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:

Reconstitution of the 26S proteasome reveals functional asymmetries in its AAA+ unfoldase

Nature Structural & Molecular Biology volume 20, pages 1164–1172 (2013)Cite this article

Subjects

Abstract

The 26S proteasome is the major eukaryotic ATP-dependent protease, yet the detailed mechanisms used by the proteasomal heterohexameric AAA+ unfoldase to drive substrate degradation remain poorly understood. To perform systematic mutational analyses of individual ATPase subunits, we heterologously expressed the unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro. Our studies demonstrate that the six ATPases have distinct roles in degradation, corresponding to their positions in the spiral staircases adopted by the AAA+ domains in the absence or presence of substrate. ATP hydrolysis in subunits at the top of the staircases is critical for substrate engagement and translocation. Whereas the unfoldase relies on this vertical asymmetry for substrate processing, interaction with the peptidase exhibits three-fold symmetry with contributions from alternate subunits. These diverse functional asymmetries highlight how the 26S proteasome deviates from simpler, homomeric AAA+ proteases.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Expression of yeast base subcomplexes in E. coli and reconstitution of the 26S proteasome.
Figure 2: Proteasomes reconstituted with endogenous or heterologously expressed base exhibit similar degradation activities for a polyubiquitinated substrate.
Figure 3: Degradation activities for base variants with a Walker-B E→Q mutation in individual Rpt subunits correlate with the subunit's position in the spiral staircase arrangement of the base.
Figure 4: Degradation activities for base variants containing single-subunit pore-loop mutations.
Figure 5: Rpt C-terminal tails contribute to gate opening differentially and in a nucleotide-independent manner.
Figure 6: Affinities of core-peptidase binding for base variants lacking individual C-terminal tails.
Figure 7: Model for the contribution of individual ATPase subunits to substrate processing and core particle interaction.

Similar content being viewed by others

References

  1. Finley, D. Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513 (2009).

    Article  CAS  Google Scholar 

  2. Hochstrasser, M. Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 30, 405–439 (1996).

    Article  CAS  Google Scholar 

  3. Groll, M. et al. A gated channel into the proteasome core particle. Nat. Struct. Biol. 7, 1062–1067 (2000).

    Article  CAS  Google Scholar 

  4. Liu, C.W. et al. ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome. Mol. Cell 24, 39–50 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Glickman, M.H. et al. A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell 94, 615–623 (1998).

    Article  CAS  Google Scholar 

  7. Smith, D.M. et al. Docking of the proteasomal ATPases' carboxyl termini in the 20S proteasome's α ring opens the gate for substrate entry. Mol. Cell 27, 731–744 (2007).

    Article  CAS  Google Scholar 

  8. Tomko, R.J. Jr. & Hochstrasser, M. Order of the proteasomal ATPases and eukaryotic proteasome assembly. Cell Biochem. Biophys. 60, 13–20 (2011).

    Article  CAS  Google Scholar 

  9. Lander, G.C. et al. Complete subunit architecture of the proteasome regulatory particle. Nature 482, 186–191 (2012).

    Article  CAS  Google Scholar 

  10. Beck, F. et al. Near-atomic resolution structural model of the yeast 26S proteasome. Proc. Natl. Acad. Sci. USA 109, 14870–14875 (2012).

    Article  CAS  Google Scholar 

  11. Hamazaki, J. et al. A novel proteasome interacting protein recruits the deubiquitinating enzyme UCH37 to 26S proteasomes. EMBO J. 25, 4524–4536 (2006).

    Article  CAS  Google Scholar 

  12. Yao, T. & Cohen, R.E. A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419, 403–407 (2002).

    Article  CAS  Google Scholar 

  13. Verma, R. et al. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298, 611–615 (2002).

    Article  CAS  Google Scholar 

  14. Sakata, E. et al. Localization of the proteasomal ubiquitin receptors Rpn10 and Rpn13 by electron cryomicroscopy. Proc. Natl. Acad. Sci. USA 109, 1479–1484 (2012).

    Article  CAS  Google Scholar 

  15. Martin, A., Baker, T.A. & Sauer, R.T. Rebuilt AAA+ motors reveal operating principles for ATP-fuelled machines. Nature 437, 1115–1120 (2005).

    Article  CAS  Google Scholar 

  16. Rabl, J. et al. Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases. Mol. Cell 30, 360–368 (2008).

    Article  CAS  Google Scholar 

  17. Kim, Y.C. & DeMartino, G.N. C termini of proteasomal ATPases play nonequivalent roles in cellular assembly of mammalian 26 S proteasome. J. Biol. Chem. 286, 26652–26666 (2011).

    Article  CAS  Google Scholar 

  18. Gillette, T.G., Kumar, B., Thompson, D., Slaughter, C.A. & DeMartino, G.N. Differential roles of the COOH termini of AAA subunits of PA700 (19 S regulator) in asymmetric assembly and activation of the 26 S proteasome. J. Biol. Chem. 283, 31813–31822 (2008).

    Article  CAS  Google Scholar 

  19. Sledź, P. et al. Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation. Proc. Natl. Acad. Sci. USA 110, 7264–7269 (2013).

    Article  Google Scholar 

  20. Matyskiela, M.E., Lander, G.C. & Martin, A. Conformational switching of the 26S proteasome enables substrate degradation. Nat. Struct. Mol. Biol. 20, 781–788 (2013).

    Article  CAS  Google Scholar 

  21. Rubin, D.M., Glickman, M.H., Larsen, C.N., Dhruvakumar, S. & Finley, D. Active site mutants in the six regulatory particle ATPases reveal multiple roles for ATP in the proteasome. EMBO J. 17, 4909–4919 (1998).

    Article  CAS  Google Scholar 

  22. Erales, J., Hoyt, M.A., Troll, F. & Coffino, P. Functional asymmetries of proteasome translocase pore. J. Biol. Chem. 287, 18535–18543 (2012).

    Article  CAS  Google Scholar 

  23. Kim, Y.C., Li, X., Thompson, D. & Demartino, G.N. ATP binding by proteasomal ATPases regulates cellular assembly and substrate-induced functions of the 26S proteasome. J. Biol. Chem. 288, 3334–3345 (2013).

    Article  CAS  Google Scholar 

  24. Lee, S.H., Moon, J.H., Yoon, S.K. & Yoon, J.B. Stable incorporation of ATPase subunits into 19 S regulatory particle of human proteasome requires nucleotide binding and C-terminal tails. J. Biol. Chem. 287, 9269–9279 (2012).

    Article  CAS  Google Scholar 

  25. Köhler, A. et al. The axial channel of the proteasome core particle is gated by the Rpt2 ATPase and controls both substrate entry and product release. Mol. Cell 7, 1143–1152 (2001).

    Article  Google Scholar 

  26. Park, S. et al. Hexameric assembly of the proteasomal ATPases is templated through their C termini. Nature 459, 866–870 (2009).

    Article  CAS  Google Scholar 

  27. Kumar, B., Kim, Y.C. & DeMartino, G.N. The C terminus of Rpt3, an ATPase subunit of PA700 (19 S) regulatory complex, is essential for 26 S proteasome assembly but not for activation. J. Biol. Chem. 285, 39523–39535 (2010).

    Article  CAS  Google Scholar 

  28. Thompson, D., Hakala, K. & DeMartino, G.N. Subcomplexes of PA700, the 19 S regulator of the 26 S proteasome, reveal relative roles of AAA subunits in 26 S proteasome assembly and activation and ATPase activity. J. Biol. Chem. 284, 24891–24903 (2009).

    Article  CAS  Google Scholar 

  29. Funakoshi, M., Tomko, R.J. Jr., Kobayashi, H. & Hochstrasser, M. Multiple assembly chaperones govern biogenesis of the proteasome regulatory particle base. Cell 137, 887–899 (2009).

    Article  CAS  Google Scholar 

  30. Roelofs, J. et al. Chaperone-mediated pathway of proteasome regulatory particle assembly. Nature 459, 861–865 (2009).

    Article  CAS  Google Scholar 

  31. Saeki, Y., Toh, E.A., Kudo, T., Kawamura, H. & Tanaka, K. Multiple proteasome-interacting proteins assist the assembly of the yeast 19S regulatory particle. Cell 137, 900–913 (2009).

    Article  CAS  Google Scholar 

  32. Kaneko, T. et al. Assembly pathway of the mammalian proteasome base subcomplex is mediated by multiple specific chaperones. Cell 137, 914–925 (2009).

    Article  CAS  Google Scholar 

  33. Park, S. et al. Reconfiguration of the proteasome during chaperone-mediated assembly. Nature 497, 512–516 (2013).

    Article  CAS  Google Scholar 

  34. Smith, D.M. et al. ATP binding to PAN or the 26S ATPases causes association with the 20S proteasome, gate opening, and translocation of unfolded proteins. Mol. Cell 20, 687–698 (2005).

    Article  CAS  Google Scholar 

  35. Verma, R., Oania, R., Graumann, J. & Deshaies, R.J. Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system. Cell 118, 99–110 (2004).

    Article  CAS  Google Scholar 

  36. Hersch, G.L., Burton, R.E., Bolon, D.N., Baker, T.A. & Sauer, R.T. Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: allosteric control of a protein machine. Cell 121, 1017–1027 (2005).

    Article  CAS  Google Scholar 

  37. Weibezahn, J., Bukau, B. & Mogk, A. Unscrambling an egg: protein disaggregation by AAA+ proteins. Microb. Cell Fact. 3, 1 (2004).

    Article  Google Scholar 

  38. Gómez, E.B., Catlett, M.G. & Forsburg, S.L. Different phenotypes in vivo are associated with ATPase motif mutations in Schizosaccharomyces pombe minichromosome maintenance proteins. Genetics 160, 1305–1318 (2002).

    PubMed  PubMed Central  Google Scholar 

  39. Wang, J. et al. Crystal structures of the HslVU peptidase–ATPase complex reveal an ATP-dependent proteolysis mechanism. Structure 9, 177–184 (2001).

    Article  CAS  Google Scholar 

  40. Martin, A., Baker, T.A. & Sauer, R.T. Pore loops of the AAA+ ClpX machine grip substrates to drive translocation and unfolding. Nat. Struct. Mol. Biol. 15, 1147–1151 (2008).

    Article  CAS  Google Scholar 

  41. Martin, A., Baker, T.A. & Sauer, R.T. Diverse pore loops of the AAA+ ClpX machine mediate unassisted and adaptor-dependent recognition of ssrA-tagged substrates. Mol. Cell 29, 441–450 (2008).

    Article  CAS  Google Scholar 

  42. Yamada-Inagawa, T., Okuno, T., Karata, K., Yamanaka, K. & Ogura, T. Conserved pore residues in the AAA protease FtsH are important for proteolysis and its coupling to ATP hydrolysis. J. Biol. Chem. 278, 50182–50187 (2003).

    Article  CAS  Google Scholar 

  43. Aubin-Tam, M.E., Olivares, A.O., Sauer, R.T., Baker, T.A. & Lang, M.J. Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine. Cell 145, 257–267 (2011).

    Article  CAS  Google Scholar 

  44. Maillard, R.A. et al. ClpX(P) generates mechanical force to unfold and translocate its protein substrates. Cell 145, 459–469 (2011).

    Article  CAS  Google Scholar 

  45. Park, E. et al. Role of the GYVG pore motif of HslU ATPase in protein unfolding and translocation for degradation by HslV peptidase. J. Biol. Chem. 280, 22892–22898 (2005).

    Article  CAS  Google Scholar 

  46. Hinnerwisch, J., Fenton, W.A., Furtak, K.J., Farr, G.W. & Horwich, A.L. Loops in the central channel of ClpA chaperone mediate protein binding, unfolding, and translocation. Cell 121, 1029–1041 (2005).

    Article  CAS  Google Scholar 

  47. Glynn, S.E., Martin, A., Nager, A.R., Baker, T.A. & Sauer, R.T. Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine. Cell 139, 744–756 (2009).

    Article  CAS  Google Scholar 

  48. Glynn, S.E., Nager, A.R., Baker, T.A. & Sauer, R.T. Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine. Nat. Struct. Mol. Biol. 19, 616–622 (2012).

    Article  CAS  Google Scholar 

  49. Martin, A., Baker, T.A. & Sauer, R.T. Distinct static and dynamic interactions control ATPase-peptidase communication in a AAA+ protease. Mol. Cell 27, 41–52 (2007).

    Article  CAS  Google Scholar 

  50. Barthelme, D. & Sauer, R.T. Bipartite determinants mediate an evolutionarily conserved interaction between Cdc48 and the 20S peptidase. Proc. Natl. Acad. Sci. USA 110, 3327–3332 (2013).

    Article  CAS  Google Scholar 

  51. Tian, G. et al. An asymmetric interface between the regulatory and core particles of the proteasome. Nat. Struct. Mol. Biol. 18, 1259–1267 (2011).

    Article  CAS  Google Scholar 

  52. Smith, D.M., Fraga, H., Reis, C., Kafri, G. & Goldberg, A.L. ATP binds to proteasomal ATPases in pairs with distinct functional effects, implying an ordered reaction cycle. Cell 144, 526–538 (2011).

    Article  CAS  Google Scholar 

  53. Li, X. & Demartino, G.N. Variably modulated gating of the 26S proteasome by ATP and polyubiquitin. Biochem. J. 421, 397–404 (2009).

    Article  CAS  Google Scholar 

  54. Seong, I.S. et al. The C-terminal tails of HslU ATPase act as a molecular switch for activation of HslV peptidase. J. Biol. Chem. 277, 25976–25982 (2002).

    Article  CAS  Google Scholar 

  55. Kim, D.Y. & Kim, K.K. Crystal structure of ClpX molecular chaperone from Helicobacter pylori. J. Biol. Chem. 278, 50664–50670 (2003).

    Article  CAS  Google Scholar 

  56. Guo, F., Maurizi, M.R., Esser, L. & Xia, D. Crystal structure of ClpA, an Hsp100 chaperone and regulator of ClpAP protease. J. Biol. Chem. 277, 46743–46752 (2002).

    Article  CAS  Google Scholar 

  57. Singleton, M.R., Sawaya, M.R., Ellenberger, T. & Wigley, D.B. Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell 101, 589–600 (2000).

    Article  CAS  Google Scholar 

  58. Thomsen, N.D. & Berger, J.M. Running in reverse: the structural basis for translocation polarity in hexameric helicases. Cell 139, 523–534 (2009).

    Article  CAS  Google Scholar 

  59. Enemark, E.J. & Joshua-Tor, L. Mechanism of DNA translocation in a replicative hexameric helicase. Nature 442, 270–275 (2006).

    Article  CAS  Google Scholar 

  60. Costa, A. et al. The structural basis for MCM2–7 helicase activation by GINS and Cdc45. Nat. Struct. Mol. Biol. 18, 471–477 (2011).

    Article  CAS  Google Scholar 

  61. Leggett, D.S., Glickman, M.H. & Finley, D. Purification of proteasomes, proteasome subcomplexes, and proteasome-associated proteins from budding yeast. Methods Mol. Biol. 301, 57–70 (2005).

    CAS  PubMed  Google Scholar 

  62. Saeki, Y., Isono, E. & Toh-E, A. Preparation of ubiquitinated substrates by the PY motif-insertion method for monitoring 26S proteasome activity. Methods Enzymol. 399, 215–227 (2005).

    Article  CAS  Google Scholar 

  63. Verma, R. et al. Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol. Biol. Cell 11, 3425–3439 (2000).

    Article  CAS  Google Scholar 

  64. Glickman, M.H., Rubin, D.M., Fried, V.A. & Finley, D. The regulatory particle of the Saccharomyces cerevisiae proteasome. Mol. Cell Biol. 18, 3149–3162 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the members of the Martin lab for helpful discussions. E.J.W. acknowledges support from a National Science Foundation Graduate Research Fellowship. This research was funded in part by the Searle Scholars Program (A.M.), US National Institutes of Health grant R01-GM094497-01A1 (A.M.) and start-up funds from the Molecular and Cell Biology Department at University of California, Berkeley (A.M.).

Author information

Authors and Affiliations

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

    Robyn Beckwith, Eric Estrin, Evan J Worden & Andreas Martin

  2. California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, California, USA

    Andreas Martin

Authors
  1. Robyn Beckwith
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eric Estrin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Evan J Worden
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andreas Martin
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.B., E.E., E.J.W. and A.M. designed experiments, expressed and purified proteasome constructs, performed biochemical experiments and analyzed data. R.B. and A.M. prepared the manuscript.

Corresponding author

Correspondence to Andreas Martin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Note (PDF 4742 kb)

Supplementary Table 1

Source data for Table 1 (XLSX 58 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Cite this article

Beckwith, R., Estrin, E., Worden, E. et al. Reconstitution of the 26S proteasome reveals functional asymmetries in its AAA+ unfoldase. Nat Struct Mol Biol 20, 1164–1172 (2013). https://doi.org/10.1038/nsmb.2659

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2659

Search

Advanced 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