Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Jul 1;109(1):51–60. doi: 10.1083/jcb.109.1.51

Spatial segregation of the regulated and constitutive secretory pathways

PMCID: PMC2115468  PMID: 2545730

Abstract

Recent experiments using DNA transfection have shown that secretory proteins in AtT-20 cells are sorted into two biochemically distinct secretory pathways. These two pathways differ in the temporal regulation of exocytosis. Proteins secreted by the regulated pathway are stored in dense-core granules until release is stimulated by secretagogues. In contrast, proteins secreted by the constitutive pathway are exported continuously, without storage. It is not known whether there are mechanisms to segregate regulated and constitutive secretory vesicles spatially. In this study, we examined the site of insertion of constitutive vesicles and compared it with that of regulated secretory granules. Regulated granules accumulate at tips of processes in these cells. To determine whether constitutively externalized membrane proteins are inserted into plasma membrane at the cell body or at process tips, AtT-20 cells were infected with ts-O45, a temperature-sensitive mutant of vesicular stomatitis virus in which transport of the surface glycoprotein G is conditionally blocked in the ER. After switching to the permissive temperature, insertion of G protein was detected at the cell body, not at process tips. Targeting of constitutive and regulated secretory vesicles to distinct areas of the plasma membrane appears to be mediated by microtubules. We found that while disruption of microtubules by colchicine had no effect on constitutive secretion, it completely blocked the accumulation of regulated granules at special release sites. Colchicine also affected the proper packaging of regulated secretory proteins. We conclude that regulated and constitutive secretory vesicles are targeted to different areas of the plasma membrane, most probably by differential interactions with microtubules. These results imply that regulated secretory granules may have unique membrane receptors for selective attachment to microtubules.

Full Text

The Full Text of this article is available as a PDF (3.7 MB).

Selected References

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

  1. Arnheiter H., Dubois-Dalcq M., Lazzarini R. A. Direct visualization of protein transport and processing in the living cell by microinjection of specific antibodies. Cell. 1984 Nov;39(1):99–109. doi: 10.1016/0092-8674(84)90195-8. [DOI] [PubMed] [Google Scholar]
  2. Beckers C. J., Keller D. S., Balch W. E. Semi-intact cells permeable to macromolecules: use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex. Cell. 1987 Aug 14;50(4):523–534. doi: 10.1016/0092-8674(87)90025-0. [DOI] [PubMed] [Google Scholar]
  3. Bergmann J. E., Tokuyasu K. T., Singer S. J. Passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the Golgi apparatus en route to the plasma membrane. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1746–1750. doi: 10.1073/pnas.78.3.1746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buckley K., Kelly R. B. Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells. J Cell Biol. 1985 Apr;100(4):1284–1294. doi: 10.1083/jcb.100.4.1284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burgess T. L., Craik C. S., Kelly R. B. The exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer. J Cell Biol. 1985 Aug;101(2):639–645. doi: 10.1083/jcb.101.2.639. [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. Carbonetto S., Fambrough D. M. Synthesis, insertion into the plasma membrane, and turnover of alpha-bungarotoxin receptors in chick sympathetic neurons. J Cell Biol. 1979 Jun;81(3):555–569. doi: 10.1083/jcb.81.3.555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doms R. W., Ruusala A., Machamer C., Helenius J., Helenius A., Rose J. K. Differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus G protein. J Cell Biol. 1988 Jul;107(1):89–99. doi: 10.1083/jcb.107.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ellisman M. H., Levinson S. R. Immunocytochemical localization of sodium channel distributions in the excitable membranes of Electrophorus electricus. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6707–6711. doi: 10.1073/pnas.79.21.6707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fan D. P., Sefton B. M. The entry into host cells of Sindbis virus, vesicular stomatitis virus and Sendai virus. Cell. 1978 Nov;15(3):985–992. doi: 10.1016/0092-8674(78)90282-9. [DOI] [PubMed] [Google Scholar]
  11. Goldberg D. J., Burmeister D. W. Stages in axon formation: observations of growth of Aplysia axons in culture using video-enhanced contrast-differential interference contrast microscopy. J Cell Biol. 1986 Nov;103(5):1921–1931. doi: 10.1083/jcb.103.5.1921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hammerschlag R. How do neuronal proteins know where they are going? . . . Speculations on the role of molecular address markers. Dev Neurosci. 1983;6(1):2–17. doi: 10.1159/000112327. [DOI] [PubMed] [Google Scholar]
  13. Kelly R. B., Buckley K. M., Burgess T. L., Carlson S. S., Caroni P., Hooper J. E., Katzen A., Moore H. P., Pfeffer S. R., Schroer T. A. Membrane traffic in neurons and peptide-secreting cells. Cold Spring Harb Symp Quant Biol. 1983;48(Pt 2):697–705. doi: 10.1101/sqb.1983.048.01.073. [DOI] [PubMed] [Google Scholar]
  14. Kelly R. B. Pathways of protein secretion in eukaryotes. Science. 1985 Oct 4;230(4721):25–32. doi: 10.1126/science.2994224. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Lipscombe D., Madison D. V., Poenie M., Reuter H., Tsien R. Y., Tsien R. W. Spatial distribution of calcium channels and cytosolic calcium transients in growth cones and cell bodies of sympathetic neurons. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2398–2402. doi: 10.1073/pnas.85.7.2398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lipsky N. G., Pagano R. E. Sphingolipid metabolism in cultured fibroblasts: microscopic and biochemical studies employing a fluorescent ceramide analogue. Proc Natl Acad Sci U S A. 1983 May;80(9):2608–2612. doi: 10.1073/pnas.80.9.2608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lombardi T., Montesano R., Wohlwend A., Amherdt M., Vassalli J. D., Orci L. Evidence for polarization of plasma membrane domains in pancreatic endocrine cells. Nature. 1985 Feb 21;313(6004):694–696. doi: 10.1038/313694a0. [DOI] [PubMed] [Google Scholar]
  20. Lomedico P. T., McAndrew S. J. Eukaryotic ribosomes can recognize preproinsulin initiation codons irrespective of their position relative to the 5' end of mRNA. Nature. 1982 Sep 16;299(5880):221–226. doi: 10.1038/299221a0. [DOI] [PubMed] [Google Scholar]
  21. Martial J. A., Hallewell R. A., Baxter J. D., Goodman H. M. Human growth hormone: complementary DNA cloning and expression in bacteria. Science. 1979 Aug 10;205(4406):602–607. doi: 10.1126/science.377496. [DOI] [PubMed] [Google Scholar]
  22. Matlin K. S. The sorting of proteins to the plasma membrane in epithelial cells. J Cell Biol. 1986 Dec;103(6 Pt 2):2565–2568. doi: 10.1083/jcb.103.6.2565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Matsuuchi L., Buckley K. M., Lowe A. W., Kelly R. B. Targeting of secretory vesicles to cytoplasmic domains in AtT-20 and PC-12 cells. J Cell Biol. 1988 Feb;106(2):239–251. doi: 10.1083/jcb.106.2.239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moore H. H., Kelly R. B. Re-routing of a secretory protein by fusion with human growth hormone sequences. Nature. 1986 May 22;321(6068):443–446. doi: 10.1038/321443a0. [DOI] [PubMed] [Google Scholar]
  25. Moore H. P. Factors controlling packaging of peptide hormones into secretory granules. Ann N Y Acad Sci. 1987;493:50–61. doi: 10.1111/j.1749-6632.1987.tb27180.x. [DOI] [PubMed] [Google Scholar]
  26. Moore H. P., Kelly R. B. Secretory protein targeting in a pituitary cell line: differential transport of foreign secretory proteins to distinct secretory pathways. J Cell Biol. 1985 Nov;101(5 Pt 1):1773–1781. doi: 10.1083/jcb.101.5.1773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moore H. P., Walker M. D., Lee F., Kelly R. B. Expressing a human proinsulin cDNA in a mouse ACTH-secreting cell. Intracellular storage, proteolytic processing, and secretion on stimulation. Cell. 1983 Dec;35(2 Pt 1):531–538. doi: 10.1016/0092-8674(83)90187-3. [DOI] [PubMed] [Google Scholar]
  28. Pfenninger K. H., Maylié-Pfenninger M. F. Lectin labeling of sprouting neurons. I. Regional distribution of surface glycoconjugates. J Cell Biol. 1981 Jun;89(3):536–546. doi: 10.1083/jcb.89.3.536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pfenninger K. H., Maylié-Pfenninger M. F. Lectin labeling of sprouting neurons. II. Relative movement and appearance of glycoconjugates during plasmalemmal expansion. J Cell Biol. 1981 Jun;89(3):547–559. doi: 10.1083/jcb.89.3.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rogalski A. A., Bergmann J. E., Singer S. J. Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane. J Cell Biol. 1984 Sep;99(3):1101–1109. doi: 10.1083/jcb.99.3.1101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rogalski A. A., Singer S. J. Associations of elements of the Golgi apparatus with microtubules. J Cell Biol. 1984 Sep;99(3):1092–1100. doi: 10.1083/jcb.99.3.1092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Ross W. N., Aréchiga H., Nicholls J. G. Influence of substrate on the distribution of calcium channels in identified leech neurons in culture. Proc Natl Acad Sci U S A. 1988 Jun;85(11):4075–4078. doi: 10.1073/pnas.85.11.4075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rotundo R. L., Carbonetto S. T. Neurons segregate clusters of membrane-bound acetylcholinesterase along their neurites. Proc Natl Acad Sci U S A. 1987 Apr;84(7):2063–2067. doi: 10.1073/pnas.84.7.2063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schulze E., Kirschner M. Microtubule dynamics in interphase cells. J Cell Biol. 1986 Mar;102(3):1020–1031. doi: 10.1083/jcb.102.3.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Singer S. J., Kupfer A. The directed migration of eukaryotic cells. Annu Rev Cell Biol. 1986;2:337–365. doi: 10.1146/annurev.cb.02.110186.002005. [DOI] [PubMed] [Google Scholar]
  38. Small R. K., Pfenninger K. H. Components of the plasma membrane of growing axons. I. Size and distribution of intramembrane particles. J Cell Biol. 1984 Apr;98(4):1422–1433. doi: 10.1083/jcb.98.4.1422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tamkun M. M., Fambrough D. M. The (Na+ + K+)-ATPase of chick sensory neurons. Studies on biosynthesis and intracellular transport. J Biol Chem. 1986 Jan 25;261(3):1009–1019. [PubMed] [Google Scholar]
  40. Thyberg J., Moskalewski S. Microtubules and the organization of the Golgi complex. Exp Cell Res. 1985 Jul;159(1):1–16. doi: 10.1016/s0014-4827(85)80032-x. [DOI] [PubMed] [Google Scholar]
  41. Tooze J., Burke B. Accumulation of adrenocorticotropin secretory granules in the midbody of telophase AtT20 cells: evidence that secretory granules move anterogradely along microtubules. J Cell Biol. 1987 Apr;104(4):1047–1057. doi: 10.1083/jcb.104.4.1047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Waxman S. G., Black J. A. Membrane structure of vesiculotubular complexes in developing axons in rat optic nerve: freeze-fracture evidence for sequential membrane assembly. Proc R Soc Lond B Biol Sci. 1985 Sep 23;225(1240):357–363. doi: 10.1098/rspb.1985.0066. [DOI] [PubMed] [Google Scholar]
  43. 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]

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

RESOURCES