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. 1987 Dec 1;105(6):2735–2743. doi: 10.1083/jcb.105.6.2735

Constitutive apical secretion of an 80-kD sulfated glycoprotein complex in the polarized epithelial Madin-Darby canine kidney cell line

PMCID: PMC2114694  PMID: 3693398

Abstract

The biosynthesis, processing, and apical secretion of a group of polypeptides (Kondor-Koch, C., R. Bravo, S. D. Fuller, D. Cutler, and H. Garoff. 1985. Cell. 43:297-306) are studied in MDCK cells using a specific polyclonal antiserum. These polypeptides are synthesized as a precursor protein which has an apparent Mr of 65,000 in its high mannose form. This precursor is converted into a protein with an apparent Mr of 80,000 containing complex carbohydrates and sulfate. After intracellular cleavage of the 80-kD protein, the 35-45-kD subunits are secreted as an 80-kD glycoprotein complex (gp 80) linked together by disulfide bonds. Secretion of the protein complex occurs by a constitutive pathway at the apical surface of the epithelial monolayer. Since the immediate post-translational precursor, the 65-kD protein, is hydrophilic in nature as shown by its partitioning behavior in a phase-separated Triton X-114 solution, gp 80 is segregated into the apical exocytotic pathway as a soluble molecule. The proteolytic maturation of gp 80 is blocked in the presence of chloroquine and its secretion is retarded. The 80-kD precursor is released at the apical cell surface, demonstrating that proteolytic processing is not necessary for the apical secretion of this protein. If N-glycosylation is inhibited by tunicamycin treatment the protein is secreted in equal amounts at both cell surfaces, indicating a role of the carbohydrate moieties in the vectorial transport of this protein.

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Selected References

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  1. Anderson R. G., Pathak R. K. Vesicles and cisternae in the trans Golgi apparatus of human fibroblasts are acidic compartments. Cell. 1985 Mar;40(3):635–643. doi: 10.1016/0092-8674(85)90212-0. [DOI] [PubMed] [Google Scholar]
  2. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  3. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  4. Caplan M. J., Anderson H. C., Palade G. E., Jamieson J. D. Intracellular sorting and polarized cell surface delivery of (Na+,K+)ATPase, an endogenous component of MDCK cell basolateral plasma membranes. Cell. 1986 Aug 15;46(4):623–631. doi: 10.1016/0092-8674(86)90888-3. [DOI] [PubMed] [Google Scholar]
  5. Cidon S., Ben-David H., Nelson N. ATP-driven proton fluxes across membranes of secretory organelles. J Biol Chem. 1983 Oct 10;258(19):11684–11688. [PubMed] [Google Scholar]
  6. Dean R. T., Jessup W., Roberts C. R. Effects of exogenous amines on mammalian cells, with particular reference to membrane flow. Biochem J. 1984 Jan 1;217(1):27–40. doi: 10.1042/bj2170027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fuller S. D., Bravo R., Simons K. An enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late Golgi compartments. EMBO J. 1985 Feb;4(2):297–307. doi: 10.1002/j.1460-2075.1985.tb03629.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fuller S., von Bonsdorff C. H., Simons K. Vesicular stomatitis virus infects and matures only through the basolateral surface of the polarized epithelial cell line, MDCK. Cell. 1984 Aug;38(1):65–77. doi: 10.1016/0092-8674(84)90527-0. [DOI] [PubMed] [Google Scholar]
  9. Garoff H., Kondor-Koch C., Riedel H. Structure and assembly of alphaviruses. Curr Top Microbiol Immunol. 1982;99:1–50. doi: 10.1007/978-3-642-68528-6_1. [DOI] [PubMed] [Google Scholar]
  10. Gottlieb T. A., Beaudry G., Rizzolo L., Colman A., Rindler M., Adesnik M., Sabatini D. D. Secretion of endogenous and exogenous proteins from polarized MDCK cell monolayers. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2100–2104. doi: 10.1073/pnas.83.7.2100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Green R. F., Meiss H. K., Rodriguez-Boulan E. Glycosylation does not determine segregation of viral envelope proteins in the plasma membrane of epithelial cells. J Cell Biol. 1981 May;89(2):230–239. doi: 10.1083/jcb.89.2.230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heifetz A., Keenan R. W., Elbein A. D. Mechanism of action of tunicamycin on the UDP-GlcNAc:dolichyl-phosphate Glc-NAc-1-phosphate transferase. Biochemistry. 1979 May 29;18(11):2186–2192. doi: 10.1021/bi00578a008. [DOI] [PubMed] [Google Scholar]
  14. Kondor-Koch C., Bravo R., Fuller S. D., Cutler D., Garoff H. Exocytotic pathways exist to both the apical and the basolateral cell surface of the polarized epithelial cell MDCK. Cell. 1985 Nov;43(1):297–306. doi: 10.1016/0092-8674(85)90035-2. [DOI] [PubMed] [Google Scholar]
  15. Ledford B. E., Davis D. F. Kinetics of serum protein secretion by cultured hepatoma cells. Evidence for multiple secretory pathways. J Biol Chem. 1983 Mar 10;258(5):3304–3308. [PubMed] [Google Scholar]
  16. Lodish H. F., Kong N., Snider M., Strous G. J. Hepatoma secretory proteins migrate from rough endoplasmic reticulum to Golgi at characteristic rates. Nature. 1983 Jul 7;304(5921):80–83. doi: 10.1038/304080a0. [DOI] [PubMed] [Google Scholar]
  17. Louvard D. Apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4132–4136. doi: 10.1073/pnas.77.7.4132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Matlin K. S., Simons K. Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation. Cell. 1983 Aug;34(1):233–243. doi: 10.1016/0092-8674(83)90154-x. [DOI] [PubMed] [Google Scholar]
  19. Matlin K. S., Simons K. Sorting of an apical plasma membrane glycoprotein occurs before it reaches the cell surface in cultured epithelial cells. J Cell Biol. 1984 Dec;99(6):2131–2139. doi: 10.1083/jcb.99.6.2131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Maxfield F. R. Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts. J Cell Biol. 1982 Nov;95(2 Pt 1):676–681. doi: 10.1083/jcb.95.2.676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Misek D. E., Bard E., Rodriguez-Boulan E. Biogenesis of epithelial cell polarity: intracellular sorting and vectorial exocytosis of an apical plasma membrane glycoprotein. Cell. 1984 Dec;39(3 Pt 2):537–546. doi: 10.1016/0092-8674(84)90460-4. [DOI] [PubMed] [Google Scholar]
  22. Misfeldt D. S., Hamamoto S. T., Pitelka D. R. Transepithelial transport in cell culture. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1212–1216. doi: 10.1073/pnas.73.4.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Mostov K. E., Deitcher D. L. Polymeric immunoglobulin receptor expressed in MDCK cells transcytoses IgA. Cell. 1986 Aug 15;46(4):613–621. doi: 10.1016/0092-8674(86)90887-1. [DOI] [PubMed] [Google Scholar]
  25. Olden K., Pratt R. M., Yamada K. M. Role of carbohydrates in protein secretion and turnover: effects of tunicamycin on the major cell surface glycoprotein of chick embryo fibroblasts. Cell. 1978 Mar;13(3):461–473. doi: 10.1016/0092-8674(78)90320-3. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  28. Pfeiffer S., Fuller S. D., Simons K. Intracellular sorting and basolateral appearance of the G protein of vesicular stomatitis virus in Madin-Darby canine kidney cells. J Cell Biol. 1985 Aug;101(2):470–476. doi: 10.1083/jcb.101.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Reggio H., Coudrier E., Louvard D. Surface and cytoplasmic domains in polarized epithelial cells. Prog Clin Biol Res. 1982;91:89–105. [PubMed] [Google Scholar]
  30. Rodriguez Boulan E., Sabatini D. D. Asymmetric budding of viruses in epithelial monlayers: a model system for study of epithelial polarity. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5071–5075. doi: 10.1073/pnas.75.10.5071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Roth M. G., Fitzpatrick J. P., Compans R. W. Polarity of influenza and vesicular stomatitis virus maturation in MDCK cells: lack of a requirement for glycosylation of viral glycoproteins. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6430–6434. doi: 10.1073/pnas.76.12.6430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Russell J. T. Delta pH, H+ diffusion potentials, and Mg2+ ATPase in neurosecretory vesicles isolated from bovine neurohypophyses. J Biol Chem. 1984 Aug 10;259(15):9496–9507. [PubMed] [Google Scholar]
  33. Saraste J., von Bonsdorff C. H., Hashimoto K., Käriäinen L., Keränen S. Semliki forest virus mutants with temperature-sensitive transport defect of envelope proteins. Virology. 1980 Jan 30;100(2):229–245. doi: 10.1016/0042-6822(80)90516-4. [DOI] [PubMed] [Google Scholar]
  34. Schwarz R. T., Rohrschneider J. M., Schmidt M. F. Suppression of glycoprotein formation of Semliki Forest, influenza, and avian sarcoma virus by tunicamycin. J Virol. 1976 Sep;19(3):782–791. doi: 10.1128/jvi.19.3.782-791.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Simons K., Fuller S. D. Cell surface polarity in epithelia. Annu Rev Cell Biol. 1985;1:243–288. doi: 10.1146/annurev.cb.01.110185.001331. [DOI] [PubMed] [Google Scholar]
  36. Steiner D. F., Docherty K., Carroll R. Golgi/granule processing of peptide hormone and neuropeptide precursors: a minireview. J Cell Biochem. 1984;24(2):121–130. doi: 10.1002/jcb.240240204. [DOI] [PubMed] [Google Scholar]
  37. Strous G. J., Du Maine A., Zijderhand-Bleekemolen J. E., Slot J. W., Schwartz A. L. Effect of lysosomotropic amines on the secretory pathway and on the recycling of the asialoglycoprotein receptor in human hepatoma cells. J Cell Biol. 1985 Aug;101(2):531–539. doi: 10.1083/jcb.101.2.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Strous G. J., Lodish H. F. Intracellular transport of secretory and membrane proteins in hepatoma cells infected by vesicular stomatitis virus. Cell. 1980 Dec;22(3):709–717. doi: 10.1016/0092-8674(80)90547-4. [DOI] [PubMed] [Google Scholar]
  39. Taub M., Chuman L., Saier M. H., Jr, Sato G. Growth of Madin-Darby canine kidney epithelial cell (MDCK) line in hormone-supplemented, serum-free medium. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3338–3342. doi: 10.1073/pnas.76.7.3338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Walter P., Jackson R. C., Marcus M. M., Lingappa V. R., Blobel G. Tryptic dissection and reconstitution of translocation activity for nascent presecretory proteins across microsomal membranes. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1795–1799. doi: 10.1073/pnas.76.4.1795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zilberstein A., Snider M. D., Porter M., Lodish H. F. Mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein. Cell. 1980 Sep;21(2):417–427. doi: 10.1016/0092-8674(80)90478-x. [DOI] [PubMed] [Google Scholar]

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