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. 1995 Apr 3;14(7):1340–1348. doi: 10.1002/j.1460-2075.1995.tb07120.x

Folding and oligomerization of influenza hemagglutinin in the ER and the intermediate compartment.

U Tatu 1, C Hammond 1, A Helenius 1
PMCID: PMC398219  PMID: 7729412

Abstract

Influenza hemagglutinin (HA) was used to analyze the stepwise folding and oligomeric assembly of glycoproteins in the early secretory pathway of living cells. In addition to mature trimers, six distinct maturation intermediates were identified. Of these, all the incompletely oxidized forms were located in the endoplasmic reticulum (ER) and associated with calnexin, a membrane-bound, lectin-like ER chaperone. Once fully oxidized, the HA dissociated from calnexin as a monomer, which rapidly became resistant to dithiothreitol (DTT) reduction. Part of these extensively folded molecules moved as monomers into the intermediate compartment between the ER and the Golgi complex. Assembly of homotrimers occurred without calnexin-involvement within the ER and in the intermediate compartment. When anchor-free HA molecules were analyzed, it was found that they reach the DTT-resistant monomeric conformation but fail to trimerize. Taken together, the results provide a definition and intracellular localization of several intermediates in the conformational maturation of HA, including the immediate precursor for trimer assembly.

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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 K. S., Cresswell P. A role for calnexin (IP90) in the assembly of class II MHC molecules. EMBO J. 1994 Feb 1;13(3):675–682. doi: 10.1002/j.1460-2075.1994.tb06306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anfinsen C. B., Scheraga H. A. Experimental and theoretical aspects of protein folding. Adv Protein Chem. 1975;29:205–300. doi: 10.1016/s0065-3233(08)60413-1. [DOI] [PubMed] [Google Scholar]
  3. Balch W. E., McCaffery J. M., Plutner H., Farquhar M. G. Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum. Cell. 1994 Mar 11;76(5):841–852. doi: 10.1016/0092-8674(94)90359-x. [DOI] [PubMed] [Google Scholar]
  4. Balch W. E., Wagner K. R., Keller D. S. Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system. J Cell Biol. 1987 Mar;104(3):749–760. doi: 10.1083/jcb.104.3.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bergeron J. J., Brenner M. B., Thomas D. Y., Williams D. B. Calnexin: a membrane-bound chaperone of the endoplasmic reticulum. Trends Biochem Sci. 1994 Mar;19(3):124–128. doi: 10.1016/0968-0004(94)90205-4. [DOI] [PubMed] [Google Scholar]
  6. Bergman L. W., Kuehl W. M. Formation of an intrachain disulfide bond on nascent immunoglobulin light chains. J Biol Chem. 1979 Sep 25;254(18):8869–8876. [PubMed] [Google Scholar]
  7. Bergman L. W., Kuehl W. M. Formation of intermolecular disulfide bonds on nascent immunoglobulin polypeptides. J Biol Chem. 1979 Jul 10;254(13):5690–5694. [PubMed] [Google Scholar]
  8. Boulay F., Doms R. W., Webster R. G., Helenius A. Posttranslational oligomerization and cooperative acid activation of mixed influenza hemagglutinin trimers. J Cell Biol. 1988 Mar;106(3):629–639. doi: 10.1083/jcb.106.3.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Braakman I., Helenius J., Helenius A. Role of ATP and disulphide bonds during protein folding in the endoplasmic reticulum. Nature. 1992 Mar 19;356(6366):260–262. doi: 10.1038/356260a0. [DOI] [PubMed] [Google Scholar]
  10. Braakman I., Hoover-Litty H., Wagner K. R., Helenius A. Folding of influenza hemagglutinin in the endoplasmic reticulum. J Cell Biol. 1991 Aug;114(3):401–411. doi: 10.1083/jcb.114.3.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ceriotti A., Colman A. Trimer formation determines the rate of influenza virus haemagglutinin transport in the early stages of secretion in Xenopus oocytes. J Cell Biol. 1990 Aug;111(2):409–420. doi: 10.1083/jcb.111.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Copeland C. S., Doms R. W., Bolzau E. M., Webster R. G., Helenius A. Assembly of influenza hemagglutinin trimers and its role in intracellular transport. J Cell Biol. 1986 Oct;103(4):1179–1191. doi: 10.1083/jcb.103.4.1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Copeland C. S., Zimmer K. P., Wagner K. R., Healey G. A., Mellman I., Helenius A. Folding, trimerization, and transport are sequential events in the biogenesis of influenza virus hemagglutinin. Cell. 1988 Apr 22;53(2):197–209. doi: 10.1016/0092-8674(88)90381-9. [DOI] [PubMed] [Google Scholar]
  14. Creighton T. E. Intermediates in the refolding of reduced pancreatic trypsin inhibitor. J Mol Biol. 1974 Aug 15;87(3):579–602. doi: 10.1016/0022-2836(74)90105-3. [DOI] [PubMed] [Google Scholar]
  15. Creighton T. E. Protein folding. Biochem J. 1990 Aug 15;270(1):1–16. doi: 10.1042/bj2700001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Daniels P. S., Jeffries S., Yates P., Schild G. C., Rogers G. N., Paulson J. C., Wharton S. A., Douglas A. R., Skehel J. J., Wiley D. C. The receptor-binding and membrane-fusion properties of influenza virus variants selected using anti-haemagglutinin monoclonal antibodies. EMBO J. 1987 May;6(5):1459–1465. doi: 10.1002/j.1460-2075.1987.tb02387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Daniels R. S., Douglas A. R., Skehel J. J., Wiley D. C. Analyses of the antigenicity of influenza haemagglutinin at the pH optimum for virus-mediated membrane fusion. J Gen Virol. 1983 Aug;64(Pt 8):1657–1662. doi: 10.1099/0022-1317-64-8-1657. [DOI] [PubMed] [Google Scholar]
  18. Daniels R. S., Douglas A. R., Skehel J. J., Wiley D. C., Naeve C. W., Webster R. G., Rogers G. N., Paulson J. C. Antigenic analyses of influenza virus haemagglutinins with different receptor-binding specificities. Virology. 1984 Oct 15;138(1):174–177. doi: 10.1016/0042-6822(84)90158-2. [DOI] [PubMed] [Google Scholar]
  19. David V., Hochstenbach F., Rajagopalan S., Brenner M. B. Interaction with newly synthesized and retained proteins in the endoplasmic reticulum suggests a chaperone function for human integral membrane protein IP90 (calnexin). J Biol Chem. 1993 May 5;268(13):9585–9592. [PubMed] [Google Scholar]
  20. Degen E., Cohen-Doyle M. F., Williams D. B. Efficient dissociation of the p88 chaperone from major histocompatibility complex class I molecules requires both beta 2-microglobulin and peptide. J Exp Med. 1992 Jun 1;175(6):1653–1661. doi: 10.1084/jem.175.6.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Doms R. W., Helenius A. Quaternary structure of influenza virus hemagglutinin after acid treatment. J Virol. 1986 Dec;60(3):833–839. doi: 10.1128/jvi.60.3.833-839.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Doxsey S. J., Sambrook J., Helenius A., White J. An efficient method for introducing macromolecules into living cells. J Cell Biol. 1985 Jul;101(1):19–27. doi: 10.1083/jcb.101.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Doyle C., Sambrook J., Gething M. J. Analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin. J Cell Biol. 1986 Oct;103(4):1193–1204. doi: 10.1083/jcb.103.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gething M. J., McCammon K., Sambrook J. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell. 1986 Sep 12;46(6):939–950. doi: 10.1016/0092-8674(86)90076-0. [DOI] [PubMed] [Google Scholar]
  25. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  26. Gliniak B. C., Kozak S. L., Jones R. T., Kabat D. Disulfide bonding controls the processing of retroviral envelope glycoproteins. J Biol Chem. 1991 Dec 5;266(34):22991–22997. [PubMed] [Google Scholar]
  27. Green W. N., Claudio T. Acetylcholine receptor assembly: subunit folding and oligomerization occur sequentially. Cell. 1993 Jul 16;74(1):57–69. doi: 10.1016/0092-8674(93)90294-z. [DOI] [PubMed] [Google Scholar]
  28. Hammond C., Braakman I., Helenius A. Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):913–917. doi: 10.1073/pnas.91.3.913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hammond C., Helenius A. Quality control in the secretory pathway: retention of a misfolded viral membrane glycoprotein involves cycling between the ER, intermediate compartment, and Golgi apparatus. J Cell Biol. 1994 Jul;126(1):41–52. doi: 10.1083/jcb.126.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hartl F. U., Hlodan R., Langer T. Molecular chaperones in protein folding: the art of avoiding sticky situations. Trends Biochem Sci. 1994 Jan;19(1):20–25. doi: 10.1016/0968-0004(94)90169-4. [DOI] [PubMed] [Google Scholar]
  31. Hauri H. P., Schweizer A. The endoplasmic reticulum-Golgi intermediate compartment. Curr Opin Cell Biol. 1992 Aug;4(4):600–608. doi: 10.1016/0955-0674(92)90078-Q. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Helenius A. How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum. Mol Biol Cell. 1994 Mar;5(3):253–265. doi: 10.1091/mbc.5.3.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Helenius A., Marquardt T., Braakman I. The endoplasmic reticulum as a protein-folding compartment. Trends Cell Biol. 1992 Aug;2(8):227–231. doi: 10.1016/0962-8924(92)90309-b. [DOI] [PubMed] [Google Scholar]
  34. Hochstenbach F., David V., Watkins S., Brenner M. B. Endoplasmic reticulum resident protein of 90 kilodaltons associates with the T- and B-cell antigen receptors and major histocompatibility complex antigens during their assembly. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4734–4738. doi: 10.1073/pnas.89.10.4734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Hurtley S. M., Bole D. G., Hoover-Litty H., Helenius A., Copeland C. S. Interactions of misfolded influenza virus hemagglutinin with binding protein (BiP). J Cell Biol. 1989 Jun;108(6):2117–2126. doi: 10.1083/jcb.108.6.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Hurtley S. M., Helenius A. Protein oligomerization in the endoplasmic reticulum. Annu Rev Cell Biol. 1989;5:277–307. doi: 10.1146/annurev.cb.05.110189.001425. [DOI] [PubMed] [Google Scholar]
  37. Jackson M. R., Cohen-Doyle M. F., Peterson P. A., Williams D. B. Regulation of MHC class I transport by the molecular chaperone, calnexin (p88, IP90). Science. 1994 Jan 21;263(5145):384–387. doi: 10.1126/science.8278813. [DOI] [PubMed] [Google Scholar]
  38. Jaenicke R. Folding and association of proteins. Prog Biophys Mol Biol. 1987;49(2-3):117–237. doi: 10.1016/0079-6107(87)90011-3. [DOI] [PubMed] [Google Scholar]
  39. Kaji E. H., Lodish H. F. In vitro unfolding of retinol-binding protein by dithiothreitol. Endoplasmic reticulum-associated factors. J Biol Chem. 1993 Oct 15;268(29):22195–22202. [PubMed] [Google Scholar]
  40. Krijnse-Locker J., Ericsson M., Rottier P. J., Griffiths G. Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step. J Cell Biol. 1994 Jan;124(1-2):55–70. doi: 10.1083/jcb.124.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Lenter M., Vestweber D. The integrin chains beta 1 and alpha 6 associate with the chaperone calnexin prior to integrin assembly. J Biol Chem. 1994 Apr 22;269(16):12263–12268. [PubMed] [Google Scholar]
  42. Lotti L. V., Torrisi M. R., Pascale M. C., Bonatti S. Immunocytochemical analysis of the transfer of vesicular stomatitis virus G glycoprotein from the intermediate compartment to the Golgi complex. J Cell Biol. 1992 Jul;118(1):43–50. doi: 10.1083/jcb.118.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Musil L. S., Goodenough D. A. Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell. 1993 Sep 24;74(6):1065–1077. doi: 10.1016/0092-8674(93)90728-9. [DOI] [PubMed] [Google Scholar]
  44. Saraste J., Kuismanen E. Pre- and post-Golgi vacuoles operate in the transport of Semliki Forest virus membrane glycoproteins to the cell surface. Cell. 1984 Sep;38(2):535–549. doi: 10.1016/0092-8674(84)90508-7. [DOI] [PubMed] [Google Scholar]
  45. Singh I., Doms R. W., Wagner K. R., Helenius A. Intracellular transport of soluble and membrane-bound glycoproteins: folding, assembly and secretion of anchor-free influenza hemagglutinin. EMBO J. 1990 Mar;9(3):631–639. doi: 10.1002/j.1460-2075.1990.tb08155.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sitia R., Meldolesi J. Endoplasmic reticulum: a dynamic patchwork of specialized subregions. Mol Biol Cell. 1992 Oct;3(10):1067–1072. doi: 10.1091/mbc.3.10.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sousa M. C., Ferrero-Garcia M. A., Parodi A. J. Recognition of the oligosaccharide and protein moieties of glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. Biochemistry. 1992 Jan 14;31(1):97–105. doi: 10.1021/bi00116a015. [DOI] [PubMed] [Google Scholar]
  48. Tooze J., Tooze S., Warren G. Replication of coronavirus MHV-A59 in sac- cells: determination of the first site of budding of progeny virions. Eur J Cell Biol. 1984 Mar;33(2):281–293. [PubMed] [Google Scholar]
  49. Tsou C. L. Folding of the nascent peptide chain into a biologically active protein. Biochemistry. 1988 Mar 22;27(6):1809–1812. doi: 10.1021/bi00406a001. [DOI] [PubMed] [Google Scholar]
  50. Wada I., Ou W. J., Liu M. C., Scheele G. Chaperone function of calnexin for the folding intermediate of gp80, the major secretory protein in MDCK cells. Regulation by redox state and ATP. J Biol Chem. 1994 Mar 11;269(10):7464–7472. [PubMed] [Google Scholar]
  51. Wagner D. D., Marder V. J. Biosynthesis of von Willebrand protein by human endothelial cells: processing steps and their intracellular localization. J Cell Biol. 1984 Dec;99(6):2123–2130. doi: 10.1083/jcb.99.6.2123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wilson I. A., Skehel J. J., Wiley D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981 Jan 29;289(5796):366–373. doi: 10.1038/289366a0. [DOI] [PubMed] [Google Scholar]
  53. Yewdell J. W., Yellen A., Bächi T. Monoclonal antibodies localize events in the folding, assembly, and intracellular transport of the influenza virus hemagglutinin glycoprotein. Cell. 1988 Mar 25;52(6):843–852. doi: 10.1016/0092-8674(88)90426-6. [DOI] [PubMed] [Google Scholar]

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