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Complete subunit architecture of the proteasome regulatory particle

Nature volume 482, pages 186–191 (2012)Cite this article

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Abstract

The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes polyubiquitinated substrates. Here we used electron microscopy and a new heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle from yeast. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes and the protein unfolding machinery at subnanometre resolution, outlining the substrate’s path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes.

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Figure 1: The lid subcomplex within the holoenzyme assembly.
Figure 2: Three-dimensional reconstructions of the recombinant lid subcomplex and the yeast 26S proteasome.
Figure 3: Localization of Rpn1 and Rpn2, and ubiquitin-interacting subunits.
Figure 4: Conformational rearrangements of the lid subcomplex upon integration into the holoenzyme.
Figure 5: Structural features of the base ATPase subunits.
Figure 6: Model for the recognition, deubiquitination and engagement of a polyubiquitinated substrate by the 26S proteasome.

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Accession codes

Data deposits

The cryoelectron microscopy density map for the 26S proteasome can be found at the Electron Microscopy Data Bank under accession number EMD-1992. The negative stain reconstructions of the recombinantly expressed and yeast-purified lid have been assigned accession numbers EMD-1993 and EMD-1994, respectively.

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Acknowledgements

We thank the members of the Martin and Nogales labs for helpful discussions, and G. Cardone for help with local resolution calculations. G.C.L. acknowledges support from Damon Runyon Cancer Research Foundation, M.E.M. acknowledges support by the American Cancer Society grant 121453-PF-11-178-01-TBE, C.B. acknowledges support from the NSF Graduate Research Fellowship. This research was funded in part by the Searle Scholars Program (A.M.), start-up funds from the UC Berkeley MCB Department (A.M.), the NIH grant R01-GM094497-01A1 (A.M.), the Lawrence Berkeley National Laboratory (G.C.L.), and the Howard Hughes Medical Institute (E.N.). Some of the work presented here was conducted at the National Resource for Automated Molecular Microscopy, which is supported by the NIH through the NCRR P41 program (RR017573).

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Author notes

  1. Gabriel C. Lander, Eric Estrin and Mary E. Matyskiela: These authors contributed equally to this work.

Authors and Affiliations

  1. Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, 94720, California, USA

    Gabriel C. Lander & Eva Nogales

  2. Department of Molecular and Cell Biology, University of California, Berkeley, 94720, California, USA

    Eric Estrin, Mary E. Matyskiela, Charlene Bashore & Andreas Martin

  3. Howard Hughes Medical Institute, University of California, Berkeley, 94720, California, USA

    Eva Nogales

  4. QB3 Institute, University of California, Berkeley, 94720, California, USA

    Eva Nogales & Andreas Martin

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Contributions

E.E., M.E.M. and C.B. designed, expressed and purified proteasome constructs, and performed biochemical experiments. G.C.L. performed the electron microscopy, processing and segmentation analysis. All authors contributed to experimental design, data analysis and manuscript preparation.

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Correspondence to Andreas Martin.

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Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-12 with legends and Supplementary Table 1. (PDF 9249 kb)

Supplementary Movie 1

The movie shows the 3D reconstruction of the yeast 26S proteasome and the spatial arrangement of all subunits within its regulatory particle, including the ubiquitin receptors and the deubiquitinating enzyme. Docking of crystal structures from homologous proteins reveals a spiral stair case orientation of the six ATPase subunits within the base unfoldase and a horseshoe-shaped arrangement of PCI domains in the lid subcomplex. (MOV 27606 kb)

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Lander, G., Estrin, E., Matyskiela, M. et al. Complete subunit architecture of the proteasome regulatory particle. Nature 482, 186–191 (2012). https://doi.org/10.1038/nature10774

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