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Polyubiquitin substrates allosterically activate their own degradation by the 26S proteasome

Nature Structural & Molecular Biology volume 16, pages 219–225 (2009)Cite this article

Abstract

The 26S proteasome degrades polyubiquitylated (polyUb) proteins by an ATP-dependent mechanism. Here we show that binding of model polyUb substrates to the 19S regulator of mammalian and yeast 26S proteasomes enhances the peptidase activities of the 20S proteasome about two-fold in a process requiring ATP hydrolysis. Monoubiquitylated proteins or tetraubiquitin alone exert no effect. However, 26S proteasomes from the yeast α3ΔN open-gate mutant and the rpt2YA and rpt5YA mutants with impaired gating can still be activated (approximately 1.3-fold to 1.8-fold) by polyUb-protein binding. Thus, binding of polyUb substrates to the 19S regulator stabilizes gate opening of the 20S proteasome and induces conformational changes of the 20S proteasome that facilitate channeling of substrates and their access to active sites. In consequence, polyUb substrates will allosterically stimulate their own degradation.

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Figure 1: MUC1-derived model substrates for 26S proteasomes.
Figure 2: PolyUb proteins stimulate the activity of the 26S proteasome.
Figure 3: Ub5-MUC4 binding stimulates all peptidase activities.
Figure 4: Proteasome activation correlates with Ub5-MUC4 binding affinity.
Figure 5: PolyUb-mediated stimulation requires ATP hydrolysis.
Figure 6: Conformational changes of the proteasome caused by Ub5-MUC4.

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Acknowledgements

We would like to thank D. Finley (Harvard Medical School) for the yeast strains rpt2YA and rpt5YA, J. Dohmen (University of Cologne) for the rpn10 mutant strain and A. Lehmann for excellent technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB421 and SFB 740 to P.-M.K.).

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  1. Dawadschargal Bech-Otschir and Annett Helfrich: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut für Biochemie, Charité—Universitätsmedizin Berlin, Monbijoustrasse 2, 10117, Berlin, Germany

    Dawadschargal Bech-Otschir, Annett Helfrich, Cordula Enenkel, Gesa Consiglieri, Michael Seeger, Hermann-Georg Holzhütter, Burkhardt Dahlmann & Peter-Michael Kloetzel

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  1. Dawadschargal Bech-Otschir
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Contributions

D.B.-O. and A.H. performed all biochemical experiments; C.E. purified yeast proteasomes; G.C. generated the MUC1 peptides; M.S. and H.-G.H. supervised the biochemical and enzyme kinetic experiments; B.D. purified the mammalian proteasomes; D.B.-O., B.D. and P.-M.K. prepared the manuscript; P.-M.K. designed the project.

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Correspondence to Peter-Michael Kloetzel.

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Bech-Otschir, D., Helfrich, A., Enenkel, C. et al. Polyubiquitin substrates allosterically activate their own degradation by the 26S proteasome. Nat Struct Mol Biol 16, 219–225 (2009). https://doi.org/10.1038/nsmb.1547

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