Pulse-Chase Labeling Techniques for the Analysis of Protein Maturation and Degradation

  • Annemieke Jansens
  • Ineke Braakman
Part of the Methods in Molecular Biology™ book series (MIMB, volume 232)


Pulse-chase experiments have proved to be a powerful tool to study protein folding, maturation, and degradation in mammalian cells. When short pulses are applied, a fraction of the total protein pool can be followed from synthesis to degradation in its natural environment.


Basic Protocol Iodoacetic Acid Stop Buffer Pulse Chase Nonreducing Sample Buffer 
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  1. 1.
    Solari, R. and Kraehenbuhl, J. P. (1984) Biosynthesis of the IgA antibody receptor: a model for the transepithelial sorting of a membrane glycoprotein. Cell 36, 61–71.PubMedCrossRefGoogle Scholar
  2. 2.
    Kim, P. S. and Arvan, P. (1991) Folding and assembly of newly synthesized thyroglobulin occurs in a pre-Golgi compartment. J. Biol. Chem. 266, 12,412–12,418.PubMedGoogle Scholar
  3. 3.
    Braakman, I., Hoover-Litty, H., Wagner, K. R., and Helenius, A. (1991) Folding of influenza hemagglutinin in the endoplasmic reticulum. J. Cell Biol. 114, 401–411.PubMedCrossRefGoogle Scholar
  4. 4.
    Doms, R. W., Ruusala, A., Machamer, C., Helenius, J., Helenius, A., and Rose, J. K. (1988) Differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus G protein. J. Cell Biol. 107, 89–99.PubMedCrossRefGoogle Scholar
  5. 5.
    Earl, P. L., Moss, B., and Doms, R. W. (1991) Folding, interaction with GRP78-BiP, assembly, and transport of the human immunodeficiency virus type 1 envelope protein. J. Virol. 65, 2047–2055.PubMedGoogle Scholar
  6. 6.
    Fuerst, T. R., Niles, E. G., Studier, F. W., and Moss, B. (1986) Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 83, 8122–8126.PubMedCrossRefGoogle Scholar
  7. 7.
    Bastaki, M., Braiterman, L. T., Johns, D. C., Chen, Y. H., and Hubbard, A. L. (2002) Absence of direct delivery for single transmembrane apical proteins or their “Secretory” forms in polarized hepatic cells. Mol. Biol. Cell 13, 225–237.PubMedCrossRefGoogle Scholar
  8. 8.
    Braakman, I., Helenius, J., and Helenius, A. (1992) Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum. EMBO J. 11, 1717–1722.PubMedGoogle Scholar
  9. 9.
    Hollecker, M. (1989) Counting integral numbers of residues by chemical modification, in Protein Structure, A Practical Approach (Creighton, T. E., ed.), IRL Press, Oxford University Press, Oxford, pp. 145–153.Google Scholar
  10. 10.
    Hurtley, S. M., Bole, D. G., Hoover-Litty, H., Helenius, A., and Copeland, C. S. (1989) Interactions of misfolded influenza virus hemagglutinin with binding protein (BiP). J. Cell Biol. 108, 2117–2126.PubMedCrossRefGoogle Scholar
  11. 11.
    Tatu, U., Braakman, I., and Helenius, A. (1993) Membrane glycoprotein folding, oligomerisation and intracellular transport: effects of dithiothreitol in living cells. EMBO J. 12, 2151–2157.PubMedGoogle Scholar
  12. 12.
    Copeland, C. S., Doms, R. W., Bolzau, E. M., Webster, R. G., and Helenius, A. (1986) Assembly of influenza hemagglutinin trimers and its role in intracellular transport. J. Cell Biol. 103, 1179–1191.PubMedCrossRefGoogle Scholar
  13. 13.
    Doms, R. W., Keller, D. S., Helenius, A., and Balch, W. E. (1987) Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers. J. Cell Biol. 105, 1957–1969.PubMedCrossRefGoogle Scholar
  14. 14.
    Wong, S. S. and Wong, L. J. (1992) Chemical crosslinking and the stabilisation of proteins and enzymes. Enzyme Microb. Technol. 14(11), 866–874.PubMedCrossRefGoogle Scholar
  15. 15.
    Tatu, U. and Helenius, A. (1997) Interactions between newly synthesized glycoproteins, calnexin and a network of resident chaperones in the endoplasmic reticulum. J. Cell Biol. 136, 555–565.PubMedCrossRefGoogle Scholar
  16. 16.
    Gelman, M. S., Kannegaard, E. S., and Kopito, R. R. (2002) A principal role for the proteasome in ER-associated degradation of misfolded intracellular Cystic Fibrosis Transmembrane Conductance Regulator. J. Biol. Chem. 277, 11,709–11,714.PubMedCrossRefGoogle Scholar
  17. 17.
    Thevenod, F. and Friedmann, J. M. (1999) Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na+/K(+)-ATPase through proteasomal and endo-/lysosomal proteolytic pathways. FASEB J. 13, 1751–1761.PubMedGoogle Scholar
  18. 18.
    Tarentino, A. L., Trimble, R. B., and Maley, F. (1978) Endo-beta-N-Acetylglucosaminidase from Streptomyces plicatus. Methods Enzymol. 50, 574–580.PubMedCrossRefGoogle Scholar
  19. 19.
    de Silva, A. M., Balch, W. E., and Helenius, A. (1990) Quality control in the endoplasmic reticulum: folding and misfolding of vesicular stomatitis virus G protein in cells and in vitro. J. Cell Biol. 111, 857–866.PubMedCrossRefGoogle Scholar
  20. 20.
    Harter, C. and Mellman, I. (1992) Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane. J. Cell Biol. 117, 311–325.PubMedCrossRefGoogle Scholar
  21. 21.
    Hammond, C., Braakman, I., and Helenius, A. (1994) Role of N-linked oligosac-charide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. Proc. Natl. Acad. Sci. USA 91, 913–917.PubMedCrossRefGoogle Scholar
  22. 22.
    Podbilewicz, B. and Mellman, I. (1990) ATP and cytosol requirements for transferrin recycling in intact and disrupted MDCK cells. EMBO J. 9, 3477–3487.PubMedGoogle Scholar
  23. 23.
    Guest, P. C., Bailyes, E. M., and Hutton, J. C. (1997) Endoplasmic reticulum Ca2+ is important for the proteolytic processing and intracellular transport of proinsulin in the pancreatic beta-cell. Biochem. J. 323(Pt 2), 445–450.PubMedGoogle Scholar
  24. 24.
    Lodish, H. F. and Kong, N. (1990) Perturbation of cellular calcium blocks exit of secretory proteins from the rough endoplasmic reticulum. J. Biol. Chem. 265, 10,893–10,899.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2003

Authors and Affiliations

  • Annemieke Jansens
    • 1
  • Ineke Braakman
    • 2
  1. 1.Department of Bio-Organic Chemistry, Bijvoet Center for Biomolecular ResearchUtrecht UniversityUtrechtThe Netherlands
  2. 2.Bijvoet Center for Biomolecular Research, Department of Bio-Organic ChemistryUtrecht UniversityUtrechtThe Netherlands

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