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

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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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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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.

Corresponding author

Correspondence to Andreas Martin.

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The authors declare no competing financial interests.

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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