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Stability of the proteasome can be regulated allosterically through engagement of its proteolytic active sites

Nature Structural & Molecular Biology volume 14, pages 1180–1188 (2007)Cite this article

Abstract

The 26S proteasome holoenzyme is formed by the association of a 20S core particle (CP) with a 19S regulatory particle (RP). The CP-RP interaction is labile and subject to regulation in vivo, but the factors controlling this association are poorly understood. Here we describe an in vitro proteasome reconstitution assay and a high-resolution, two-dimensional gel electrophoresis system. Using these techniques, we find that a yeast CP–RP complex can contain a substoichiometric amount of tightly bound, essentially non-exchangeable ATP. However, this nucleotide is dispensable for gating of the CP channel, provided that the CP–RP complex is preserved by the Ecm29 protein. Unexpectedly, proteasome inhibitors are potent in stabilizing proteasomes against the dissociation of CP–RP. These data indicate that active sites of the CP communicate with bound RP, despite their spatial separation. We propose that ongoing protein degradation may suppress proteasome disassembly, thereby enhancing the processivity of proteolysis.

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Figure 1: Analysis of the CP-RP interaction by purification and reconstitution.
Figure 2: Nucleotide dependence of the CP-RP interaction.
Figure 3: CP-RP dissociation in the absence of nucleotide.
Figure 4: Ecm29 can substitute for nucleotide.
Figure 5: Proteasome inhibitors stabilize the CP-RP interaction.
Figure 6: Stabilization depends on the reactive groups of inhibitors.
Figure 7: Dose dependence of proteasome stabilization and inhibition.

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Acknowledgements

We thank M. Hochstrasser (Yale University) for yeast strains; Millennium Pharmaceuticals for PS-341; M. Maurizi, D.S. Newburg, and members of the Finley laboratory, especially J. Hanna and N. Sofaer, for reading the manuscript and for insightful comments; and the ICCB screening facility at Harvard Medical School for use of their facility. This research was supported by grants from the US National Institutes of Health grants to D.F. (GM43601) and R.W.K. (GM66492).

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  1. Maurits F Kleijnen and Jeroen Roelofs: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, 02115, Massachusetts, USA.

    Maurits F Kleijnen, Jeroen Roelofs, Soyeon Park, Nathaniel A Hathaway, Randall W King & Daniel Finley

  2. Department of Biology, Technion–Israel Institute of Technology, Haifa, 32000, Israel

    Michael Glickman

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Kleijnen, M., Roelofs, J., Park, S. et al. Stability of the proteasome can be regulated allosterically through engagement of its proteolytic active sites. Nat Struct Mol Biol 14, 1180–1188 (2007). https://doi.org/10.1038/nsmb1335

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