Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Sep 1;126(5):1149–1156. doi: 10.1083/jcb.126.5.1149

pH-independent and -dependent cleavage of proinsulin in the same secretory vesicle

PMCID: PMC2120168  PMID: 8063854

Abstract

By quantitative immunoelectron microscopy and HPLC, we have studied the effect of disrupting pH gradients, by ammonium chloride, on proinsulin conversion in the insulin-producing B-cells of the islets of langerhans. Proinsulin content and pH in single secretory vesicles were measured on consecutive serial sections immunostained alternately with anti-proinsulin or anti-dinitrophenol (to reveal the pH-sensitive probe DAMP) antibodies. Radioactivity labeled proinsulin, proinsulin cleavage intermediates, and insulin were quantitated by HPLC analysis of extracts of islets treated in the same conditions. Cleavage at the C- peptide/A-chain junction is significantly less sensitive to pH gradient disruption than that of the B-chain/C-peptide junction, but the range of pH and proinsulin content in individual vesicles indicate that both cleavages occur in the same vesicle released from the TGN.

Full Text

The Full Text of this article is available as a PDF (2.6 MB).

Selected References

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

  1. Al-Awqati Q. Proton-translocating ATPases. Annu Rev Cell Biol. 1986;2:179–199. doi: 10.1146/annurev.cb.02.110186.001143. [DOI] [PubMed] [Google Scholar]
  2. Anderson R. G., Falck J. R., Goldstein J. L., Brown M. S. Visualization of acidic organelles in intact cells by electron microscopy. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4838–4842. doi: 10.1073/pnas.81.15.4838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson R. G., Orci L. A view of acidic intracellular compartments. J Cell Biol. 1988 Mar;106(3):539–543. doi: 10.1083/jcb.106.3.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Bailyes E. M., Hutton J. C. Kinetic analysis of the type-1 proinsulin endopeptidase by a monoclonal antibody-based immunoadsorbent assay. Biochem J. 1992 Aug 15;286(Pt 1):223–229. doi: 10.1042/bj2860223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bailyes E. M., Shennan K. I., Seal A. J., Smeekens S. P., Steiner D. F., Hutton J. C., Docherty K. A member of the eukaryotic subtilisin family (PC3) has the enzymic properties of the type 1 proinsulin-converting endopeptidase. Biochem J. 1992 Jul 15;285(Pt 2):391–394. doi: 10.1042/bj2850391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Barasch J., Kiss B., Prince A., Saiman L., Gruenert D., al-Awqati Q. Defective acidification of intracellular organelles in cystic fibrosis. Nature. 1991 Jul 4;352(6330):70–73. doi: 10.1038/352070a0. [DOI] [PubMed] [Google Scholar]
  8. Bennett D. L., Bailyes E. M., Nielsen E., Guest P. C., Rutherford N. G., Arden S. D., Hutton J. C. Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family. J Biol Chem. 1992 Jul 25;267(21):15229–15236. [PubMed] [Google Scholar]
  9. Davidson H. W., Rhodes C. J., Hutton J. C. Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic beta cell via two distinct site-specific endopeptidases. Nature. 1988 May 5;333(6168):93–96. doi: 10.1038/333093a0. [DOI] [PubMed] [Google Scholar]
  10. Gross D., Skvorak A., Hendrick G., Weir G., Villa-Komaroff L., Halban P. Oxidation of rat insulin II, but not I, leads to anomalous elution profiles upon HPLC analysis of insulin-related peptides. FEBS Lett. 1988 Dec 5;241(1-2):205–208. doi: 10.1016/0014-5793(88)81062-7. [DOI] [PubMed] [Google Scholar]
  11. Halban P. A., Rhodes C. J., Shoelson S. E. High-performance liquid chromatography (HPLC): a rapid, flexible and sensitive method for separating islet proinsulin and insulin. Diabetologia. 1986 Dec;29(12):893–896. doi: 10.1007/BF00870146. [DOI] [PubMed] [Google Scholar]
  12. Lacy P. E., Kostianovsky M. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes. 1967 Jan;16(1):35–39. doi: 10.2337/diab.16.1.35. [DOI] [PubMed] [Google Scholar]
  13. Madsen O. D., Frank B. H., Steiner D. F. Human proinsulin-specific antigenic determinants identified by monoclonal antibodies. Diabetes. 1984 Oct;33(10):1012–1016. doi: 10.2337/diab.33.10.1012. [DOI] [PubMed] [Google Scholar]
  14. Mains R. E., May V. The role of a low pH intracellular compartment in the processing, storage, and secretion of ACTH and endorphin. J Biol Chem. 1988 Jun 5;263(16):7887–7894. [PubMed] [Google Scholar]
  15. Mallya S. K., Partin J. S., Valdizan M. C., Lennarz W. J. Proteolysis of the major yolk glycoproteins is regulated by acidification of the yolk platelets in sea urchin embryos. J Cell Biol. 1992 Jun;117(6):1211–1221. doi: 10.1083/jcb.117.6.1211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Maxwell M. H. Two rapid and simple methods used for the removal of resins from 1.0 micron thick epoxy sections. J Microsc. 1978 Mar;112(2):253–255. doi: 10.1111/j.1365-2818.1978.tb01174.x. [DOI] [PubMed] [Google Scholar]
  17. Mellman I., Fuchs R., Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986;55:663–700. doi: 10.1146/annurev.bi.55.070186.003311. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Neerman-Arbez M., Cirulli V., Halban P. A. Levels of the conversion endoproteases PC1 (PC3) and PC2 distinguish between insulin-producing pancreatic islet beta cells and non-beta cells. Biochem J. 1994 May 15;300(Pt 1):57–61. doi: 10.1042/bj3000057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Neerman-Arbez M., Sizonenko S. V., Halban P. A. Slow cleavage at the proinsulin B-chain/connecting peptide junction associated with low levels of endoprotease PC1/3 in transformed beta cells. J Biol Chem. 1993 Aug 5;268(22):16098–16100. [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. Rhodes C. J., Halban P. A. The intracellular handling of insulin-related peptides in isolated pancreatic islets. Evidence for differential rates of degradation of insulin and C-peptide. Biochem J. 1988 Apr 1;251(1):23–30. doi: 10.1042/bj2510023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rhodes C. J., Lincoln B., Shoelson S. E. Preferential cleavage of des-31,32-proinsulin over intact proinsulin by the insulin secretory granule type II endopeptidase. Implication of a favored route for prohormone processing. J Biol Chem. 1992 Nov 15;267(32):22719–22727. [PubMed] [Google Scholar]
  27. 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]
  28. Sizonenko S. V., Halban P. A. Differential rates of conversion of rat proinsulins I and II. Evidence for slow cleavage at the B-chain/C-peptide junction of proinsulin II. Biochem J. 1991 Sep 15;278(Pt 3):621–625. doi: 10.1042/bj2780621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sizonenko S., Irminger J. C., Buhler L., Deng S., Morel P., Halban P. A. Kinetics of proinsulin conversion in human islets. Diabetes. 1993 Jun;42(6):933–936. doi: 10.2337/diab.42.6.933. [DOI] [PubMed] [Google Scholar]
  30. Steiner D. F., Michael J., Houghten R., Mathieu M., Gardner P. R., Ravazzola M., Orci L. Use of a synthetic peptide antigen to generate antisera reactive with a proteolytic processing site in native human proinsulin: demonstration of cleavage within clathrin-coated (pro)secretory vesicles. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6184–6188. doi: 10.1073/pnas.84.17.6184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Steiner D. F., Smeekens S. P., Ohagi S., Chan S. J. The new enzymology of precursor processing endoproteases. J Biol Chem. 1992 Nov 25;267(33):23435–23438. [PubMed] [Google Scholar]

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

RESOURCES