: 10.1083/jcb.115.3.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mizoguchi A., Kim S., Ueda T., Takai Y. Tissue distribution of smg p25A, a ras p21-like GTP-binding protein, studied by use of a specific monoclonal antibody. Biochem Biophys Res Commun. 1989 Aug 15;162(3):1438–1445. doi: 10.1016/0006-291x(89)90835-8. [DOI] [PubMed] [Google Scholar]
  23. Novick P. J., Goud B., Salminen A., Walworth N. C., Nair J., Potenza M. Regulation of vesicular traffic by a GTP-binding protein on the cytoplasmic surface of secretory vesicles in yeast. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 2):637–647. doi: 10.1101/sqb.1988.053.01.073. [DOI] [PubMed] [Google Scholar]
  24. Padfield P. J., Balch W. E., Jamieson J. D. A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1656–1660. doi: 10.1073/pnas.89.5.1656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Padfield P. J., Jamieson J. D. Low molecular weight GTP-binding proteins associated with zymogen granule membranes from rat pancreas. Biochem Biophys Res Commun. 1991 Jan 31;174(2):600–605. doi: 10.1016/0006-291x(91)91459-p. [DOI] [PubMed] [Google Scholar]
  26. Pfeffer S. R. GTP-binding proteins in intracellular transport. Trends Cell Biol. 1992 Feb;2(2):41–46. doi: 10.1016/0962-8924(92)90161-f. [DOI] [PubMed] [Google Scholar]
  27. Regazzi R., Kikuchi A., Takai Y., Wollheim C. B. The small GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP dissociation inhibitor proteins. J Biol Chem. 1992 Sep 5;267(25):17512–17519. [PubMed] [Google Scholar]
  28. Takai Y., Kaibuchi K., Kikuchi A., Kawata M. Small GTP-binding proteins. Int Rev Cytol. 1992;133:187–230. doi: 10.1016/s0074-7696(08)61861-6. [DOI] [PubMed] [Google Scholar]
  29. Tooze J., Kern H. F., Fuller S. D., Howell K. E. Condensation-sorting events in the rough endoplasmic reticulum of exocrine pancreatic cells. J Cell Biol. 1989 Jul;109(1):35–50. doi: 10.1083/jcb.109.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Touchot N., Chardin P., Tavitian A. Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8210–8214. doi: 10.1073/pnas.84.23.8210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Walworth N. C., Goud B., Kabcenell A. K., Novick P. J. Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic. EMBO J. 1989 Jun;8(6):1685–1693. doi: 10.1002/j.1460-2075.1989.tb03560.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zahraoui A., Touchot N., Chardin P., Tavitian A. The human Rab genes encode a family of GTP-binding proteins related to yeast YPT1 and SEC4 products involved in secretion. J Biol Chem. 1989 Jul 25;264(21):12394–12401. [PubMed] [Google Scholar]

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