ABSTRACT The 26S proteasome degrades polyubiquitylated (polyUb) proteins by an ATP-dependent mechanism. Here we show that binding of model polyUb substrates to the 19S regulator of mammalian

and yeast 26S proteasomes enhances the peptidase activities of the 20S proteasome about two-fold in a process requiring ATP hydrolysis. Monoubiquitylated proteins or tetraubiquitin alone

exert no effect. However, 26S proteasomes from the yeast α3ΔN open-gate mutant and the _rpt2YA_ and _rpt5YA_ mutants with impaired gating can still be activated (approximately 1.3-fold to

1.8-fold) by polyUb-protein binding. Thus, binding of polyUb substrates to the 19S regulator stabilizes gate opening of the 20S proteasome and induces conformational changes of the 20S

print issues and online access $209.00 per year only $17.42 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be

subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR

CONTENT BEING VIEWED BY OTHERS ALLOSTERIC CONTROL OF UBP6 AND THE PROTEASOME VIA A BIDIRECTIONAL SWITCH Article Open access 11 February 2022 MECHANISMS AND REGULATION OF SUBSTRATE

DEGRADATION BY THE 26S PROTEASOME Article 03 October 2024 ALLOSTERIC COUPLING BETWEEN Α-RINGS OF THE 20S PROTEASOME Article Open access 11 September 2020 REFERENCES * Voges, D., Zwickl, P.

& Baumeister, W. The 26S proteasome: a molecular machine designed for controlled proteolysis. _Annu. Rev. Biochem._ 68, 1015–1068 (1999). Article  CAS  Google Scholar  * Groll, M. et al.

A gated channel into the proteasome core particle. _Nat. Struct. Biol._ 7, 1062–1067 (2000). Article  CAS  Google Scholar  * Rechsteiner, M. & Hill, C.P. Mobilizing the proteolytic

machine: cell biological roles of proteasome activators and inhibitors. _Trends Cell Biol._ 15, 27–33 (2005). Article  CAS  Google Scholar  * 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  * 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  * Yao, T. & Cohen, R.E. A cryptic protease couples deubiquitination and degradation by the

proteasome. _Nature_ 419, 403–407 (2002). Article  CAS  Google Scholar  * 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  * Braun, B.C. et al. The base of the proteasome

regulatory particle exhibits chaperone-like activity. _Nat. Cell Biol._ 1, 221–226 (1999). Article  CAS  Google Scholar  * Kohler, 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  CAS  Google Scholar  * 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  * Lam, Y.A.,

Lawson, T.G., Velayutham, M., Zweier, J.L. & Pickart, C.M. A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal. _Nature_ 416, 763–767 (2002). Article  CAS 

Google Scholar  * Pickart, C.M. & Cohen, R.E. Proteasomes and their kin: proteases in the machine age. _Nat. Rev. Mol. Cell Biol._ 5, 177–187 (2004). Article  CAS  Google Scholar  *

Seeger, M., Ferrell, K., Frank, R. & Dubiel, W. HIV-1 tat inhibits the 20 S proteasome and its 11 S regulator-mediated activation. _J. Biol. Chem._ 272, 8145–8148 (1997). Article  CAS 

Google Scholar  * Ferrell, K., Wilkinson, C.R., Dubiel, W. & Gordon, C. Regulatory subunit interactions of the 26S proteasome, a complex problem. _Trends Biochem. Sci._ 25, 83–88 (2000).

Article  CAS  Google Scholar  * Babbitt, S.E. et al. ATP hydrolysis-dependent disassembly of the 26S proteasome is part of the catalytic cycle. _Cell_ 121, 553–565 (2005). Article  CAS 

Google Scholar  * Kleijnen, M.F. 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). Article  CAS  Google Scholar  * Hetfeld, B.K. et al. The zinc finger of the CSN-associated deubiquitinating enzyme USP15 is essential to rescue the E3 ligase Rbx1. _Curr.

Biol._ 15, 1217–1221 (2005). Article  CAS  Google Scholar  * 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  * Fenteany, G. et al. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin.

_Science_ 268, 726–731 (1995). Article  CAS  Google Scholar  * Schmidtke, G., Emch, S., Groettrup, M. & Holzhutter, H.G. Evidence for the existence of a non-catalytic modifier site of

peptide hydrolysis by the 20 S proteasome. _J. Biol. Chem._ 275, 22056–22063 (2000). Article  CAS  Google Scholar  * Kisselev, A.F., Kaganovich, D. & Goldberg, A.L. Binding of

hydrophobic peptides to several non-catalytic sites promotes peptide hydrolysis by all active sites of 20 S proteasomes. Evidence for peptide-induced channel opening in the α-rings. _J.

Biol. Chem._ 277, 22260–22270 (2002). Article  CAS  Google Scholar  * Glickman, M.H. Getting in and out of the proteasome. _Semin. Cell Dev. Biol._ 11, 149–158 (2000). Article  CAS  Google

Scholar  * Kohler, A. et al. The substrate translocation channel of the proteasome. _Biochimie_ 83, 325–332 (2001). Article  CAS  Google Scholar  * 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  *

Benaroudj, N., Zwickl, P., Seemuller, E., Baumeister, W. & Goldberg, A.L. ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation. _Mol.

Cell_ 11, 69–78 (2003). Article  CAS  Google Scholar  * Navon, A. & Goldberg, A.L. Proteins are unfolded on the surface of the ATPase ring before transport into the proteasome. _Mol.

Cell_ 8, 1339–1349 (2001). Article  CAS  Google Scholar  * Amerik, A.Y. & Hochstrasser, M. Mechanism and function of deubiquitinating enzymes. _Biochim. Biophys. Acta_ 1695, 189–207

(2004). Article  CAS  Google Scholar  * Stohwasser, R., Salzmann, U., Giesebrecht, J., Kloetzel, P.M. & Holzhutter, H.G. Kinetic evidences for facilitation of peptide channelling by the

proteasome activator PA28. _Eur. J. Biochem._ 267, 6221–6230 (2000). Article  CAS  Google Scholar  * Kopp, F., Dahlmann, B. & Kuehn, L. Reconstitution of hybrid proteasomes from purified

PA700–20 S complexes and PA28αβ activator: ultrastructure and peptidase activities. _J. Mol. Biol._ 313, 465–471 (2001). Article  CAS  Google Scholar  * Walz, J. et al. 26S proteasome

structure revealed by three-dimensional electron microscopy. _J. Struct. Biol._ 121, 19–29 (1998). Article  CAS  Google Scholar  * da Fonseca, P.C. & Morris, E.P. Structure of the human

26S proteasome: Subunit radial displacements open the gate into the proteolytic core. _J. Biol. Chem._ 283, 23305–23314 (2008). Article  CAS  Google Scholar  * Groettrup, M. et al. The

interferon-γ-inducible 11 S regulator (PA28) and the LMP2/LMP7 subunits govern the peptide production by the 20 S proteasome _in vitro_. _J. Biol. Chem._ 270, 23808–23815 (1995). Article 

CAS  Google Scholar  * Dahlmann, B., Kuehn, L. & Reinauer, H. Studies on the activation by ATP of the 26 S proteasome complex from rat skeletal muscle. _Biochem. J._ 309, 195–202 (1995).

Article  CAS  Google Scholar  * Wendler, P., Lehmann, A., Janek, K., Baumgart, S. & Enenkel, C. The bipartite nuclear localization sequence of Rpn2 is required for nuclear import of

proteasomal base complexes via karyopherin αβ and proteasome functions. _J. Biol. Chem._ 279, 37751–37762 (2004). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We would

like to thank D. Finley (Harvard Medical School) for the yeast strains _rpt2YA_ and _rpt5YA_, J. Dohmen (University of Cologne) for the _rpn10_ mutant strain and A. Lehmann for excellent

technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB421 and SFB 740 to P.-M.K.). AUTHOR INFORMATION Author notes * Dawadschargal Bech-Otschir

and Annett Helfrich: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Institut für Biochemie, Charité—Universitätsmedizin Berlin, Monbijoustrasse 2, 10117, Berlin,

Germany Dawadschargal Bech-Otschir, Annett Helfrich, Cordula Enenkel, Gesa Consiglieri, Michael Seeger, Hermann-Georg Holzhütter, Burkhardt Dahlmann & Peter-Michael Kloetzel Authors *

Dawadschargal Bech-Otschir View author publications You can also search for this author inPubMed Google Scholar * Annett Helfrich View author publications You can also search for this author

inPubMed Google Scholar * Cordula Enenkel View author publications You can also search for this author inPubMed Google Scholar * Gesa Consiglieri View author publications You can also

search for this author inPubMed Google Scholar * Michael Seeger View author publications You can also search for this author inPubMed Google Scholar * Hermann-Georg Holzhütter View author

publications You can also search for this author inPubMed Google Scholar * Burkhardt Dahlmann View author publications You can also search for this author inPubMed Google Scholar *

Peter-Michael Kloetzel View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS D.B.-O. and A.H. performed all biochemical experiments; C.E.

purified yeast proteasomes; G.C. generated the MUC1 peptides; M.S. and H.-G.H. supervised the biochemical and enzyme kinetic experiments; B.D. purified the mammalian proteasomes; D.B.-O.,

B.D. and P.-M.K. prepared the manuscript; P.-M.K. designed the project. CORRESPONDING AUTHOR Correspondence to Peter-Michael Kloetzel. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND

FIGURES Supplementary Figures 1–4 and Supplementary Methods (PDF 297 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Bech-Otschir, D., Helfrich, A.,

Enenkel, C. _et al._ Polyubiquitin substrates allosterically activate their own degradation by the 26S proteasome. _Nat Struct Mol Biol_ 16, 219–225 (2009). https://doi.org/10.1038/nsmb.1547

Download citation * Received: 02 July 2008 * Accepted: 29 December 2008 * Published: 25 January 2009 * Issue Date: February 2009 * DOI: https://doi.org/10.1038/nsmb.1547 SHARE THIS ARTICLE

Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided

by the Springer Nature SharedIt content-sharing initiative