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Ubp6 deubiquitinase controls conformational dynamics and substrate degradation of the 26S proteasome

Nature Structural & Molecular Biology volume 22, pages 712–719 (2015)Cite this article

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Abstract

Substrates are targeted for proteasomal degradation through the attachment of ubiquitin chains that need to be removed by proteasomal deubiquitinases before substrate processing. In budding yeast, the deubiquitinase Ubp6 trims ubiquitin chains and affects substrate processing by the proteasome, but the underlying mechanisms and the location of Ubp6 within the holoenzyme have been elusive. Here we show that Ubp6 activity strongly responds to interactions with the base ATPase and the conformational state of the proteasome. Electron microscopy analyses reveal that ubiquitin-bound Ubp6 contacts the N ring and AAA+ ring of the ATPase hexamer and is in proximity to the deubiquitinase Rpn11. Ubiquitin-bound Ubp6 inhibits substrate deubiquitination by Rpn11, stabilizes the substrate-engaged conformation of the proteasome and allosterically interferes with the engagement of a subsequent substrate. Ubp6 may thus act as a ubiquitin-dependent 'timer' to coordinate individual processing steps at the proteasome and modulate substrate degradation.

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Figure 1: Ubp6 deubiquitination activity responds to the conformational state of the proteasome.
Figure 2: Ubp6 allostery is connected to substrate engagement.
Figure 3: Ubiquitin-bound Ubp6 interacts with the Rpt hexamer of the base.
Figure 4: Ubiquitin-bound Ubp6 stabilizes the substrate-engaged conformation of the proteasome, and ubiquitin-independent substrate delivery to the proteasome reveals that ubiquitin-bound Ubp6 allosterically inhibits multiple-turnover but not single-turnover degradation.
Figure 5: Ubp6 affects ubiquitin-dependent degradation.
Figure 6: Model for Ubp6 acting as a ubiquitin-dependent timer to allosterically control proteasome conformational changes, Rpn11 deubiquitination and substrate degradation.

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References

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Goldberg, A.L. Protein degradation and protection against misfolded or damaged proteins. Nature 426, 895–899 (2003).

    CAS  PubMed  Google Scholar 

  3. Goldberg, A.L. Functions of the proteasome: from protein degradation and immune surveillance to cancer therapy. Biochem. Soc. Trans. 35, 12–17 (2007).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  6. Groll, M. et al. Structure of 20S proteasome from yeast at 2.4Å resolution. Nature 386, 463–471 (1997).

    CAS  PubMed  Google Scholar 

  7. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Xu, P. et al. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137, 133–145 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Saeki, Y. et al. Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome. EMBO J. 28, 359–371 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhang, N. et al. Structure of the s5a:k48-linked diubiquitin complex and its interactions with rpn13. Mol. Cell 35, 280–290 (2009).

    PubMed  PubMed Central  Google Scholar 

  14. Riedinger, C. et al. Structure of Rpn10 and its interactions with polyubiquitin chains and the proteasome subunit Rpn12. J. Biol. Chem. 285, 33992–34003 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Elsasser, S., Chandler-Militello, D., Müller, B., Hanna, J. & Finley, D. Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome. J. Biol. Chem. 279, 26817–26822 (2004).

    CAS  PubMed  Google Scholar 

  16. Zhang, D. et al. Together, Rpn10 and Dsk2 can serve as a polyubiquitin chain-length sensor. Mol. Cell 36, 1018–1033 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Mayor, T., Graumann, J., Bryan, J., MacCoss, M.J. & Deshaies, R.J. Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway. Mol. Cell. Proteomics 6, 1885–1895 (2007).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Beckwith, R., Estrin, E., Worden, E.J. & Martin, A. Reconstitution of the 26S proteasome reveals functional asymmetries in its AAA+ unfoldase. Nat. Struct. Mol. Biol. 20, 1164–1172 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Śledź, P. & Unverdorben, P. 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).

    PubMed  PubMed Central  Google Scholar 

  23. Hu, M. et al. Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14. EMBO J. 24, 3747–3756 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Leggett, D.S. et al. Multiple associated proteins regulate proteasome structure and function. Mol. Cell 10, 495–507 (2002).

    CAS  PubMed  Google Scholar 

  25. Elsasser, S. et al. Proteasome subunit Rpn1 binds ubiquitin-like protein domains. Nat. Cell Biol. 4, 725–730 (2002).

    CAS  PubMed  Google Scholar 

  26. Hanna, J. et al. Deubiquitinating enzyme Ubp6 functions noncatalytically to delay proteasomal degradation. Cell 127, 99–111 (2006).

    CAS  PubMed  Google Scholar 

  27. Peth, A., Besche, H.C. & Goldberg, A.L. Ubiquitinated proteins activate the proteasome by binding to Usp14/Ubp6, which causes 20S gate opening. Mol. Cell 36, 794–804 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Peth, A., Kukushkin, N., Bossé, M. & Goldberg, A.L. Ubiquitinated proteins activate the proteasomal ATPases by binding to Usp14 or Uch37 homologs. J. Biol. Chem. 288, 7781–7790 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Lee, B.-H. et al. Enhancement of proteasome activity by a small-molecule inhibitor of USP14. Nature 467, 179–184 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Torres, E.M. et al. Identification of aneuploidy-tolerating mutations. Cell 143, 71–83 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Dephoure, N. et al. Quantitative proteomic analysis reveals posttranslational responses to aneuploidy in yeast. eLife 3, e03023 (2014).

    PubMed  PubMed Central  Google Scholar 

  32. Walters, B.J. et al. A catalytic independent function of the deubiquitinating enzyme USP14 regulates hippocampal synaptic short-term plasticity and vesicle number. J. Physiol. (Lond.) 592, 571–586 (2014).

    CAS  Google Scholar 

  33. Levchenko, I. A specificity-enhancing factor for the ClpXP degradation machine. Science 289, 2354–2356 (2000).

    CAS  PubMed  Google Scholar 

  34. Dong, K.C. et al. Preparation of distinct ubiquitin chain reagents of high purity and yield. Structure 19, 1053–1063 (2011).

    CAS  PubMed  Google Scholar 

  35. Borodovsky, A. et al. A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J. 20, 5187–5196 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Asano, S. et al. A molecular census of 26S proteasomes in intact neurons. Science 347, 439–442 (2015).

    CAS  PubMed  Google Scholar 

  37. Chu, B.W. et al. The E3 ubiquitin ligase UBE3C enhances proteasome processivity by ubiquitinating partially proteolyzed substrates. J. Biol. Chem. 288, 34575–34587 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Aufderheide, A. et al. Structural characterization of the interaction of Ubp6 with the 26S proteasome. Proc. Natl. Acad. Sci. USA 112, 8626–8631 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Marshall, A.G. et al. Genetic background alters the severity and onset of neuromuscular disease caused by the loss of ubiquitin-specific protease 14 (Usp14). PLoS ONE 8, e84042 (2013).

    PubMed  PubMed Central  Google Scholar 

  40. Chen, P.-C. et al. The proteasome-associated deubiquitinating enzyme Usp14 is essential for the maintenance of synaptic ubiquitin levels and the development of neuromuscular junctions. J. Neurosci. 29, 10909–10919 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Crosas, B. et al. Ubiquitin chains are remodeled at the proteasome by opposing ubiquitin ligase and deubiquitinating activities. Cell 127, 1401–1413 (2006).

    CAS  PubMed  Google Scholar 

  42. Aviram, S. & Kornitzer, D. The ubiquitin ligase Hul5 promotes proteasomal processivity. Mol. Cell. Biol. 30, 985–994 (2010).

    CAS  PubMed  Google Scholar 

  43. Inobe, T., Fishbain, S., Prakash, S. & Matouschek, A. Defining the geometry of the two-component proteasome degron. Nat. Chem. Biol. 7, 161–167 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Gomez, T.A., Kolawa, N., Gee, M., Sweredoski, M.J. & Deshaies, R.J. Identification of a functional docking site in the Rpn1 LRR domain for the UBA-UBL domain protein Ddi1. BMC Biol. 9, 33 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Unverdorben, P. et al. Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome. Proc. Natl. Acad. Sci. USA 111, 5544–5549 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Worden, E.J., Padovani, C. & Martin, A. Structure of the Rpn11–Rpn8 dimer reveals mechanisms of substrate deubiquitination during proteasomal degradation. Nat. Struct. Mol. Biol. 21, 220–227 (2014).

    CAS  PubMed  Google Scholar 

  47. Lander, G.C. et al. Appion: an integrated, database-driven pipeline to facilitate EM image processing. J. Struct. Biol. 166, 95–102 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 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).

    CAS  PubMed  Google Scholar 

  49. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Kather, I., Bippes, C.A. & Schmid, F.X. A stable disulfide-free gene-3-protein of phage fd generated by in vitro evolution. J. Mol. Biol. 354, 666–678 (2005).

    CAS  PubMed  Google Scholar 

  51. Pickart, C.M. & Raasi, S. Controlled synthesis of polyubiquitin chains. Methods Enzymol. 399, 21–36 (2005).

    CAS  PubMed  Google Scholar 

  52. Carragher, B. et al. Leginon: an automated system for acquisition of images from vitreous ice specimens. J. Struct. Biol. 132, 33–45 (2000).

    CAS  PubMed  Google Scholar 

  53. Roseman, A.M. FindEM: a fast, efficient program for automatic selection of particles from electron micrographs. J. Struct. Biol. 145, 91–99 (2004).

    CAS  PubMed  Google Scholar 

  54. Scheres, S.H.W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Goddard, T.D., Huang, C.C. & Ferrin, T.E. Visualizing density maps with UCSF Chimera. J. Struct. Biol. 157, 281–287 (2007).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank the members of the Martin laboratory for helpful discussions, C. Padovani and R. Beckwith (both in A.M.'s laboratory) for purified ubiquitin dimers and proteasome base subcomplexes, respectively. We are also grateful to T. Wandless (Stanford School of Medicine) for providing the lysineless GFP construct, K. Nyquist for cloning the GFP model substrate used in degradation assays and the D.O. Morgan laboratory (University of California, San Francisco) for ubiquitin reagents. C.B. acknowledges support from the US National Science Foundation Graduate Research Fellowship, and M.E.M. acknowledges support from the American Cancer Society (grant 121453-PF-11-178-01-TBE). This research was also funded in part by the Damon Runyon Cancer Research Foundation (DFS-#07-13), the Pew Scholars program, the Searle Scholars program and the US National Institutes of Health (grant DP2 EB020402-01) to G.C.L. A.M. acknowledges support from the Searle Scholars Program, start-up funds from the Molecular & Cell Biology Department at the University of California, Berkeley, the US National Institutes of Health (grant R01-GM094497) and the US National Science Foundation CAREER Program (NSF-MCB-1150288).

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  1. Mary E Matyskiela

    Present address: Present address: Celgene Corporation, San Diego, California, USA.,

Authors and Affiliations

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

    Charlene Bashore, Ellen A Goodall, Mary E Matyskiela & Andreas Martin

  2. Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, California, USA

    Corey M Dambacher & Gabriel C Lander

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

    Andreas Martin

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Contributions

C.B., E.A.G., M.E.M. and A.M. designed, expressed, and purified proteasome components and performed biochemical experiments. C.M.D. and G.C.L. performed EM, data processing and segmentation analyses. All authors contributed to experimental design, data analyses and manuscript preparation.

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Correspondence to Andreas Martin.

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Integrated supplementary information

Supplementary Figure 1 ATP-γS–bound holoenzymes resemble a substrate-translocating proteasome conformation.

EM reconstruction of the ATPγS-bound proteasome agrees with EM reconstruction of substrate-bound, translocating proteasome. Font and back views of reconstructions of ATPγS-bound proteasome (EMDB 2596, gray) and substrate-bound proteasome (EMDB 5669, cyan) that were aligned in UCSF Chimera using the “fit in map tool”.

Supplementary Figure 2 Functional cross-talk between Ubp6 and the base ATPase.

(a) Proteasomes reconstituted with SspB2-Rpt2 base are competent to degrade ubiquitinated substrate. 200 nM WT or SspB2-Rpt2 base was assembled into proteasomes with 600nM Lid, Rpn10, and CP. Degradation of 2µM ubiquitinated EGFP substrate20 was measured by loss of fluorescence at 511nm. (b) Ubp6 stimulates ATPase rate of the isolated base subcomplex. ATPase rates of purified recombinant base were measured in the presence of Ubp6 C118A, di-ubiquitin, and Ubp6 C118A and di-ubiquitin. Error bars show SEM of at least three independent experiments. (c) Ubp6 similarly stimulates ATPase rate of proteasomes reconstituted with WT base or SspB2-Rpt2 base. Proteasomes were reconstituted with 200nM WT or SspB2-Rpt2 base and 600nM Lid, Rpn10, and CP. 900nM Ubp6 and/or 20µM di-ubiquitin were added to show similar stimulations of ATPase rate with the wild-type or SspB2-Rpt2 base. Di-ubiquitin with Ubp6 stimulated proteasomes similarly to UbVS-Ubp6. Error bars represent SEM of at least three independent experiments. (d) Substrate translocation by the proteasome stimulates Ubp6 deubiquitination. Wild-type Ubp6 activity was measured with proteasomes reconstituted with SspB2-Rpt2 base. Addition of saturating amounts of an unfolded substrate increases Ubp6 deubiquitination, although not as much as the addition of ATPγS. Error bars represent s.e.m of three independent experiments. (e) Ubp6-free holoenzymes were preincubated with buffer, 10% DMSO, or o-phenanthroline (o-PA) for 10 minutes before Ub-AMC measurements. Averages of three technical replicates are plotted with corresponding linear regression.

Supplementary Figure 3 3D classes in ATP-bound Ub–Ubp6 proteasomes.

(a) Front and (b) back views of different proteasome conformations seen by negative stain EM.

Supplementary Figure 4 Ubp6–UbVS proteasome structure with ATP.

(a) Raw micrograph of the grids. (b) Sharpened reconstruction shows holoenzymes in an apo state. (c) Differential projections of the 3D model to match the 2D projections shown. (d) 2D projections calculated from the 3D model. (e) Reference-free 2D classes from actual data set. (f) FSC curve. (g) Angular distribution plot (Euler plot).

Supplementary Figure 5 Ubp6–UbVS proteasome structure with ATP-γS.

(a) Raw micrograph of the grids. (b) Sharpened reconstruction shows holoenzymes in an engaged state. (c) Differential projections of the 3D model to match the 2D projections shown. (d) 2D projections calculated from the 3D model. (e) Reference-free 2D classes from actual data set. (f) FSC curve. (g) Angular distribution plot (Euler plot).

Supplementary Figure 6 Zoomed-in view of docked Ubp6–UbVS model.

Docked Ubp6-UbVS model (see methods, PDB 1VJV) in EM density places the N-terminus of the catalytic domain of Ubp6 near the connecting density between Ubp6 (red) and Rpn1 (purple, PDB 4CR4 chain Z). The N-terminal region of the Ubp6 catalytic domain (residues 104 to 114) are shown in green.

Supplementary Figure 7 Ubp6 is not degraded by the proteasome.

100nM purified holoenzymes lacking Ubp6 were mixed with His6-tagged wild-type, C118A or Ub-VS treated wild-type Ubp6 and incubated at 30˚ C with ATP regeneration system. (a) Ubp6 was visualized by western blot detecting His6. (b) Holoenzyme levels were detected by blotting against Flag-tagged Rpn11.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 1093 kb)

Supplementary Data Set 1

Representative gels for SDS-PAGE degradation assay of unfolded substrate (Supplement to Fig. 4) (PDF 4604 kb)

Supplementary Data Set 2

Compiled experimental data (XLSX 55 kb)

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Bashore, C., Dambacher, C., Goodall, E. et al. Ubp6 deubiquitinase controls conformational dynamics and substrate degradation of the 26S proteasome. Nat Struct Mol Biol 22, 712–719 (2015). https://doi.org/10.1038/nsmb.3075

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