Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A ubiquitin-based tagging system for controlled modulation of protein stability

Abstract

Many biotechnology applications depend on the expression of exogenous proteins in a predictable and controllable manner. A key determinant of the intracellular concentration of a given protein is its stability or “half-life.” We have developed a versatile and reliable system for producing short half-life forms of proteins expressed in mammalian cells. The system consists of a series of destabilization domains composed of varying numbers of a mutant form of ubiquitin (UbG76V) that cannot be cleaved by ubiquitin hydrolases. We show that increasing the number of UbG76V moieties within the destabilization domain results in a graded decrease in protein half-life and steady-state levels when fused to heterologous reporter proteins as well as cellular proteins. Cells expressing a destabilized β-lactamase reporter act as a robust, high-throughput screening (HTS)-compatible assay for proteasome activity within cells.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: β-lactamase has a short half-life in vitro when fused to polyUb destabilization domains.
Figure 2: PolyUb destabilization domains confer short half-life to GFP and procaspase-3 in vitro.
Figure 3: Kinetics of degradation and steady-state levels of Ub–β-lactamase fusion proteins in intact cells.
Figure 4: Cells expressing polyUb–β-lactamase reporter serve as a robust cell-based assay for proteasome activity.

Similar content being viewed by others

References

  1. Lee, D.H. & Goldberg, A.L. Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol. 8, 397–403 (1998).

    Article  CAS  PubMed  Google Scholar 

  2. Hershko, A. & Ciechanover, A. The ubiquitin system . Ann. Rev. Biochem. 67, 425– 479 (1998).

    Article  CAS  PubMed  Google Scholar 

  3. Thrower, J.S., Hoffman, L., Rechsteiner, M. & Pickart, C.M. Recognition of the polyubiquitin proteolytic signal. EMBO J. 19, 94–102 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Varshavsky, A. The N-end rule: functions, mysteries, uses. Proc. Natl. Acad. Sci. USA 93, 12142–12149 ( 1996).

    Article  Google Scholar 

  5. Koepp, D.M., Harper, J.W. & Elledge, S.J. How the cyclin became a cyclin: regulated proteolysis in the cell cycle. Cell 97, 431– 434 (1999).

    Article  CAS  PubMed  Google Scholar 

  6. Laney, J.D. & Hochstrasser, M. Substrate targeting in the ubiquitin as a degradation signal. Cell 97, 427–430 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Johnson, E.S., Bartel, B., Seufert, W. & Varshavsky, A. Ubiquitin as a degradation signal. EMBO J. 11, 497 –505 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Johnson, E.S., Ma, P.C., Ota, I.M. & Varshavsky, A. A proteolytic pathway that recognizes ubiquitin as a degradation signal. J. Biol. Chem. 270, 17442–17456 ( 1995).

    Article  CAS  PubMed  Google Scholar 

  9. Corish, P. & Tyler-Smith, C. Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng. 12, 1035–1040 ( 1999).

    Article  CAS  PubMed  Google Scholar 

  10. Li, X. et al. Generation of destabilized green fluorescent protein as a transcription reporter . J. Biol. Chem. 273, 34970– 34975 (1998).

    Article  CAS  PubMed  Google Scholar 

  11. Thompson, J.F., Hayes, L.S. & Lloyd, D.B. Modulation of firefly luciferase stability and impact on studies of gene regulation. Gene 103, 171–177 (1991).

    Article  CAS  PubMed  Google Scholar 

  12. Worley, C.K., Ling, R. & Callis, J. Engineering in vivo instability of firefly luciferase and Escherichia coli beta-glucuronidase in higher plants using recognition elements from the ubiquitin pathway. Plant Mol. Biol. 37, 337–347 (1998).

    Article  CAS  PubMed  Google Scholar 

  13. Butt, T.R., Khan, M.I., Marsh, J., Ecker, D.J. & Crooke, S.T. Ubiquitin–metallothionein fusion protein expression in yeast. A genetic approach for analysis of ubiquitin functions. J. Biol. Chem. 263, 16364– 16371 (1988).

    CAS  PubMed  Google Scholar 

  14. Bachmair, A. & Varshavsky, A. The degradation signal in a short-lived protein. Cell 56, 1019–1032 (1989).

    Article  CAS  PubMed  Google Scholar 

  15. Zlokarnik, G. et al. Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. Science 279, 84–88 (1998).

    Article  CAS  PubMed  Google Scholar 

  16. Whitney, M. et al. A genome-wide functional assay of signal transduction in living mammalian cells. Nat. Biotechnol. 16, 1329– 1333 (1998).

    Article  CAS  PubMed  Google Scholar 

  17. Bachmair, A., Finley, D. & Varshavsky, A. In vivo half-life of a protein is a function of its amino-terminal residue. Science 234, 179 –186 (1986).

    Article  CAS  PubMed  Google Scholar 

  18. Gonda, D.K. et al. Universality and structure of the N-end rule. J. Biol. Chem. 264, 16700–16712 ( 1989).

    CAS  PubMed  Google Scholar 

  19. Dantuma, N.P., Lindsten, K., Glas, R., Jellne, M. & Masucci, M.G. Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells. Nat. Biotechnol. 18, 538– 543 (2000).

    Article  CAS  PubMed  Google Scholar 

  20. Deichsel, H. et al. Green fluorescent proteins with short half-lives as reporters in Dictyostelium discoideum. Dev. Genes Evol. 209, 63–68 (1999).

    Article  CAS  PubMed  Google Scholar 

  21. Ciechanover, A. The ubiquitin–proteasome pathway: on protein death and cell life. EMBO J. 17, 1751–1760 ( 1998).

    Article  Google Scholar 

  22. Li, X. et al. Characterization of NFkappaB activation by detection of green fluorescent protein-tagged IkappaB degradation in living cells. J. Biol. Chem. 274, 21244–21250 ( 1999).

    Article  CAS  PubMed  Google Scholar 

  23. Tsien, R.Y. The green fluorescent protein. Ann. Rev. Biochem. 67 , 509–544 (1998).

    Article  CAS  PubMed  Google Scholar 

  24. Kunkel, T.A., Bebenek, K. & McClary, J. Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol. 204, 125– 139 (1991).

    Article  CAS  PubMed  Google Scholar 

  25. Hershko, A. & Heller, H. Occurrence of a polyubiquitin structure in ubiquitin–protein conjugates. Biochem. Biophys. Res. Commun. 128, 1079–1086 (1985).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful for helpful discussions with many scientists at Aurora Biosciences, particularly Andrew Cubitt and Gregor Zlokarnik. Richard Yuan and Jay Jones provided excellent technical support, and Tom Knapp and Eric Hare performed the FACS analyses. We thank S. Mobashery for purified TEM-1 β-lactamase and Steve Xanthoudakis and Don Nicholson (Merck Frosst Centre for Therapeutic Research) for the procaspase-3 cDNA.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jeffrey H. Stack.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stack, J., Whitney, M., Rodems, S. et al. A ubiquitin-based tagging system for controlled modulation of protein stability. Nat Biotechnol 18, 1298–1302 (2000). https://doi.org/10.1038/82422

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/82422

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing