Skip to main content

Analyzing folding and degradation of metabolically labelled polypeptides by conventional and diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Abstract

Efficient protein folding and quality control are essential for unperturbed cell viability. Defects in these processes may lead to production of aberrant polypeptides that are either degraded leading to “loss-of-function” phenotypes, or deposited in or outside cells leading to “gain-of-toxic-function” phenotypes. Elucidation of molecular mechanisms regulating folding and quality control of newly synthesized polypeptides is therefore of greatest interest. Here we describe protocols for metabolic labelling of transfected/infected mammalian cells with [35S]-methionine and [35S]-cysteine, for immunoisolation from detergent extracts of the selected model proteins and for the investigation of the model polypeptide’s intracellular fate in response to chaperone-deletions or to cell exposure to folding or degradation inhibitors.

Abbreviations

BACE:

beta secretase

CHAPS:

3-(3-cholamydopropyl)dimethylamino)-1-propanesulphonate

Cnx:

calnexin

ER:

endoplasmic reticulum

ERAD:

ER-associated protein degradation

HA:

influenza virus hemagglutinin

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

References

  1. Gething MJ, Sambrook J. Protein folding in the cell. Nature 1992; 355:33–45.

    Article  PubMed  CAS  Google Scholar 

  2. Parodi AJ. Role of N-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation. Biochem J 2000; 348:1–13.

    Article  PubMed  CAS  Google Scholar 

  3. Ellgaard L, Ruddock LW. The human protein disulphide isomerase family: substrate interactions and functional properties. EMBO Rep 2005; 6:28–32.

    Article  PubMed  CAS  Google Scholar 

  4. Ellgaard L, Molinari M, Helenius A. Setting the standards: quality control in the secretory pathway. Science 1999; 286:1882–1888.

    Article  PubMed  CAS  Google Scholar 

  5. Hebert DN, Garman SC, Molinari M. The glycan code of the endoplasmic reticulum: asparagine-linked carbohydrates as protein maturation and quality-control tags. Trends Cell Biol 2005; 15:364–370.

    Article  PubMed  CAS  Google Scholar 

  6. Schroder M, Kaufman RJ. The Mammalian unfolded protein response. Annu Rev Biochem 2005; 74:739–789.

    Article  PubMed  Google Scholar 

  7. Meusser B, Hirsch C, Jarosch E, Sommer T. ERAD: the long road to destruction. Nat Cell Biol 2005; 7:766–772.

    Article  PubMed  CAS  Google Scholar 

  8. McCracken AA, Brodsky JL. Evolving questions and paradigm shifts in endoplasmic-reticulum-associated degradation (ERAD). Bioessays 2003; 25:868–877.

    Article  PubMed  CAS  Google Scholar 

  9. Ou WJ, Cameron PH, Thomas DY, Bergeron JJ. Association of folding intermediates of glycoproteins with calnexin during protein maturation. Nature 1993; 364:771–776.

    Article  PubMed  CAS  Google Scholar 

  10. Hammond C, Helenius A. Folding of VSV G protein: sequential interaction with BiP and calnexin. Science 1994; 266:456–458.

    Article  PubMed  CAS  Google Scholar 

  11. Nauseef WM, McCormick SJ, Clark RA. Calreticulin functions as a molecular chaperone in the biosynthesis of myeloperoxidase. J Biol Chem 1995; 270:4741–4747.

    Article  PubMed  CAS  Google Scholar 

  12. Molinari M, Helenius A. Glycoproteins form mixed disulphides with oxidoreductases during folding in living cells. Nature 1999; 402:90–93.

    Article  PubMed  CAS  Google Scholar 

  13. Braakman I, Hoover-Litty H, Wagner KR, Helenius A. Folding of influenza hemagglutinin in the endoplasmic reticulum. J Cell Biol 1991; 114:401–411.

    Article  PubMed  CAS  Google Scholar 

  14. Chen W, Helenius J, Braakman I, Helenius A. Cotranslational folding and calnexin binding during glycoprotein synthesis. Proc Natl Acad Sci USA 1995; 92:6229–6233.

    Article  PubMed  CAS  Google Scholar 

  15. Cabibbo A, Pagani M, Fabbri M, Rocchi M, Farmery MR, Bulleid NJ, Sitia R. ERO1-L, a human protein that favors disulfide bond formation in the endoplasmic reticulum. J Biol Chem 2000; 275:4827–4833.

    Article  PubMed  CAS  Google Scholar 

  16. Liu Y, Choudhury P, Cabral CM, Sifers RN. Oligosaccharide modification in the early secretory pathway directs the selection of a misfolded glycoprotein for degradation by the proteasome. J Biol Chem 1999; 274:5861–5867.

    Article  PubMed  CAS  Google Scholar 

  17. Molinari M, Eriksson KK, Calanca V, Galli C, Cresswell P, Michalak M, Helenius A. Contrasting Functions of Calreticulin and Calnexin in Glycoprotein Folding and ER Quality Control. Mol Cell 2004; 13:125–135.

    Article  PubMed  CAS  Google Scholar 

  18. Gallione CJ, Rose JK. A single amino acid substitution in a hydrophobic domain causes temperature-sensitive cell-surface transport of a mutant viral glycoprotein. J Virol 1985; 54:374–382.

    PubMed  CAS  Google Scholar 

  19. Helenius A, Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta 1975; 415:29–79.

    PubMed  CAS  Google Scholar 

  20. Sommer A, Traut RR. Diagonal polyacrylamidedodecyl sulfate gel electrophoresis for the identification of ribosomal proteins crosslinked with methyl-4- mercaptobutyrimidate. Proc Natl Acad Sci USA 1974; 71:3946–3950.

    Article  PubMed  CAS  Google Scholar 

  21. Molinari M, Helenius A. Analyzing cotranslational protein folding and disulfide formation by diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methods Enzymol 2002; 348:35–42.

    Article  PubMed  CAS  Google Scholar 

  22. Molinari M, Calanca V, Galli C, Lucca P, Paganetti P. Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle. Science 2003; 299:1397–1400.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Maurizio Molinari.

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Soldà, T., Olivari, S. & Molinari, M. Analyzing folding and degradation of metabolically labelled polypeptides by conventional and diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biol. Proced. Online 7, 136–143 (2005). https://doi.org/10.1251/bpo111

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1251/bpo111

Indexing terms