Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Mar 1;110(3):721–730. doi: 10.1083/jcb.110.3.721

Sorting during transport to the surface of PC12 cells: divergence of synaptic vesicle and secretory granule proteins

PMCID: PMC2116026  PMID: 2307706

Abstract

PC12 cells, a cell line derived from a rat pheochromocytoma, have both regulated and constitutive secretory pathways. Regulated secretion occurs via large dense core granules, which are related to chromaffin granules and are abundant in these cells. In addition, PC12 cells also contain small electron-lucent vesicles, whose numbers increase in response to nerve growth factor and which may be related to cholinergic synaptic vesicles. These could characterize a second regulated secretory pathway. We have investigated the trafficking of protein markers for both these organelles. We have purified and characterized the large dense core granules from these cells using sequential velocity and equilibrium gradients. We demonstrate the copurification of the major PC12 soluble regulated secretory protein (secretogranin II) with this organelle. As a marker for the synaptic vesicle-like organelles in this system, we have used the integral membrane glycoprotein p38 or synaptophysin. We show that the p38-enriched fraction of PC12 cells comigrates with rat brain synaptic vesicles on an equilibrium gradient. We also demonstrate that p38 purifies away from the dense core granules; less than 5% of this protein is found in our dense granule fraction. Finally we show that p38 does not pass through the dense granule fraction in pulse-chase experiments. These results rule out the possibility of p38 reaching the small clear vesicles via mature dense granules and imply that these cells may have two independently derived regulated pathways.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

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

  1. 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]
  2. Buckley K. M., Floor E., Kelly R. B. Cloning and sequence analysis of cDNA encoding p38, a major synaptic vesicle protein. J Cell Biol. 1987 Dec;105(6 Pt 1):2447–2456. doi: 10.1083/jcb.105.6.2447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Burke B., Griffiths G., Reggio H., Louvard D., Warren G. A monoclonal antibody against a 135-K Golgi membrane protein. EMBO J. 1982;1(12):1621–1628. doi: 10.1002/j.1460-2075.1982.tb01364.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Fischer-Colbrie R., Schober M. Isolation and characterization of chromogranins A, B, and C from bovine chromaffin granules and a rat pheochromocytoma. J Neurochem. 1987 Jan;48(1):262–270. doi: 10.1111/j.1471-4159.1987.tb13157.x. [DOI] [PubMed] [Google Scholar]
  7. Franke W. W., Grund C., Achtstätter T. Co-expression of cytokeratins and neurofilament proteins in a permanent cell line: cultured rat PC12 cells combine neuronal and epithelial features. J Cell Biol. 1986 Nov;103(5):1933–1943. doi: 10.1083/jcb.103.5.1933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Greene L. A., Rein G. Synthesis, storage and release of acetylcholine by a noradrenergic pheochromocytoma cell line. Nature. 1977 Jul 28;268(5618):349–351. doi: 10.1038/268349a0. [DOI] [PubMed] [Google Scholar]
  9. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gumbiner B., Kelly R. B. Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells. Cell. 1982 Jan;28(1):51–59. doi: 10.1016/0092-8674(82)90374-9. [DOI] [PubMed] [Google Scholar]
  11. Hell J. W., Maycox P. R., Stadler H., Jahn R. Uptake of GABA by rat brain synaptic vesicles isolated by a new procedure. EMBO J. 1988 Oct;7(10):3023–3029. doi: 10.1002/j.1460-2075.1988.tb03166.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jahn R., Schiebler W., Ouimet C., Greengard P. A 38,000-dalton membrane protein (p38) present in synaptic vesicles. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4137–4141. doi: 10.1073/pnas.82.12.4137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Lagercrantz H. On the composition and function of large dense cored vesicles in sympathetic nerves. Neuroscience. 1976;1(2):81–92. doi: 10.1016/0306-4522(76)90002-6. [DOI] [PubMed] [Google Scholar]
  16. Leube R. E., Kaiser P., Seiter A., Zimbelmann R., Franke W. W., Rehm H., Knaus P., Prior P., Betz H., Reinke H. Synaptophysin: molecular organization and mRNA expression as determined from cloned cDNA. EMBO J. 1987 Nov;6(11):3261–3268. doi: 10.1002/j.1460-2075.1987.tb02644.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lowe A. W., Madeddu L., Kelly R. B. Endocrine secretory granules and neuronal synaptic vesicles have three integral membrane proteins in common. J Cell Biol. 1988 Jan;106(1):51–59. doi: 10.1083/jcb.106.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Luckenbill-Edds L., Van Horn C., Greene L. A. Fine structure of initial outgrowth of processes induced in a pheochromocytoma cell line (PC12) by nerve growth factor. J Neurocytol. 1979 Aug;8(4):493–511. doi: 10.1007/BF01214805. [DOI] [PubMed] [Google Scholar]
  19. Navone F., Jahn R., Di Gioia G., Stukenbrok H., Greengard P., De Camilli P. Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells. J Cell Biol. 1986 Dec;103(6 Pt 1):2511–2527. doi: 10.1083/jcb.103.6.2511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. PENNINGTON R. J. Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem J. 1961 Sep;80:649–654. doi: 10.1042/bj0800649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rehm H., Wiedenmann B., Betz H. Molecular characterization of synaptophysin, a major calcium-binding protein of the synaptic vesicle membrane. EMBO J. 1986 Mar;5(3):535–541. doi: 10.1002/j.1460-2075.1986.tb04243.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Roda L. G., Nolan J. A., Kim S. U., Hogue-Angeletti R. A. Isolation and characterization of chromaffin granules from a pheochromocytoma (PC 12) cell line. Exp Cell Res. 1980 Jul;128(1):103–109. doi: 10.1016/0014-4827(80)90392-4. [DOI] [PubMed] [Google Scholar]
  23. Rosa P., Hille A., Lee R. W., Zanini A., De Camilli P., Huttner W. B. Secretogranins I and II: two tyrosine-sulfated secretory proteins common to a variety of cells secreting peptides by the regulated pathway. J Cell Biol. 1985 Nov;101(5 Pt 1):1999–2011. doi: 10.1083/jcb.101.5.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. SELLINGER O. Z., BEAUFAY H., JACQUES P., DOYEN A., DE DUVE C. Tissue fractionation studies. 15. Intracellular distribution and properties of beta-N-acetylglucosaminidase and beta-galactosidase in rat liver. Biochem J. 1960 Mar;74:450–456. doi: 10.1042/bj0740450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schubert D., Klier F. G. Storage and release of acetylcholine by a clonal cell line. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5184–5188. doi: 10.1073/pnas.74.11.5184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schweitzer E. S., Kelly R. B. Selective packaging of human growth hormone into synaptic vesicles in a rat neuronal (PC12) cell line. J Cell Biol. 1985 Aug;101(2):667–676. doi: 10.1083/jcb.101.2.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tischler A. S., Greene L. A. Morphologic and cytochemical properties of a clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Lab Invest. 1978 Aug;39(2):77–89. [PubMed] [Google Scholar]
  28. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Valtorta F., Jahn R., Fesce R., Greengard P., Ceccarelli B. Synaptophysin (p38) at the frog neuromuscular junction: its incorporation into the axolemma and recycling after intense quantal secretion. J Cell Biol. 1988 Dec;107(6 Pt 2):2717–2727. doi: 10.1083/jcb.107.6.2717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wagner J. A., Carlson S. S., Kelly R. B. Chemical and physical characterization of cholinergic synaptic vesicles. Biochemistry. 1978 Apr 4;17(7):1199–1206. doi: 10.1021/bi00600a010. [DOI] [PubMed] [Google Scholar]
  31. Wagner J. A. Structure of catecholamine secretory vesicles from PC12 cells. J Neurochem. 1985 Oct;45(4):1244–1253. doi: 10.1111/j.1471-4159.1985.tb05549.x. [DOI] [PubMed] [Google Scholar]
  32. Wiedenmann B., Franke W. W. Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles. Cell. 1985 Jul;41(3):1017–1028. doi: 10.1016/s0092-8674(85)80082-9. [DOI] [PubMed] [Google Scholar]
  33. Wiedenmann B., Franke W. W., Kuhn C., Moll R., Gould V. E. Synaptophysin: a marker protein for neuroendocrine cells and neoplasms. Proc Natl Acad Sci U S A. 1986 May;83(10):3500–3504. doi: 10.1073/pnas.83.10.3500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wiedenmann B., Rehm H., Knierim M., Becker C. M. Fractionation of synaptophysin-containing vesicles from rat brain and cultured PC12 pheochromocytoma cells. FEBS Lett. 1988 Nov 21;240(1-2):71–77. doi: 10.1016/0014-5793(88)80342-9. [DOI] [PubMed] [Google Scholar]

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

RESOURCES