Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Aug 1;349(Pt 3):843–852. doi: 10.1042/bj3490843

Involvement of the membrane lipid bilayer in sorting prohormone convertase 2 into the regulated secretory pathway.

M Blázquez 1, C Thiele 1, W B Huttner 1, K Docherty 1, K I Shennan 1
PMCID: PMC1221213  PMID: 10903147

Abstract

Prohormone convertase 2 (PC2) is a neuroendocrine-specific protease involved in the intracellular maturation of prohormones and proneuropeptides. PC2 is synthesised as a proprotein (proPC2) that undergoes proteolysis, aggregation and membrane association during its transit through the regulated secretory pathway. We have previously shown that the pro region of proPC2 plays a key role in its aggregation and membrane association. To investigate this further, we determined the binding properties of a peptide containing amino acids 45-84 of proPC2 (proPC2(45-84)) to trans-Golgi network/granule-enriched membranes from the AtT20 cell line. Removal of peripheral membrane proteins or hydrolysis of integral membrane proteins did not affect the binding properties of proPC2(45-84). Rather, proPC2(45-84) was shown to bind to protein-free liposomes in a pH- and Ca(2+)-dependent manner. To identify the component of the lipid bilayer involved in this membrane association, we used chromaffin-granule membranes and studied the binding properties of the endogenous PC2. Treatment of the membranes with saponin, a cholesterol-depleting detergent, failed to extract PC2 from the membranes, whereas chromogranin A (CgA) was removed. Treatment of the membranes with Triton X-100 yielded a low-density detergent-insoluble fraction enriched in PC2, but not CgA. The detergent-insoluble fraction also contained glycoprotein III, known to be part of the lipid rafts (membrane microdomains rich in sphingolipids). Finally, sphingolipid depletion of AtT20 cells resulted in the mis-sorting of PC2, suggestive of a link between the association of PC2 with lipid rafts in the membrane and its sorting into the regulated secretory pathway.

Full Text

The Full Text of this article is available as a PDF (308.2 KB).

Selected References

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

  1. 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]
  2. Arvan P., Castle D. Sorting and storage during secretory granule biogenesis: looking backward and looking forward. Biochem J. 1998 Jun 15;332(Pt 3):593–610. doi: 10.1042/bj3320593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  4. Braks J. A., Martens G. J. 7B2 is a neuroendocrine chaperone that transiently interacts with prohormone convertase PC2 in the secretory pathway. Cell. 1994 Jul 29;78(2):263–273. doi: 10.1016/0092-8674(94)90296-8. [DOI] [PubMed] [Google Scholar]
  5. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [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. Chanat E., Huttner W. B. Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network. J Cell Biol. 1991 Dec;115(6):1505–1519. doi: 10.1083/jcb.115.6.1505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chanat E., Weiss U., Huttner W. B. The disulfide bond in chromogranin B, which is essential for its sorting to secretory granules, is not required for its aggregation in the trans-Golgi network. FEBS Lett. 1994 Sep 5;351(2):225–230. doi: 10.1016/0014-5793(94)00865-5. [DOI] [PubMed] [Google Scholar]
  9. Chanat E., Weiss U., Huttner W. B., Tooze S. A. Reduction of the disulfide bond of chromogranin B (secretogranin I) in the trans-Golgi network causes its missorting to the constitutive secretory pathways. EMBO J. 1993 May;12(5):2159–2168. doi: 10.1002/j.1460-2075.1993.tb05864.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Christie D. L., Batchelor D. C., Palmer D. J. Identification of kex2-related proteases in chromaffin granules by partial amino acid sequence analysis. J Biol Chem. 1991 Aug 25;266(24):15679–15683. [PubMed] [Google Scholar]
  11. Cool D. R., Loh Y. P. Identification of a sorting signal for the regulated secretory pathway at the N-terminus of pro-opiomelanocortin. Biochimie. 1994;76(3-4):265–270. doi: 10.1016/0300-9084(94)90156-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cool D. R., Normant E., Shen F., Chen H. C., Pannell L., Zhang Y., Loh Y. P. Carboxypeptidase E is a regulated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in Cpe(fat) mice. Cell. 1997 Jan 10;88(1):73–83. doi: 10.1016/s0092-8674(00)81860-7. [DOI] [PubMed] [Google Scholar]
  13. Creemers J. W., Usac E. F., Bright N. A., Van de Loo J. W., Jansen E., Van de Ven W. J., Hutton J. C. Identification of a transferable sorting domain for the regulated pathway in the prohormone convertase PC2. J Biol Chem. 1996 Oct 11;271(41):25284–25291. doi: 10.1074/jbc.271.41.25284. [DOI] [PubMed] [Google Scholar]
  14. Fischer-Colbrie R., Zangerle R., Frischenschlager I., Weber A., Winkler H. Isolation and immunological characterization of a glycoprotein from adrenal chromaffin granules. J Neurochem. 1984 Apr;42(4):1008–1016. doi: 10.1111/j.1471-4159.1984.tb12704.x. [DOI] [PubMed] [Google Scholar]
  15. Fricker L. D., Das B., Angeletti R. H. Identification of the pH-dependent membrane anchor of carboxypeptidase E (EC 3.4.17.10). J Biol Chem. 1990 Feb 15;265(5):2476–2482. [PubMed] [Google Scholar]
  16. Glombik M. M., Krömer A., Salm T., Huttner W. B., Gerdes H. H. The disulfide-bonded loop of chromogranin B mediates membrane binding and directs sorting from the trans-Golgi network to secretory granules. EMBO J. 1999 Feb 15;18(4):1059–1070. doi: 10.1093/emboj/18.4.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Guest P. C., Arden S. D., Bennett D. L., Clark A., Rutherford N. G., Hutton J. C. The post-translational processing and intracellular sorting of PC2 in the islets of Langerhans. J Biol Chem. 1992 Nov 5;267(31):22401–22406. [PubMed] [Google Scholar]
  18. Irminger J. C., Verchere C. B., Meyer K., Halban P. A. Proinsulin targeting to the regulated pathway is not impaired in carboxypeptidase E-deficient Cpefat/Cpefat mice. J Biol Chem. 1997 Oct 31;272(44):27532–27534. doi: 10.1074/jbc.272.44.27532. [DOI] [PubMed] [Google Scholar]
  19. Jan G., Taylor N. A., Scougall K. T., Docherty K., Shennan K. I. The propeptide of prohormone convertase PC2 acts as a transferable aggregation and membrane-association signal. Eur J Biochem. 1998 Oct 1;257(1):41–46. doi: 10.1046/j.1432-1327.1998.2570041.x. [DOI] [PubMed] [Google Scholar]
  20. Krömer A., Glombik M. M., Huttner W. B., Gerdes H. H. Essential role of the disulfide-bonded loop of chromogranin B for sorting to secretory granules is revealed by expression of a deletion mutant in the absence of endogenous granin synthesis. J Cell Biol. 1998 Mar 23;140(6):1331–1346. doi: 10.1083/jcb.140.6.1331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kuliawat R., Arvan P. Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells. J Cell Biol. 1994 Jul;126(1):77–86. doi: 10.1083/jcb.126.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lainé J., Lebel D. Efficient binding of regulated secretory protein aggregates to membrane phospholipids at acidic pH. Biochem J. 1999 Mar 1;338(Pt 2):289–294. [PMC free article] [PubMed] [Google Scholar]
  23. Lamango N. S., Apletalina E., Liu J., Lindberg I. The proteolytic maturation of prohormone convertase 2 (PC2) is a pH-driven process. Arch Biochem Biophys. 1999 Feb 15;362(2):275–282. doi: 10.1006/abbi.1998.1033. [DOI] [PubMed] [Google Scholar]
  24. Laslop A., Steiner H. J., Egger C., Wolkersdorfer M., Kapelari S., Hogue-Angeletti R., Erickson J. D., Fischer-Colbrie R., Winkler H. Glycoprotein III (clusterin, sulfated glycoprotein 2) in endocrine, nervous, and other tissues: immunochemical characterization, subcellular localization, and regulation of biosynthesis. J Neurochem. 1993 Oct;61(4):1498–1505. doi: 10.1111/j.1471-4159.1993.tb13645.x. [DOI] [PubMed] [Google Scholar]
  25. Ledesma M. D., Simons K., Dotti C. G. Neuronal polarity: essential role of protein-lipid complexes in axonal sorting. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3966–3971. doi: 10.1073/pnas.95.7.3966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matthews G., Colman A. A highly efficient, cell-free translation/translocation system prepared from Xenopus eggs. Nucleic Acids Res. 1991 Dec 11;19(23):6405–6412. doi: 10.1093/nar/19.23.6405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Matthews G., Shennan K. I., Seal A. J., Taylor N. A., Colman A., Docherty K. Autocatalytic maturation of the prohormone convertase PC2. J Biol Chem. 1994 Jan 7;269(1):588–592. [PubMed] [Google Scholar]
  28. 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]
  29. Normant E., Loh Y. P. Depletion of carboxypeptidase E, a regulated secretory pathway sorting receptor, causes misrouting and constitutive secretion of proinsulin and proenkephalin, but not chromogranin A. Endocrinology. 1998 Apr;139(4):2137–2145. doi: 10.1210/endo.139.4.5951. [DOI] [PubMed] [Google Scholar]
  30. Palmer D. J., Christie D. L. Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H), and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules. J Biol Chem. 1992 Oct 5;267(28):19806–19812. [PubMed] [Google Scholar]
  31. 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]
  32. Scheiffele P., Roth M. G., Simons K. Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain. EMBO J. 1997 Sep 15;16(18):5501–5508. doi: 10.1093/emboj/16.18.5501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shennan K. I. Intracellular targeting of secretory proteins in neuroendocrine cells. Biochem Soc Trans. 1996 May;24(2):535–539. doi: 10.1042/bst0240535. [DOI] [PubMed] [Google Scholar]
  34. Shennan K. I., Taylor N. A., Docherty K. Calcium- and pH-dependent aggregation and membrane association of the precursor of the prohormone convertase PC2. J Biol Chem. 1994 Jul 15;269(28):18646–18650. [PubMed] [Google Scholar]
  35. Shennan K. I., Taylor N. A., Jermany J. L., Matthews G., Docherty K. Differences in pH optima and calcium requirements for maturation of the prohormone convertases PC2 and PC3 indicates different intracellular locations for these events. J Biol Chem. 1995 Jan 20;270(3):1402–1407. doi: 10.1074/jbc.270.3.1402. [DOI] [PubMed] [Google Scholar]
  36. Simons K., Ikonen E. Functional rafts in cell membranes. Nature. 1997 Jun 5;387(6633):569–572. doi: 10.1038/42408. [DOI] [PubMed] [Google Scholar]
  37. Smith A. D., Winkler H. A simple method for the isolation of adrenal chromaffin granules on a large scale. Biochem J. 1967 May;103(2):480–482. doi: 10.1042/bj1030480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stulnig T. M., Berger M., Sigmund T., Raederstorff D., Stockinger H., Waldhäusl W. Polyunsaturated fatty acids inhibit T cell signal transduction by modification of detergent-insoluble membrane domains. J Cell Biol. 1998 Nov 2;143(3):637–644. doi: 10.1083/jcb.143.3.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Szoka F., Jr, Papahadjopoulos D. Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4194–4198. doi: 10.1073/pnas.75.9.4194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Taylor N. A., Jan G., Scougall K. T., Docherty K., Shennan K. I. Sorting of PC2 to the regulated secretory pathway in AtT20 cells. J Mol Endocrinol. 1998 Oct;21(2):209–216. doi: 10.1677/jme.0.0210209. [DOI] [PubMed] [Google Scholar]
  41. Taylor N. A., Shennan K. I., Cutler D. F., Docherty K. Mutations within the propeptide, the primary cleavage site or the catalytic site, or deletion of C-terminal sequences, prevents secretion of proPC2 from transfected COS-7 cells. Biochem J. 1997 Jan 15;321(Pt 2):367–373. doi: 10.1042/bj3210367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Thiele C., Gerdes H. H., Huttner W. B. Protein secretion: puzzling receptors. Curr Biol. 1997 Aug 1;7(8):R496–R500. doi: 10.1016/s0960-9822(06)00247-8. [DOI] [PubMed] [Google Scholar]
  43. Thiele C., Huttner W. B. The disulfide-bonded loop of chromogranins, which is essential for sorting to secretory granules, mediates homodimerization. J Biol Chem. 1998 Jan 9;273(2):1223–1231. doi: 10.1074/jbc.273.2.1223. [DOI] [PubMed] [Google Scholar]
  44. Varlamov O., Fricker L. D. The C-terminal region of carboxypeptidase E involved in membrane binding is distinct from the region involved with intracellular routing. J Biol Chem. 1996 Mar 15;271(11):6077–6083. doi: 10.1074/jbc.271.11.6077. [DOI] [PubMed] [Google Scholar]
  45. Wang E., Norred W. P., Bacon C. W., Riley R. T., Merrill A. H., Jr Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme. J Biol Chem. 1991 Aug 5;266(22):14486–14490. [PubMed] [Google Scholar]
  46. Westhead E. W. Lipid composition and orientation in secretory vesicles. Ann N Y Acad Sci. 1987;493:92–100. doi: 10.1111/j.1749-6632.1987.tb27186.x. [DOI] [PubMed] [Google Scholar]
  47. Zegers M. M., Hoekstra D. Mechanisms and functional features of polarized membrane traffic in epithelial and hepatic cells. Biochem J. 1998 Dec 1;336(Pt 2):257–269. doi: 10.1042/bj3360257. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES