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Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells

Nature Biotechnology volume 18, pages 538–543 (2000)Cite this article

Abstract

The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics because of its critical involvement in cell cycle progression and antigen presentation. However, dissection of the pathway and development of modulators are hampered by the complexity of the system and the lack of easily detectable authentic substrates. We have developed a convenient reporter system by producing N-end rule and ubiquitin fusion degradation (UFD)-targeted green fluorescent proteins that allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Accumulation of these reporters serves as an early predictor of G2/M arrest and apoptosis in cells treated with proteasome inhibitors. Comparison of reporter accumulation and cleavage of fluorogenic substrates demonstrates that the rate-limiting chymotrypsin-like activity of the proteasome can be substantially curtailed without significant effect on ubiquitin-dependent proteolysis. These reporters provide a new powerful tool for elucidation of the ubiquitin/proteasome pathway and for high throughput screening of compounds that selectively modify proteolysis in vivo.

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Figure 1: Targeting of GFP for proteasomal degradation.
Figure 2: Ub-X-GFP expression monitored by fluorescence intensity.
Figure 3: Accumulation of GFP reporter upon treatment with inhibitor.
Figure 4: Correlation between proteasomal degradation and cell growth.
Figure 5: Effect of different inhibitors on degradation of reporter.

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Acknowledgements

We thank Hidde Ploegh and Benedikt Kessler for providing the peptide-vinyl sulfones, Bo Öberg for the ritonavir, Stefan Jentsch for the Ub-Pro-βGal plasmid, and Anatoly Sharipo and Teresa Frisan for helpful discussions. This work was supported by grants awarded by the Swedish Cancer Society, the Swedish Foundation of Strategy Research, and the Hedlund Foundation, Stockholm, Sweden. N.P.D. is supported by a postdoctoral fellowship awarded by the European Commission Training and Mobility Program on “The central role of the ubiquitin-proteasome system in regulatory processes involved in immunological, inflammatory, endocrinological and malignant disorders,” contract no. ERBFMRXCT960026.

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  1. Microbiology and Tumor Biology Center, Karolinska Institutet , S-171 77, Stockholm, Sweden

    Nico P. Dantuma, Kristina Lindsten, Rickard Glas, Marianne Jellne & Maria G. Masucci

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  1. Nico P. Dantuma
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Correspondence to Maria G. Masucci.

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Dantuma, N., Lindsten, K., Glas, R. et al. Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells. Nat Biotechnol 18, 538–543 (2000). https://doi.org/10.1038/75406

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