Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Jul 1;111(1):31–44. doi: 10.1083/jcb.111.1.31

Intracellular processing and transport of NH2-terminally truncated forms of a hemagglutinin-neuraminidase type II glycoprotein

PMCID: PMC2116159  PMID: 2164031

Abstract

Six amino-terminal deletion mutants of the NH2-terminally anchored (type II orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza virus type 3 were expressed in tissue culture by recombinant SV-40 viruses. The mutations consisted of progressive deletions of the cytoplasmic domain and, in some cases, of the hydrophobic signal/anchor. Three activities were dissociated for the signal/anchor: membrane insertion, translocation, and anchoring/transport. HN protein lacking the entire cytoplasmic tail was inserted efficiently into the membrane of the endoplasmic reticulum but was translocated inefficiently into the lumen. However, the small amounts that were successfully translocated appeared to be processed subsequently in a manner indistinguishable from that of parental HN. Thus, the cytoplasmic domain was not required for maturation of this type II glycoprotein. Progressive deletions into the membrane anchor restored efficient translocation, indicating that the NH2-terminal 44 amino acids were fully dispensable for membrane insertion and translocation and that a 10-amino acid hydrophobic signal sequence was sufficient for both activities. These latter HN molecules appeared to be folded authentically as assayed by hemagglutination activity, reactivity with a conformation-specific antiserum, correct formation of intramolecular disulfide bonds, and homooligomerization. However, most (85-90%) of these molecules accumulated in the ER. This showed that folding and oligomerization into a biologically active form, which presumably represents a virion spike, occurs essentially to completion within that compartment but is not sufficient for efficient transport through the exocytotic pathway. Protein transport also appeared to depend on the structure of the membrane anchor. These latter mutants were not stably integrated in the membrane, and the small proportion (10-15%) that was processed through the exocytotic pathway was secreted. The maturation steps and some of the effects of mutations described here for a type II glycoprotein resemble previous observations for prototypic type I glycoproteins and are indicative of close similarities in these processes for proteins of both membrane orientations.

Full Text

The Full Text of this article is available as a PDF (2.5 MB).

Selected References

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

  1. Blobel G. Intracellular protein topogenesis. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496–1500. doi: 10.1073/pnas.77.3.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bole D. G., Hendershot L. M., Kearney J. F. Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J Cell Biol. 1986 May;102(5):1558–1566. doi: 10.1083/jcb.102.5.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bos T. J., Davis A. R., Nayak D. P. NH2-terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2327–2331. doi: 10.1073/pnas.81.8.2327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown D. J., Hogue B. G., Nayak D. P. Redundancy of signal and anchor functions in the NH2-terminal uncharged region of influenza virus neuraminidase, a class II membrane glycoprotein. J Virol. 1988 Oct;62(10):3824–3831. doi: 10.1128/jvi.62.10.3824-3831.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Cutler D. F., Melancon P., Garoff H. Mutants of the membrane-binding region of Semliki Forest virus E2 protein. II. Topology and membrane binding. J Cell Biol. 1986 Mar;102(3):902–910. doi: 10.1083/jcb.102.3.902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davis N. G., Model P. An artificial anchor domain: hydrophobicity suffices to stop transfer. Cell. 1985 Jun;41(2):607–614. doi: 10.1016/s0092-8674(85)80033-7. [DOI] [PubMed] [Google Scholar]
  8. Doms R. W., Ruusala A., Machamer C., Helenius J., Helenius A., Rose J. K. Differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus G protein. J Cell Biol. 1988 Jul;107(1):89–99. doi: 10.1083/jcb.107.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elango N., Coligan J. E., Jambou R. C., Venkatesan S. Human parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein: nucleotide sequence of mRNA and limited amino acid sequence of the purified protein. J Virol. 1986 Feb;57(2):481–489. doi: 10.1128/jvi.57.2.481-489.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fujiki Y., Hubbard A. L., Fowler S., Lazarow P. B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. doi: 10.1083/jcb.93.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Hendricks D. A., Baradaran K., McIntosh K., Patterson J. L. Appearance of a soluble form of the G protein of respiratory syncytial virus in fluids of infected cells. J Gen Virol. 1987 Jun;68(Pt 6):1705–1714. doi: 10.1099/0022-1317-68-6-1705. [DOI] [PubMed] [Google Scholar]
  13. Hunt L. A., Davidson S. K., Golemboski D. B. Unusual heterogeneity in the glycosylation of the G protein of the hazelhurst strain of vesicular stomatitis virus. Arch Biochem Biophys. 1983 Oct 1;226(1):347–356. doi: 10.1016/0003-9861(83)90301-6. [DOI] [PubMed] [Google Scholar]
  14. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  15. Kreis T. E., Lodish H. F. Oligomerization is essential for transport of vesicular stomatitis viral glycoprotein to the cell surface. Cell. 1986 Sep 12;46(6):929–937. doi: 10.1016/0092-8674(86)90075-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laski F. A., Belagaje R., RajBhandary U. L., Sharp P. A. An amber suppressor tRNA gene derived by site-specific mutagenesis: cloning and function in mammalian cells. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5813–5817. doi: 10.1073/pnas.79.19.5813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lipp J., Dobberstein B. The membrane-spanning segment of invariant chain (I gamma) contains a potentially cleavable signal sequence. Cell. 1986 Sep 26;46(7):1103–1112. doi: 10.1016/0092-8674(86)90710-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Louvard D., Reggio H., Warren G. Antibodies to the Golgi complex and the rough endoplasmic reticulum. J Cell Biol. 1982 Jan;92(1):92–107. doi: 10.1083/jcb.92.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Markoff L., Lin B. C., Sveda M. M., Lai C. J. Glycosylation and surface expression of the influenza virus neuraminidase requires the N-terminal hydrophobic region. Mol Cell Biol. 1984 Jan;4(1):8–16. doi: 10.1128/mcb.4.1.8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Meyer D. I., Krause E., Dobberstein B. Secretory protein translocation across membranes-the role of the "docking protein'. Nature. 1982 Jun 24;297(5868):647–650. doi: 10.1038/297647a0. [DOI] [PubMed] [Google Scholar]
  21. Morrison T. G., Ward L. J. Intracellular processing of the vesicular stomatitis virus glycoprotein and the Newcastle disease virus hemagglutinin-neuraminidase glycoprotein. Virus Res. 1984;1(3):225–239. doi: 10.1016/0168-1702(84)90041-8. [DOI] [PubMed] [Google Scholar]
  22. Mottet G., Portner A., Roux L. Drastic immunoreactivity changes between the immature and mature forms of the Sendai virus HN and F0 glycoproteins. J Virol. 1986 Jul;59(1):132–141. doi: 10.1128/jvi.59.1.132-141.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Munro S., Pelham H. R. An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell. 1986 Jul 18;46(2):291–300. doi: 10.1016/0092-8674(86)90746-4. [DOI] [PubMed] [Google Scholar]
  24. Olmsted R. A., Murphy B. R., Lawrence L. A., Elango N., Moss B., Collins P. L. Processing, surface expression, and immunogenicity of carboxy-terminally truncated mutants of G protein of human respiratory syncytial virus. J Virol. 1989 Jan;63(1):411–420. doi: 10.1128/jvi.63.1.411-420.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Paterson R. G., Lamb R. A. Ability of the hydrophobic fusion-related external domain of a paramyxovirus F protein to act as a membrane anchor. Cell. 1987 Feb 13;48(3):441–452. doi: 10.1016/0092-8674(87)90195-4. [DOI] [PubMed] [Google Scholar]
  26. Randall L. L., Hardy S. J. Unity in function in the absence of consensus in sequence: role of leader peptides in export. Science. 1989 Mar 3;243(4895):1156–1159. doi: 10.1126/science.2646712. [DOI] [PubMed] [Google Scholar]
  27. Rapoport T. A. Protein translocation across and integration into membranes. CRC Crit Rev Biochem. 1986;20(1):73–137. doi: 10.3109/10409238609115901. [DOI] [PubMed] [Google Scholar]
  28. Rose J. K., Doms R. W. Regulation of protein export from the endoplasmic reticulum. Annu Rev Cell Biol. 1988;4:257–288. doi: 10.1146/annurev.cb.04.110188.001353. [DOI] [PubMed] [Google Scholar]
  29. Rosner M. R., Grinna L. S., Robbins P. W. Differences in glycosylation patterns of closely related murine leukemia viruses. Proc Natl Acad Sci U S A. 1980 Jan;77(1):67–71. doi: 10.1073/pnas.77.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sabatini D. D., Kreibich G., Morimoto T., Adesnik M. Mechanisms for the incorporation of proteins in membranes and organelles. J Cell Biol. 1982 Jan;92(1):1–22. doi: 10.1083/jcb.92.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Spriggs M. K., Murphy B. R., Prince G. A., Olmsted R. A., Collins P. L. Expression of the F and HN glycoproteins of human parainfluenza virus type 3 by recombinant vaccinia viruses: contributions of the individual proteins to host immunity. J Virol. 1987 Nov;61(11):3416–3423. doi: 10.1128/jvi.61.11.3416-3423.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sveda M. M., Markoff L. J., Lai C. J. Influenza virus hemagglutinin containing an altered hydrophobic carboxy terminus accumulates intracellularly. J Virol. 1984 Jan;49(1):223–228. doi: 10.1128/jvi.49.1.223-228.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Szczesna-Skorupa E., Browne N., Mead D., Kemper B. Positive charges at the NH2 terminus convert the membrane-anchor signal peptide of cytochrome P-450 to a secretory signal peptide. Proc Natl Acad Sci U S A. 1988 Feb;85(3):738–742. doi: 10.1073/pnas.85.3.738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tarentino A. L., Maley F. Purification and properties of an endo-beta-N-acetylglucosaminidase from Streptomyces griseus. J Biol Chem. 1974 Feb 10;249(3):811–817. [PubMed] [Google Scholar]
  35. Thompson S. D., Laver W. G., Murti K. G., Portner A. Isolation of a biologically active soluble form of the hemagglutinin-neuraminidase protein of Sendai virus. J Virol. 1988 Dec;62(12):4653–4660. doi: 10.1128/jvi.62.12.4653-4660.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vidal S., Mottet G., Kolakofsky D., Roux L. Addition of high-mannose sugars must precede disulfide bond formation for proper folding of Sendai virus glycoproteins. J Virol. 1989 Feb;63(2):892–900. doi: 10.1128/jvi.63.2.892-900.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Walter P., Blobel G. Translocation of proteins across the endoplasmic reticulum III. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes. J Cell Biol. 1981 Nov;91(2 Pt 1):557–561. doi: 10.1083/jcb.91.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Waxham M. N., Merz D. C., Wolinsky J. S. Intracellular maturation of mumps virus hemagglutinin-neuraminidase glycoprotein: conformational changes detected with monoclonal antibodies. J Virol. 1986 Aug;59(2):392–400. doi: 10.1128/jvi.59.2.392-400.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wieland F. T., Gleason M. L., Serafini T. A., Rothman J. E. The rate of bulk flow from the endoplasmic reticulum to the cell surface. Cell. 1987 Jul 17;50(2):289–300. doi: 10.1016/0092-8674(87)90224-8. [DOI] [PubMed] [Google Scholar]
  40. Wilson C., Gilmore R., Morrison T. Translation and membrane insertion of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus. Mol Cell Biol. 1987 Apr;7(4):1386–1392. doi: 10.1128/mcb.7.4.1386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zerial M., Huylebroeck D., Garoff H. Foreign transmembrane peptides replacing the internal signal sequence of transferrin receptor allow its translocation and membrane binding. Cell. 1987 Jan 16;48(1):147–155. doi: 10.1016/0092-8674(87)90365-5. [DOI] [PubMed] [Google Scholar]
  42. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]
  43. von Heijne G. Towards a comparative anatomy of N-terminal topogenic protein sequences. J Mol Biol. 1986 May 5;189(1):239–242. doi: 10.1016/0022-2836(86)90394-3. [DOI] [PubMed] [Google Scholar]

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

RESOURCES