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. 1991 Jan 1;112(1):39–54. doi: 10.1083/jcb.112.1.39

Reconstitution of constitutive secretion using semi-intact cells: regulation by GTP but not calcium

PMCID: PMC2288804  PMID: 1986006

Abstract

Regulated exocytosis in many permeabilized cells can be triggered by calcium and nonhydrolyzable GTP analogues. Here we examine the role of these effectors in exocytosis of constitutive vesicles using a system that reconstitutes transport between the trans-Golgi region and the plasma membrane. Transport is assayed by two independent methods: the movement of a transmembrane glycoprotein (vesicular stomatitis virus glycoprotein [VSV G protein]) to the cell surface; and the release of a soluble marker, sulfated glycosaminoglycan (GAG) chains, that have been synthesized and radiolabeled in the trans-Golgi. The plasma membrane of CHO cells was selectively perforated with the bacterial cytolysin streptolysin-O. These perforated cells allow exchange of ions and cytosolic proteins but retain intracellular organelles and transport vesicles. Incubation of the semi-intact cells with ATP and a cytosolic fraction results in transport of VSV G protein and GAG chains to the cell surface. The transport reaction is temperature dependent, requires hydrolyzable ATP, and is inhibited by N-ethylmaleimide. Nonhydrolyzable GTP analogs such as GTP gamma S, which stimulate the fusion of regulated secretory granules, completely abolish constitutive secretion. The rate and extent of constitutive transport between the trans-Golgi and the plasma membrane is independent of free Ca2+ concentrations. This is in marked contrast to fusion of regulated secretory granules with the plasma membrane, and transport between the ER and the cis-Golgi (Beckers, C. J. M., and W. E. Balch. 1989. J. Cell Biol. 108:1245-1256; Baker, D., L. Wuestehube, R. Schekman, and D. Botstein. 1990. Proc. Natl. Acad. Sci. USA. 87:355-359).

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Selected References

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  1. Ahnert-Hilger G., Mach W., Föhr K. J., Gratzl M. Poration by alpha-toxin and streptolysin O: an approach to analyze intracellular processes. Methods Cell Biol. 1989;31:63–90. doi: 10.1016/s0091-679x(08)61602-7. [DOI] [PubMed] [Google Scholar]
  2. Almers W. Exocytosis. Annu Rev Physiol. 1990;52:607–624. doi: 10.1146/annurev.ph.52.030190.003135. [DOI] [PubMed] [Google Scholar]
  3. Baeuerle P. A., Huttner W. B. Tyrosine sulfation is a trans-Golgi-specific protein modification. J Cell Biol. 1987 Dec;105(6 Pt 1):2655–2664. doi: 10.1083/jcb.105.6.2655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baker D., Wuestehube L., Schekman R., Botstein D., Segev N. GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction. Proc Natl Acad Sci U S A. 1990 Jan;87(1):355–359. doi: 10.1073/pnas.87.1.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Balch W. E. Biochemistry of interorganelle transport. A new frontier in enzymology emerges from versatile in vitro model systems. J Biol Chem. 1989 Oct 15;264(29):16965–16968. [PubMed] [Google Scholar]
  6. Beckers C. J., Balch W. E. Calcium and GTP: essential components in vesicular trafficking between the endoplasmic reticulum and Golgi apparatus. J Cell Biol. 1989 Apr;108(4):1245–1256. doi: 10.1083/jcb.108.4.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Beckers C. J., Block M. R., Glick B. S., Rothman J. E., Balch W. E. Vesicular transport between the endoplasmic reticulum and the Golgi stack requires the NEM-sensitive fusion protein. Nature. 1989 Jun 1;339(6223):397–398. doi: 10.1038/339397a0. [DOI] [PubMed] [Google Scholar]
  8. Bennett M. K., Wandinger-Ness A., Simons K. Release of putative exocytic transport vesicles from perforated MDCK cells. EMBO J. 1988 Dec 20;7(13):4075–4085. doi: 10.1002/j.1460-2075.1988.tb03301.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bhakdi S., Tranum-Jensen J., Sziegoleit A. Mechanism of membrane damage by streptolysin-O. Infect Immun. 1985 Jan;47(1):52–60. doi: 10.1128/iai.47.1.52-60.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Block M. R., Glick B. S., Wilcox C. A., Wieland F. T., Rothman J. E. Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7852–7856. doi: 10.1073/pnas.85.21.7852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bourne H. R. Do GTPases direct membrane traffic in secretion? Cell. 1988 Jun 3;53(5):669–671. doi: 10.1016/0092-8674(88)90081-5. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Burgess T. L., Kelly R. B. Sorting and secretion of adrenocorticotropin in a pituitary tumor cell line after perturbation of the level of a secretory granule-specific proteoglycan. J Cell Biol. 1984 Dec;99(6):2223–2230. doi: 10.1083/jcb.99.6.2223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Burgoyne R. D. Secretory vesicle-associated proteins and their role in exocytosis. Annu Rev Physiol. 1990;52:647–659. doi: 10.1146/annurev.ph.52.030190.003243. [DOI] [PubMed] [Google Scholar]
  15. Compton T., Ivanov I. E., Gottlieb T., Rindler M., Adesnik M., Sabatini D. D. A sorting signal for the basolateral delivery of the vesicular stomatitis virus (VSV) G protein lies in its luminal domain: analysis of the targeting of VSV G-influenza hemagglutinin chimeras. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4112–4116. doi: 10.1073/pnas.86.11.4112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. De Lisle R. C., Williams J. A. Regulation of membrane fusion in secretory exocytosis. Annu Rev Physiol. 1986;48:225–238. doi: 10.1146/annurev.ph.48.030186.001301. [DOI] [PubMed] [Google Scholar]
  17. Diaz R., Mayorga L. S., Weidman P. J., Rothman J. E., Stahl P. D. Vesicle fusion following receptor-mediated endocytosis requires a protein active in Golgi transport. Nature. 1989 Jun 1;339(6223):398–400. doi: 10.1038/339398a0. [DOI] [PubMed] [Google Scholar]
  18. Diaz R., Stahl P. D. Digitonin permeabilization procedures for the study of endosome acidification and function. Methods Cell Biol. 1989;31:25–43. doi: 10.1016/s0091-679x(08)61600-3. [DOI] [PubMed] [Google Scholar]
  19. Dunn L. A., Holz R. W. Catecholamine secretion from digitonin-treated adrenal medullary chromaffin cells. J Biol Chem. 1983 Apr 25;258(8):4989–4993. [PubMed] [Google Scholar]
  20. Farquhar M. G. Progress in unraveling pathways of Golgi traffic. Annu Rev Cell Biol. 1985;1:447–488. doi: 10.1146/annurev.cb.01.110185.002311. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Kimura J. H., Lohmander L. S., Hascall V. C. Studies on the biosynthesis of cartilage proteoglycan in a model system of cultured chondrocytes from the Swarm rat chondrosarcoma. J Cell Biochem. 1984;26(4):261–278. doi: 10.1002/jcb.240260406. [DOI] [PubMed] [Google Scholar]
  24. Knight D. E., Baker P. F. Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields. J Membr Biol. 1982;68(2):107–140. doi: 10.1007/BF01872259. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Kreis T. E. Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J. 1986 May;5(5):931–941. doi: 10.1002/j.1460-2075.1986.tb04306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lefrancios L., Lyles D. S. The interactionof antiody with the major surface glycoprotein of vesicular stomatitis virus. I. Analysis of neutralizing epitopes with monoclonal antibodies. Virology. 1982 Aug;121(1):157–167. [PubMed] [Google Scholar]
  28. Luikart S. D., Sackrison J. L., Thomas C. V. Altered glycosaminoglycan production by HL-60 cells treated with 4-methylumbelliferyl-beta-D-xyloside. Blood. 1985 Oct;66(4):866–872. [PubMed] [Google Scholar]
  29. Melançon P., Glick B. S., Malhotra V., Weidman P. J., Serafini T., Gleason M. L., Orci L., Rothman J. E. Involvement of GTP-binding "G" proteins in transport through the Golgi stack. Cell. 1987 Dec 24;51(6):1053–1062. doi: 10.1016/0092-8674(87)90591-5. [DOI] [PubMed] [Google Scholar]
  30. Noe D. A., Delenick J. C. Quantitative analysis of membrane and secretory protein processing and intracellular transport. J Cell Sci. 1989 Mar;92(Pt 3):449–459. doi: 10.1242/jcs.92.3.449. [DOI] [PubMed] [Google Scholar]
  31. Orci L., Ravazzola M., Amherdt M., Perrelet A., Powell S. K., Quinn D. L., Moore H. P. The trans-most cisternae of the Golgi complex: a compartment for sorting of secretory and plasma membrane proteins. Cell. 1987 Dec 24;51(6):1039–1051. doi: 10.1016/0092-8674(87)90590-3. [DOI] [PubMed] [Google Scholar]
  32. Pagano R. E. A fluorescent derivative of ceramide: physical properties and use in studying the Golgi apparatus of animal cells. Methods Cell Biol. 1989;29:75–85. doi: 10.1016/s0091-679x(08)60188-0. [DOI] [PubMed] [Google Scholar]
  33. Pfeffer S. R., Rothman J. E. Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem. 1987;56:829–852. doi: 10.1146/annurev.bi.56.070187.004145. [DOI] [PubMed] [Google Scholar]
  34. Rink T. J., Sanchez A., Hallam T. J. Diacylglycerol and phorbol ester stimulate secretion without raising cytoplasmic free calcium in human platelets. Nature. 1983 Sep 22;305(5932):317–319. doi: 10.1038/305317a0. [DOI] [PubMed] [Google Scholar]
  35. Rivas R. J., Moore H. P. Spatial segregation of the regulated and constitutive secretory pathways. J Cell Biol. 1989 Jul;109(1):51–60. doi: 10.1083/jcb.109.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rodriguez-Boulan E., Nelson W. J. Morphogenesis of the polarized epithelial cell phenotype. Science. 1989 Aug 18;245(4919):718–725. doi: 10.1126/science.2672330. [DOI] [PubMed] [Google Scholar]
  37. Rose J. K., Bergmann J. E. Expression from cloned cDNA of cell-surface secreted forms of the glycoprotein of vesicular stomatitis virus in eucaryotic cells. Cell. 1982 Oct;30(3):753–762. doi: 10.1016/0092-8674(82)90280-x. [DOI] [PubMed] [Google Scholar]
  38. Salminen A., Novick P. J. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell. 1987 May 22;49(4):527–538. doi: 10.1016/0092-8674(87)90455-7. [DOI] [PubMed] [Google Scholar]
  39. Salminen A., Novick P. J. The Sec15 protein responds to the function of the GTP binding protein, Sec4, to control vesicular traffic in yeast. J Cell Biol. 1989 Sep;109(3):1023–1036. doi: 10.1083/jcb.109.3.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schwartz N. B., Galligani L., Ho P. L., Dorfman A. Stimulation of synthesis of free chondroitin sulfate chains by beta-D-xylosides in cultured cells. Proc Natl Acad Sci U S A. 1974 Oct;71(10):4047–4051. doi: 10.1073/pnas.71.10.4047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schäfer T., Karli U. O., Gratwohl E. K., Schweizer F. E., Burger M. M. Digitonin-permeabilized cells are exocytosis competent. J Neurochem. 1987 Dec;49(6):1697–1707. doi: 10.1111/j.1471-4159.1987.tb02427.x. [DOI] [PubMed] [Google Scholar]
  42. Simons K., Fuller S. D. Cell surface polarity in epithelia. Annu Rev Cell Biol. 1985;1:243–288. doi: 10.1146/annurev.cb.01.110185.001331. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Tooze S. A., Huttner W. B. Cell-free protein sorting to the regulated and constitutive secretory pathways. Cell. 1990 Mar 9;60(5):837–847. doi: 10.1016/0092-8674(90)90097-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vallar L., Biden T. J., Wollheim C. B. Guanine nucleotides induce Ca2+-independent insulin secretion from permeabilized RINm5F cells. J Biol Chem. 1987 Apr 15;262(11):5049–5056. [PubMed] [Google Scholar]
  46. Velasco A., Hidalgo J., Pérez-Vilar J., García-Herdugo G., Navas P. Detection of glycosaminoglycans in the Golgi complex of chondrocytes. Eur J Cell Biol. 1988 Dec;47(2):241–250. [PubMed] [Google Scholar]
  47. 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]
  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]
  49. Zilberstein A., Snider M. D., Porter M., Lodish H. F. Mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein. Cell. 1980 Sep;21(2):417–427. doi: 10.1016/0092-8674(80)90478-x. [DOI] [PubMed] [Google Scholar]
  50. de Curtis I., Simons K. Dissection of Semliki Forest virus glycoprotein delivery from the trans-Golgi network to the cell surface in permeabilized BHK cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8052–8056. doi: 10.1073/pnas.85.21.8052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. de Curtis I., Simons K. Isolation of exocytic carrier vesicles from BHK cells. Cell. 1989 Aug 25;58(4):719–727. doi: 10.1016/0092-8674(89)90106-2. [DOI] [PubMed] [Google Scholar]

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