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. 1994 Jan 1;13(1):18–33. doi: 10.1002/j.1460-2075.1994.tb06231.x

Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface.

S S Molloy 1, L Thomas 1, J K VanSlyke 1, P E Stenberg 1, G Thomas 1
PMCID: PMC394775  PMID: 7508380

Abstract

Furin is a membrane-associated endoprotease that efficiently cleaves precursor proteins on the C-terminal side of the consensus sequence, Arg-X-Lys/Arg-Arg1, and has been proposed to catalyze these reactions in both exocytic and endocytic compartments. To study its biosynthesis and routing, a furin construct (designated fur/f) containing the FLAG epitope tag inserted on the C-terminal side of the enzyme's autoproteolytic maturation site was used. Introduction of the epitope tag had no effect on the expression, proteolytic maturation or activity of furin. Analysis of the localization of fur/f by immunofluorescence microscopy showed that its staining pattern largely overlapped with those of several Golgi-associated markers. Treatment of cells with brefeldin A caused the fur/f distribution to collapse around the microtubule organizing center, indicating that furin is concentrated in the trans-Golgi network (TGN). Immunoelectron microscopy showed unequivocally that furin resides in the TGN where it colocalized with TGN38. In agreement with its proposed activity in multiple compartments, antibody uptake studies showed that fur/f cycles between the cell surface and TGN. Furthermore, targeting to the TGN requires sequences in the cytoplasmic tail of the enzyme. Pulse-chase and immunofluorescence analyses demonstrated that proregion removal occurs in the endoplasmic reticulum and that cleavage may be required for exist from this compartment. Finally, we show that proregion removal is necessary but not sufficient for enzyme activation.

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Selected References

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

  1. Anderson E. D., Thomas L., Hayflick J. S., Thomas G. Inhibition of HIV-1 gp160-dependent membrane fusion by a furin-directed alpha 1-antitrypsin variant. J Biol Chem. 1993 Nov 25;268(33):24887–24891. [PubMed] [Google Scholar]
  2. Barr P. J. Mammalian subtilisins: the long-sought dibasic processing endoproteases. Cell. 1991 Jul 12;66(1):1–3. doi: 10.1016/0092-8674(91)90129-m. [DOI] [PubMed] [Google Scholar]
  3. Bentley A. K., Rees D. J., Rizza C., Brownlee G. G. Defective propeptide processing of blood clotting factor IX caused by mutation of arginine to glutamine at position -4. Cell. 1986 May 9;45(3):343–348. doi: 10.1016/0092-8674(86)90319-3. [DOI] [PubMed] [Google Scholar]
  4. Bos K., Wraight C., Stanley K. K. TGN38 is maintained in the trans-Golgi network by a tyrosine-containing motif in the cytoplasmic domain. EMBO J. 1993 May;12(5):2219–2228. doi: 10.1002/j.1460-2075.1993.tb05870.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bresnahan P. A., Leduc R., Thomas L., Thorner J., Gibson H. L., Brake A. J., Barr P. J., Thomas G. Human fur gene encodes a yeast KEX2-like endoprotease that cleaves pro-beta-NGF in vivo. J Cell Biol. 1990 Dec;111(6 Pt 2):2851–2859. doi: 10.1083/jcb.111.6.2851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chege N. W., Pfeffer S. R. Compartmentation of the Golgi complex: brefeldin-A distinguishes trans-Golgi cisternae from the trans-Golgi network. J Cell Biol. 1990 Sep;111(3):893–899. doi: 10.1083/jcb.111.3.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dahms N. M., Lobel P., Kornfeld S. Mannose 6-phosphate receptors and lysosomal enzyme targeting. J Biol Chem. 1989 Jul 25;264(21):12115–12118. [PubMed] [Google Scholar]
  8. Disdier M., Morrissey J. H., Fugate R. D., Bainton D. F., McEver R. P. Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway. Mol Biol Cell. 1992 Mar;3(3):309–321. doi: 10.1091/mbc.3.3.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Duncan J. R., Kornfeld S. Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus. J Cell Biol. 1988 Mar;106(3):617–628. doi: 10.1083/jcb.106.3.617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ehlers M. R., Riordan J. F. Membrane proteins with soluble counterparts: role of proteolysis in the release of transmembrane proteins. Biochemistry. 1991 Oct 22;30(42):10065–10074. doi: 10.1021/bi00106a001. [DOI] [PubMed] [Google Scholar]
  11. Foster D. C., Sprecher C. A., Holly R. D., Gambee J. E., Walker K. M., Kumar A. A. Endoproteolytic processing of the dibasic cleavage site in the human protein C precursor in transfected mammalian cells: effects of sequence alterations on efficiency of cleavage. Biochemistry. 1990 Jan 16;29(2):347–354. doi: 10.1021/bi00454a007. [DOI] [PubMed] [Google Scholar]
  12. Fuller R. S., Brake A., Thorner J. Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1434–1438. doi: 10.1073/pnas.86.5.1434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gotoh B., Ohnishi Y., Inocencio N. M., Esaki E., Nakayama K., Barr P. J., Thomas G., Nagai Y. Mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: superiority of furin/PACE to PC2 or PC1/PC3. J Virol. 1992 Nov;66(11):6391–6397. doi: 10.1128/jvi.66.11.6391-6397.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Green J., Griffiths G., Louvard D., Quinn P., Warren G. Passage of viral membrane proteins through the Golgi complex. J Mol Biol. 1981 Nov 15;152(4):663–698. doi: 10.1016/0022-2836(81)90122-4. [DOI] [PubMed] [Google Scholar]
  15. Griffiths G., Hoflack B., Simons K., Mellman I., Kornfeld S. The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell. 1988 Feb 12;52(3):329–341. doi: 10.1016/s0092-8674(88)80026-6. [DOI] [PubMed] [Google Scholar]
  16. Griffiths G., McDowall A., Back R., Dubochet J. On the preparation of cryosections for immunocytochemistry. J Ultrastruct Res. 1984 Oct;89(1):65–78. doi: 10.1016/s0022-5320(84)80024-6. [DOI] [PubMed] [Google Scholar]
  17. Griffiths G., Simons K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science. 1986 Oct 24;234(4775):438–443. doi: 10.1126/science.2945253. [DOI] [PubMed] [Google Scholar]
  18. Hallenberger S., Bosch V., Angliker H., Shaw E., Klenk H. D., Garten W. Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160. Nature. 1992 Nov 26;360(6402):358–361. doi: 10.1038/360358a0. [DOI] [PubMed] [Google Scholar]
  19. Hatsuzawa K., Nagahama M., Takahashi S., Takada K., Murakami K., Nakayama K. Purification and characterization of furin, a Kex2-like processing endoprotease, produced in Chinese hamster ovary cells. J Biol Chem. 1992 Aug 15;267(23):16094–16099. [PubMed] [Google Scholar]
  20. Hayflick J. S., Wolfgang W. J., Forte M. A., Thomas G. A unique Kex2-like endoprotease from Drosophila melanogaster is expressed in the central nervous system during early embryogenesis. J Neurosci. 1992 Mar;12(3):705–717. doi: 10.1523/JNEUROSCI.12-03-00705.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hosaka M., Nagahama M., Kim W. S., Watanabe T., Hatsuzawa K., Ikemizu J., Murakami K., Nakayama K. Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway. J Biol Chem. 1991 Jul 5;266(19):12127–12130. [PubMed] [Google Scholar]
  22. Humphrey J. S., Peters P. J., Yuan L. C., Bonifacino J. S. Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence. J Cell Biol. 1993 Mar;120(5):1123–1135. doi: 10.1083/jcb.120.5.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  24. Jiang Y. P., Wang H., D'Eustachio P., Musacchio J. M., Schlessinger J., Sap J. Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region. Mol Cell Biol. 1993 May;13(5):2942–2951. doi: 10.1128/mcb.13.5.2942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Julius D., Brake A., Blair L., Kunisawa R., Thorner J. Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor. Cell. 1984 Jul;37(3):1075–1089. doi: 10.1016/0092-8674(84)90442-2. [DOI] [PubMed] [Google Scholar]
  26. Kayyem J. F., Roman J. M., de la Rosa E. J., Schwarz U., Dreyer W. J. Bravo/Nr-CAM is closely related to the cell adhesion molecules L1 and Ng-CAM and has a similar heterodimer structure. J Cell Biol. 1992 Sep;118(5):1259–1270. doi: 10.1083/jcb.118.5.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Klausner R. D., Donaldson J. G., Lippincott-Schwartz J. Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol. 1992 Mar;116(5):1071–1080. doi: 10.1083/jcb.116.5.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Klimpel K. R., Molloy S. S., Thomas G., Leppla S. H. Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10277–10281. doi: 10.1073/pnas.89.21.10277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Leduc R., Molloy S. S., Thorne B. A., Thomas G. Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage. J Biol Chem. 1992 Jul 15;267(20):14304–14308. [PubMed] [Google Scholar]
  31. Luzio J. P., Brake B., Banting G., Howell K. E., Braghetta P., Stanley K. K. Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38). Biochem J. 1990 Aug 15;270(1):97–102. doi: 10.1042/bj2700097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. MacDonald R. G., Pfeffer S. R., Coussens L., Tepper M. A., Brocklebank C. M., Mole J. E., Anderson J. K., Chen E., Czech M. P., Ullrich A. A single receptor binds both insulin-like growth factor II and mannose-6-phosphate. Science. 1988 Mar 4;239(4844):1134–1137. doi: 10.1126/science.2964083. [DOI] [PubMed] [Google Scholar]
  33. McCune J. M., Rabin L. B., Feinberg M. B., Lieberman M., Kosek J. C., Reyes G. R., Weissman I. L. Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus. Cell. 1988 Apr 8;53(1):55–67. doi: 10.1016/0092-8674(88)90487-4. [DOI] [PubMed] [Google Scholar]
  34. McKeehan W. L., Ham R. G. Methods for reducing the serum requirement for growth in vitro of nontransformed diploid fibroblasts. Dev Biol Stand. 1976 Dec 13;37:97–98. [PubMed] [Google Scholar]
  35. Misumi Y., Oda K., Fujiwara T., Takami N., Tashiro K., Ikehara Y. Functional expression of furin demonstrating its intracellular localization and endoprotease activity for processing of proalbumin and complement pro-C3. J Biol Chem. 1991 Sep 5;266(25):16954–16959. [PubMed] [Google Scholar]
  36. Mizuno K., Nakamura T., Ohshima T., Tanaka S., Matsuo H. Characterization of KEX2-encoded endopeptidase from yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1989 Feb 28;159(1):305–311. doi: 10.1016/0006-291x(89)92438-8. [DOI] [PubMed] [Google Scholar]
  37. Moehring J. M., Inocencio N. M., Robertson B. J., Moehring T. J. Expression of mouse furin in a Chinese hamster cell resistant to Pseudomonas exotoxin A and viruses complements the genetic lesion. J Biol Chem. 1993 Feb 5;268(4):2590–2594. [PubMed] [Google Scholar]
  38. Molloy S. S., Bresnahan P. A., Leppla S. H., Klimpel K. R., Thomas G. Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen. J Biol Chem. 1992 Aug 15;267(23):16396–16402. [PubMed] [Google Scholar]
  39. Morgan D. O., Edman J. C., Standring D. N., Fried V. A., Smith M. C., Roth R. A., Rutter W. J. Insulin-like growth factor II receptor as a multifunctional binding protein. Nature. 1987 Sep 24;329(6137):301–307. doi: 10.1038/329301a0. [DOI] [PubMed] [Google Scholar]
  40. Pearse B. M., Robinson M. S. Clathrin, adaptors, and sorting. Annu Rev Cell Biol. 1990;6:151–171. doi: 10.1146/annurev.cb.06.110190.001055. [DOI] [PubMed] [Google Scholar]
  41. Reaves B., Banting G. Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN38. J Cell Biol. 1992 Jan;116(1):85–94. doi: 10.1083/jcb.116.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rehemtulla A., Dorner A. J., Kaufman R. J. Regulation of PACE propeptide-processing activity: requirement for a post-endoplasmic reticulum compartment and autoproteolytic activation. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8235–8239. doi: 10.1073/pnas.89.17.8235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Robinson M. S. Adaptins. Trends Cell Biol. 1992 Oct;2(10):293–297. doi: 10.1016/0962-8924(92)90118-7. [DOI] [PubMed] [Google Scholar]
  44. Slot J. W., Geuze H. J., Gigengack S., Lienhard G. E., James D. E. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol. 1991 Apr;113(1):123–135. doi: 10.1083/jcb.113.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sossin W. S., Fisher J. M., Scheller R. H. Cellular and molecular biology of neuropeptide processing and packaging. Neuron. 1989 May;2(5):1407–1417. doi: 10.1016/0896-6273(89)90186-4. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Stieneke-Gröber A., Vey M., Angliker H., Shaw E., Thomas G., Roberts C., Klenk H. D., Garten W. Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease. EMBO J. 1992 Jul;11(7):2407–2414. doi: 10.1002/j.1460-2075.1992.tb05305.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tokuyasu K. T. Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for cryoultramicrotomy. Histochem J. 1989 Mar;21(3):163–171. doi: 10.1007/BF01007491. [DOI] [PubMed] [Google Scholar]
  49. Wilcox C. A., Redding K., Wright R., Fuller R. S. Mutation of a tyrosine localization signal in the cytosolic tail of yeast Kex2 protease disrupts Golgi retention and results in default transport to the vacuole. Mol Biol Cell. 1992 Dec;3(12):1353–1371. doi: 10.1091/mbc.3.12.1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wise R. J., Barr P. J., Wong P. A., Kiefer M. C., Brake A. J., Kaufman R. J. Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9378–9382. doi: 10.1073/pnas.87.23.9378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yoshimasa Y., Paul J. I., Whittaker J., Steiner D. F. Effects of amino acid replacements within the tetrabasic cleavage site on the processing of the human insulin receptor precursor expressed in Chinese hamster ovary cells. J Biol Chem. 1990 Oct 5;265(28):17230–17237. [PubMed] [Google Scholar]

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