Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Jun 1;106(6):1843–1851. doi: 10.1083/jcb.106.6.1843

Efficient targeting to storage granules of human proinsulins with altered propeptide domain

PMCID: PMC2115124  PMID: 2838491

Abstract

In neuronal and endocrine cells, peptide hormones are selectively segregated into storage granules, while other proteins are exported continuously without storage. Sorting of hormones by cellular machinery involves the recognition of specific structural domains on prohormone molecules. Since the propeptide of insulin is known to play an important role in its three-dimensional structure, it is reasonable to speculate that targeting of proinsulin to storage granules would require a functional connecting peptide. To test this hypothesis, we constructed two mutations in human proinsulin with different predicted structures. In one mutation, Ins delta C, the entire C peptide was deleted, resulting in an altered insulin in which the B and the A chains are joined contiguously. In the other mutation, Ins/IGF, the C peptide of proinsulin was replaced with the unrelated 12-amino acid connecting peptide of human insulin-like growth factor-I; this substitution should permit correct folding of the B and A chains to form a tertiary structure similar to that of proinsulin. By several biochemical and morphological criteria, we found that Ins/IGF is efficiently targeted to storage granules, suggesting that the C peptide of proinsulin does not contain necessary sorting information. Unexpectedly, Ins delta C, which presumably cannot fold properly, is also targeted to granules at a high efficiency. These results imply that either the targeting machinery can tolerate changes in the tertiary structure of transported proteins, or that the B and A chains of insulin can form a relatively intact three-dimensional structure even in the absence of C peptide.

Full Text

The Full Text of this article is available as a PDF (2.4 MB).

Selected References

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

  1. Armbruster B. L., Carlemalm E., Chiovetti R., Garavito R. M., Hobot J. A., Kellenberger E., Villiger W. Specimen preparation for electron microscopy using low temperature embedding resins. J Microsc. 1982 Apr;126(Pt 1):77–85. doi: 10.1111/j.1365-2818.1982.tb00358.x. [DOI] [PubMed] [Google Scholar]
  2. Bedwell D. M., Klionsky D. J., Emr S. D. The yeast F1-ATPase beta subunit precursor contains functionally redundant mitochondrial protein import information. Mol Cell Biol. 1987 Nov;7(11):4038–4047. doi: 10.1128/mcb.7.11.4038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blundell T. L., Bedarkar S., Rinderknecht E., Humbel R. E. Insulin-like growth factor: a model for tertiary structure accounting for immunoreactivity and receptor binding. Proc Natl Acad Sci U S A. 1978 Jan;75(1):180–184. doi: 10.1073/pnas.75.1.180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bole D. G., Hendershot L. M., Kearney J. F. Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J Cell Biol. 1986 May;102(5):1558–1566. doi: 10.1083/jcb.102.5.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burgess T. L., Craik C. S., Matsuuchi L., Kelly R. B. In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules. J Cell Biol. 1987 Aug;105(2):659–668. doi: 10.1083/jcb.105.2.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Vroede M. A., Rechler M. M., Nissley S. P., Ogawa H., Joshi S., Burke G. T., Katsoyannis P. G. Mitogenic activity and receptor reactivity of hybrid molecules containing portions of the insulin-like growth factor I (IGF-I), IGF-II, and insulin molecules. Diabetes. 1986 Mar;35(3):355–361. doi: 10.2337/diab.35.3.355. [DOI] [PubMed] [Google Scholar]
  7. Docherty K., Steiner D. F. Post-translational proteolysis in polypeptide hormone biosynthesis. Annu Rev Physiol. 1982;44:625–638. doi: 10.1146/annurev.ph.44.030182.003205. [DOI] [PubMed] [Google Scholar]
  8. Froesch E. R., Schmid C., Schwander J., Zapf J. Actions of insulin-like growth factors. Annu Rev Physiol. 1985;47:443–467. doi: 10.1146/annurev.ph.47.030185.002303. [DOI] [PubMed] [Google Scholar]
  9. Gumbiner B., Kelly R. B. Secretory granules of an anterior pituitary cell line, AtT-20, contain only mature forms of corticotropin and beta-lipotropin. Proc Natl Acad Sci U S A. 1981 Jan;78(1):318–322. doi: 10.1073/pnas.78.1.318. [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. Hurt E. C., Pesold-Hurt B., Schatz G. The amino-terminal region of an imported mitochondrial precursor polypeptide can direct cytoplasmic dihydrofolate reductase into the mitochondrial matrix. EMBO J. 1984 Dec 20;3(13):3149–3156. doi: 10.1002/j.1460-2075.1984.tb02272.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Johnson L. M., Bankaitis V. A., Emr S. D. Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease. Cell. 1987 Mar 13;48(5):875–885. doi: 10.1016/0092-8674(87)90084-5. [DOI] [PubMed] [Google Scholar]
  13. Kahn C. R. The molecular mechanism of insulin action. Annu Rev Med. 1985;36:429–451. doi: 10.1146/annurev.me.36.020185.002241. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. King G. L., Kahn C. R. Non-parallel evolution of metabolic and growth-promoting functions of insulin. Nature. 1981 Aug 13;292(5824):644–646. doi: 10.1038/292644a0. [DOI] [PubMed] [Google Scholar]
  17. King G. L., Kahn C. R., Samuels B., Danho W., Bullesbach E. E., Gattner H. G. Synthesis and characterization of molecular hybrids of insulin and insulin-like growth factor I. The role of the A-chain extension peptide. J Biol Chem. 1982 Sep 25;257(18):10869–10873. [PubMed] [Google Scholar]
  18. Mains R. E., Eipper B. A. Biosynthesis of adrenocorticotropic hormone in mouse pituitary tumor cells. J Biol Chem. 1976 Jul 10;251(13):4115–4120. [PubMed] [Google Scholar]
  19. 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]
  20. Moore H. P., Gumbiner B., Kelly R. B. Chloroquine diverts ACTH from a regulated to a constitutive secretory pathway in AtT-20 cells. 1983 Mar 31-Apr 6Nature. 302(5907):434–436. doi: 10.1038/302434a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Ogata C., Hatada M., Tomlinson G., Shin W. C., Kim S. H. Crystal structure of the intensely sweet protein monellin. Nature. 1987 Aug 20;328(6132):739–742. doi: 10.1038/328739a0. [DOI] [PubMed] [Google Scholar]
  24. Orci L., Ravazzola M., Amherdt M., Madsen O., Perrelet A., Vassalli J. D., Anderson R. G. Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles. J Cell Biol. 1986 Dec;103(6 Pt 1):2273–2281. doi: 10.1083/jcb.103.6.2273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Orci L., Ravazzola M., Amherdt M., Madsen O., Vassalli J. D., Perrelet A. Direct identification of prohormone conversion site in insulin-secreting cells. Cell. 1985 Sep;42(2):671–681. doi: 10.1016/0092-8674(85)90124-2. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Orci L., Ravazzola M., Storch M. J., Anderson R. G., Vassalli J. D., Perrelet A. Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesicles. Cell. 1987 Jun 19;49(6):865–868. doi: 10.1016/0092-8674(87)90624-6. [DOI] [PubMed] [Google Scholar]
  28. Rhodes C. J., Halban P. A. Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway. J Cell Biol. 1987 Jul;105(1):145–153. doi: 10.1083/jcb.105.1.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roberts B. L., Richardson W. D., Smith A. E. The effect of protein context on nuclear location signal function. Cell. 1987 Jul 31;50(3):465–475. doi: 10.1016/0092-8674(87)90500-9. [DOI] [PubMed] [Google Scholar]
  30. Roth J., Bendayan M., Orci L. Ultrastructural localization of intracellular antigens by the use of protein A-gold complex. J Histochem Cytochem. 1978 Dec;26(12):1074–1081. doi: 10.1177/26.12.366014. [DOI] [PubMed] [Google Scholar]
  31. Rothman J. E. Protein sorting by selective retention in the endoplasmic reticulum and Golgi stack. Cell. 1987 Aug 14;50(4):521–522. doi: 10.1016/0092-8674(87)90024-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schatz G., Butow R. A. How are proteins imported into mitochondria? Cell. 1983 Feb;32(2):316–318. doi: 10.1016/0092-8674(83)90450-6. [DOI] [PubMed] [Google Scholar]
  34. Steiner D. F., Chan S. J., Welsh J. M., Kwok S. C. Structure and evolution of the insulin gene. Annu Rev Genet. 1985;19:463–484. doi: 10.1146/annurev.ge.19.120185.002335. [DOI] [PubMed] [Google Scholar]
  35. Steiner D. F. On the role of the proinsulin C-peptide. Diabetes. 1978;27 (Suppl 1):145–148. doi: 10.2337/diab.27.1.s145. [DOI] [PubMed] [Google Scholar]
  36. Thim L., Hansen M. T., Norris K., Hoegh I., Boel E., Forstrom J., Ammerer G., Fiil N. P. Secretion and processing of insulin precursors in yeast. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6766–6770. doi: 10.1073/pnas.83.18.6766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tooze J., Hollinshead M., Frank R., Burke B. An antibody specific for an endoproteolytic cleavage site provides evidence that pro-opiomelanocortin is packaged into secretory granules in AtT20 cells before its cleavage. J Cell Biol. 1987 Jul;105(1):155–162. doi: 10.1083/jcb.105.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Valls L. A., Hunter C. P., Rothman J. H., Stevens T. H. Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide. Cell. 1987 Mar 13;48(5):887–897. doi: 10.1016/0092-8674(87)90085-7. [DOI] [PubMed] [Google Scholar]
  39. Wollmer A., Brandenburg D., Vogt H. P., Schermutzki W. Reduction/reoxidation studies with cross-linked insulin derivatives. Hoppe Seylers Z Physiol Chem. 1974 Nov;355(11):1471–1476. [PubMed] [Google Scholar]
  40. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

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

RESOURCES