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. 1985 Dec 1;101(6):2036–2046. doi: 10.1083/jcb.101.6.2036

Newly synthesized G protein of vesicular stomatitis virus is not transported to the Golgi complex in mitotic cells

PMCID: PMC2114025  PMID: 2999158

Abstract

Newly synthesized G protein of vesicular stomatitis virus is not transported to the surface of cultured mammalian cells during mitosis (Warren et al., 1983, J. Cell Biol. 97:1623-1628). To determine where intracellular transport is inhibited, we have examined the post- translational modifications of G protein, which are indicators of specific compartments on the transport pathway. G protein in mitotic cells had only endo H-sensitive oligosaccharides containing seven or eight mannose residues, but no terminal glucose, and was not fatty acylated. These modifications were indicative of processing only by enzymes of the endoplasmic reticulum (ER). Quantitative immunocytochemistry was used as an independent method to confirm that transport of G protein out of the ER was inhibited. The density of G protein in the ER cisternae was 2.5 times greater than in infected G1 cells treated similarly. Incubation of infected mitotic cells with cycloheximide, which inhibits protein synthesis without affecting transport, did not result in a decrease in the density of G protein in the ER cisternae, demonstrating that G protein cannot be chased out of the ER. These results suggest that intracellular transport stops at or before the first vesicle-mediated step on the pathway.

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Selected References

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  1. Atkinson P. H., Lee J. T. Co-translational excision of alpha-glucose and alpha-mannose in nascent vesicular stomatitis virus G protein. J Cell Biol. 1984 Jun;98(6):2245–2249. doi: 10.1083/jcb.98.6.2245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berger M., Schmidt M. F. Cell-free fatty acid acylation of Semliki Forest viral polypeptides with microsomal membranes from eukaryotic cells. J Biol Chem. 1984 Jun 10;259(11):7245–7252. [PubMed] [Google Scholar]
  3. Bergmann J. E., Singer S. J. Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (G) of vesicular stomatitis virus in infected Chinese hamster ovary cells. J Cell Biol. 1983 Dec;97(6):1777–1787. doi: 10.1083/jcb.97.6.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bergmann J. E., Tokuyasu K. T., Singer S. J. Passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the Golgi apparatus en route to the plasma membrane. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1746–1750. doi: 10.1073/pnas.78.3.1746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berlin R. D., Oliver J. M. Surface functions during mitosis. II. Quantitation of pinocytosis and kinetic characterization of the mitotic cycle with a new fluorescence technique. J Cell Biol. 1980 Jun;85(3):660–671. doi: 10.1083/jcb.85.3.660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berlin R. D., Oliver J. M., Walter R. J. Surface functions during Mitosis I: phagocytosis, pinocytosis and mobility of surface-bound Con A. Cell. 1978 Oct;15(2):327–341. doi: 10.1016/0092-8674(78)90002-8. [DOI] [PubMed] [Google Scholar]
  7. Bischoff J., Kornfeld R. Evidence for an alpha-mannosidase in endoplasmic reticulum of rat liver. J Biol Chem. 1983 Jul 10;258(13):7907–7910. [PubMed] [Google Scholar]
  8. 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]
  9. Brandtzaeg P. Conjugates of immunoglobulin G with different fluorochromes. I. Characterization by anionic-exchange chromatography. Scand J Immunol. 1973;2(3):273–290. doi: 10.1111/j.1365-3083.1973.tb02037.x. [DOI] [PubMed] [Google Scholar]
  10. Burke B., Matlin K., Bause E., Legler G., Peyrieras N., Ploegh H. Inhibition of N-linked oligosaccharide trimming does not interfere with surface expression of certain integral membrane proteins. EMBO J. 1984 Mar;3(3):551–556. doi: 10.1002/j.1460-2075.1984.tb01845.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. FAWCETT D. W. SURFACE SPECIALIZATIONS OF ABSORBING CELLS. J Histochem Cytochem. 1965 Feb;13:75–91. doi: 10.1177/13.2.75. [DOI] [PubMed] [Google Scholar]
  12. Godelaine D., Spiro M. J., Spiro R. G. Processing of the carbohydrate units of thyroglobulin. J Biol Chem. 1981 Oct 10;256(19):10161–10168. [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Griffiths G., Quinn P., Warren G. Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus. J Cell Biol. 1983 Mar;96(3):835–850. doi: 10.1083/jcb.96.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grinna L. S., Robbins P. W. Glycoprotein biosynthesis. Rat liver microsomal glucosidases which process oligosaccharides. J Biol Chem. 1979 Sep 25;254(18):8814–8818. [PubMed] [Google Scholar]
  17. Helenius A., Kartenbeck J., Simons K., Fries E. On the entry of Semliki forest virus into BHK-21 cells. J Cell Biol. 1980 Feb;84(2):404–420. doi: 10.1083/jcb.84.2.404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hercz A., Harpaz N. Characterization of the oligosaccharides of liver Z variant alpha 1-antitrypsin. Can J Biochem. 1980 Aug;58(8):644–648. doi: 10.1139/o80-089. [DOI] [PubMed] [Google Scholar]
  19. Hesketh T. R., Beaven M. A., Rogers J., Burke B., Warren G. B. Stimulated release of histamine by a rat mast cell line is inhibited during mitosis. J Cell Biol. 1984 Jun;98(6):2250–2254. doi: 10.1083/jcb.98.6.2250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hubbard S. C., Ivatt R. J. Synthesis and processing of asparagine-linked oligosaccharides. Annu Rev Biochem. 1981;50:555–583. doi: 10.1146/annurev.bi.50.070181.003011. [DOI] [PubMed] [Google Scholar]
  21. Jamieson J. D., Palade G. E. Intracellular transport of secretory proteins in the pancreatic exocrine cell. IV. Metabolic requirements. J Cell Biol. 1968 Dec;39(3):589–603. doi: 10.1083/jcb.39.3.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Klevecz R. R. Automated cell cycle analysis. Methods Cell Biol. 1975;10:157–172. doi: 10.1016/s0091-679x(08)60735-9. [DOI] [PubMed] [Google Scholar]
  23. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  24. Matlin K. S., Reggio H., Helenius A., Simons K. Pathway of vesicular stomatitis virus entry leading to infection. J Mol Biol. 1982 Apr 15;156(3):609–631. doi: 10.1016/0022-2836(82)90269-8. [DOI] [PubMed] [Google Scholar]
  25. Mattila K., Luukkonen A., Renkonen O. Protein-bound oligosaccharides of Semliki Forest virus. Biochim Biophys Acta. 1976 Feb 6;419(3):435–444. doi: 10.1016/0005-2736(76)90257-1. [DOI] [PubMed] [Google Scholar]
  26. Quinn P., Griffiths G., Warren G. Dissection of the Golgi complex. II. Density separation of specific Golgi functions in virally infected cells treated with monensin. J Cell Biol. 1983 Mar;96(3):851–856. doi: 10.1083/jcb.96.3.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Quintart J., Bartholeyns J., Baudhuin P. Characterization of subcellular components in synchronized hepatoma cells as a function of the cell cycle. Biochem J. 1979 Oct 15;184(1):133–141. doi: 10.1042/bj1840133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Robertson M. A., Etchison J. R., Robertson J. S., Summers D. F., Stanley P. Specific changes in the oligosaccharide moieties of VSV grown in different lectin-resistnat CHO cells. Cell. 1978 Mar;13(3):515–526. doi: 10.1016/0092-8674(78)90325-2. [DOI] [PubMed] [Google Scholar]
  29. Rose J. K., Adams G. A., Gallione C. J. The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2050–2054. doi: 10.1073/pnas.81.7.2050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rosner M. R., Hubbard S. C., Ivatt R. J., Robbins P. W. N-asparagine-linked oligosaccharides: biosynthesis of the lipid-linked oligosaccharides. Methods Enzymol. 1982;83:399–408. doi: 10.1016/0076-6879(82)83037-1. [DOI] [PubMed] [Google Scholar]
  31. Roth J., Berger E. G. Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae. J Cell Biol. 1982 Apr;93(1):223–229. doi: 10.1083/jcb.93.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sager P. R., Brown P. A., Berlin R. D. Analysis of transferrin recycling in mitotic and interphase HeLa cells by quantitative fluorescence microscopy. Cell. 1984 Dec;39(2 Pt 1):275–282. doi: 10.1016/0092-8674(84)90005-9. [DOI] [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. Schlesinger S., Gottlieb C., Feil P., Gelb N., Kornfeld S. Growth of enveloped RNA viruses in a line of chinese hamster ovary cells with deficient N-acetylglucosaminyltransferase activity. J Virol. 1975 Jan;17(1):239–246. doi: 10.1128/jvi.17.1.239-246.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schmidt M. F., Schlesinger M. J. Relation of fatty acid attachment to the translation and maturation of vesicular stomatitis and Sindbis virus membrane glycoproteins. J Biol Chem. 1980 Apr 25;255(8):3334–3339. [PubMed] [Google Scholar]
  36. Tabas I., Kornfeld S. The synthesis of complex-type oligosaccharides. III. Identification of an alpha-D-mannosidase activity involved in a late stage of processing of complex-type oligosaccharides. J Biol Chem. 1978 Nov 10;253(21):7779–7786. [PubMed] [Google Scholar]
  37. Wang K., Feramisco J. R., Ash J. F. Fluorescent localization of contractile proteins in tissue culture cells. Methods Enzymol. 1982;85(Pt B):514–562. doi: 10.1016/0076-6879(82)85050-7. [DOI] [PubMed] [Google Scholar]
  38. Warren G., Davoust J., Cockcroft A. Recycling of transferrin receptors in A431 cells is inhibited during mitosis. EMBO J. 1984 Oct;3(10):2217–2225. doi: 10.1002/j.1460-2075.1984.tb02119.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Warren G., Featherstone C., Griffiths G., Burke B. Newly synthesized G protein of vesicular stomatitis virus is not transported to the cell surface during mitosis. J Cell Biol. 1983 Nov;97(5 Pt 1):1623–1628. doi: 10.1083/jcb.97.5.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wen D., Schlesinger M. J. Fatty acid-acylated proteins in secretory mutants of Saccharomyces cerevisiae. Mol Cell Biol. 1984 Apr;4(4):688–694. doi: 10.1128/mcb.4.4.688. [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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