Abstract
The 26S proteasome is a multisubunit enzyme composed of a cylindrical catalytic core (20S) and a regulatory particle (19S) that together perform the essential degradation of cellular proteins tagged by ubiquitin. To date, however, substrate trajectory within the complex remains elusive. Here we describe a previously unknown functional unit within the 19S, comprising two subunits, Rpn1 and Rpn2. These toroids physically link the site of substrate recruitment with the site of proteolysis. Rpn2 interfaces with the 20S, whereas Rpn1 sits atop Rpn2, serving as a docking site for a substrate-recruitment factor. The 19S ATPases encircle the Rpn1-Rpn2 stack, covering the remainder of the 20S surface. Both Rpn1-Rpn2 and the ATPases are required for substrate translocation and gating of the proteolytic channel. Similar pairing of units is found in unfoldases and nuclear transporters, exposing common features of these protein nanomachines.
Similar content being viewed by others
References
Maupin-Furlow, J.A. et al. Proteasomes from structure to function: perspectives from Archaea. Curr. Top. Dev. Biol. 75, 125–169 (2006).
Baker, T.A. & Sauer, R.T. ATP-dependent proteases of bacteria: recognition logic and operating principles. Trends Biochem. Sci. 31, 647–653 (2006).
Voges, D., Zwickl, P. & Baumeister, W. The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem. 68, 1015–1068 (1999).
Zwickl, P., Baumeister, W. & Steven, A. Dis-assembly lines: the proteasome and related ATPase-assisted proteases. Curr. Opin. Struct. Biol. 10, 242–250 (2000).
Glickman, M.H. & Ciechanover, A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82, 373–428 (2002).
Pickart, C.M. & Cohen, R.E. Proteasomes and their kin: proteases in the machine age. Nat. Rev. Mol. Cell Biol. 5, 177–187 (2004).
Schmidt, M., Hanna, J., Elsasser, S. & Finley, D. Proteasome-associated proteins: regulation of a proteolytic machine. Biol. Chem. 386, 725–737 (2005).
Groll, M. et al. Structure of 20S proteasome from yeast at a 2.4 resolution. Nature 386, 463–471 (1997).
Groll, M. et al. A gated channel into the core particle of the proteasome. Nat. Struct. Biol. 7, 1062–1067 (2000).
Bajorek, M., Finley, D. & Glickman, M.H. Proteasome disassembly and downregulation is correlated with viability during stationary phase. Curr. Biol. 13, 1140–1144 (2003).
Osmulski, P.A. & Gaczynska, M. Nanoenzymology of the 20S proteasome: proteasomal actions are controlled by the allosteric transition. Biochemistry 41, 7047–7053 (2002).
Forster, A., Masters, E.I., Whitby, F.G., Robinson, H. & Hill, C.P. The 1.9 structure of a proteasome-11S activator complex and implications for proteasome-PAN/PA700 interactions. Mol. Cell 18, 589–599 (2005).
Rechsteiner, M. & Hill, C.P. Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors. Trends Cell Biol. 15, 27–33 (2005).
Smith, D.M., Benaroudj, N. & Goldberg, A. Proteasomes and their associated ATPases: a destructive combination. J. Struct. Biol. 156, 72–83 (2006).
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).
Liu, C.-W. ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome. Mol. Cell 24, 39–50 (2006).
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).
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).
Kajava, A.V., Gorbea, C., Ortega, J., Rechsteiner, M. & Steven, A.C. New HEAT-like repeat motifs in proteins regulating proteasome structure and function. J. Struct. Biol. 146, 425–430 (2004).
Kajava, A.V. What curves α-solenoids? Evidence for an α-helical toroid structure of Rpn1 and Rpn2 proteins of the 26S proteasome. J. Biol. Chem. 277, 49791–49798 (2002).
Ortega, J. et al. The axial channel of the 20 S proteasome opens upon binding of the PA200 activator. J. Mol. Biol. 346, 1221–1227 (2005).
Chen, X., Barton, L.F., Chi, Y., Clurman, B.E. & Roberts, J.M. Ubiquitin-independent degradation of cell-cycle inhibitors by the REGγ proteasome. Mol. Cell 26, 843–852 (2007).
Dorn, I.T., Eschrich, R., Seemuller, E., Guckenberger, R. & Tampe, R. High-resolution AFM-imaging and mechanistic analysis of the 20S proteasome. J. Mol. Biol. 288, 1027–1036 (1999).
Thess, A. et al. Specific orientation and two-dimensional crystallization of the proteasome at metal-chelating lipid interfaces. J. Biol. Chem. 277, 36321–36328 (2002).
Glickman, M.H., Rubin, D.M., Fried, V.A. & Finley, D. The regulatory particle of the S. cerevisiae proteasome. Mol. Cell. Biol. 18, 3149–3162 (1998).
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).
Marques, A.J., Glanemann, C., Ramos, P.C. & Dohmen, R.J. The C-terminal extension of the β7 subunit and activator complexes stabilize nascent 20S proteasomes and promote their maturation. J. Biol. Chem. 282, 34869–34876 (2007).
Isono, E. et al. The assembly pathway of the 19S regulatory particle of the yeast 26S proteasome. Mol. Biol. Cell 18, 569–580 (2007).
Nickell, S. et al. Structural analysis of the 26S proteasome by cryoelectron tomography. Biochem. Biophys. Res. Commun. 353, 115–120 (2007).
Kurucz, E. et al. Assembly of the Drosophila 26 S proteasome is accompanied by extensive subunit rearrangements. Biochem. J. 365, 527–536 (2002).
Walz, J. et al. 26S proteasome structure revealed by three-dimensional electron microscopy. J. Struct. Biol. 121, 19–29 (1998).
Iwanczyk, J. et al. Structure of the Blm10–20S proteasome complex by cryo-electron microscopy. Insights into the mechanism of activation of mature yeast proteasomes. J. Mol. Biol. 363, 648–659 (2006).
Strickland, E., Hakala, K., Thomas, P.J. & DeMartino, G.N. Recognition of misfolding proteins by PA700, the regulatory subcomplex of the 26S proteasome. J. Biol. Chem. 275, 5565–5572 (2000).
Braun, B.C. et al. The base of the proteasome regulatory particle exhibits chaperone-like activity. Nat. Cell Biol. 1, 221–226 (1999).
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).
Elsasser, S. et al. Proteasome subunit Rpn1 binds ubiquitin-like protein domains. Nat. Cell Biol. 4, 725–730 (2002).
Seeger, M. et al. Interaction of the anaphase-promoting complex/cyclosome and proteasome protein complexes with multiubiquitin chain-binding proteins. J. Biol. Chem. 278, 16791–16796 (2003).
Saeki, Y., Sone, T., Toh-e, A. & Yokosawa, H. Identification of ubiquitin-like protein-binding subunits of the 26S proteasome. Biochem. Biophys. Res. Commun. 296, 813–819 (2002).
Chen, L. & Madura, K. Rad23 promotes the targeting of proteolytic substrates to the proteasome. Mol. Cell. Biol. 22, 4902–4913 (2002).
Lee, S., Choi, J.-M. & Tsai, F.T.F. Visualizing the ATPase cycle in a protein disaggregating machine: structural basis for substrate binding by ClpB. Mol. Cell 25, 261–271 (2007).
Lum, R., Tkach, J.M., Vierling, E. & Glover, J.R. Evidence for an unfolding/threading mechanism for protein disaggregation by Saccharomyces cerevisiae Hsp104. J. Biol. Chem. 279, 29139–29146 (2004).
Thibault, G., Tsitrin, Y., Davidson, T., Gribun, A. & Houry, W.A. Large nucleotide-dependent movement of the N-terminal domain of the ClpX chaperone. EMBO J. 25, 3367–3376 (2006).
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).
Zolkiewski, M. A camel passes through the eye of a needle: protein unfolding activity of Clp ATPases. Mol. Microbiol. 61, 1094–1100 (2006).
Wang, J. et al. Nucleotide-dependent conformational changes in a protease-associated ATPase HslU. Structure 9, 1107–1116 (2001).
Thirumalai, D. & Lorimer, G.H. Chaperonin-mediated protein folding. Annu. Rev. Biophys. Biomol. Struct. 30, 245–269 (2001).
Horwich, A.L., Weber-Ban, E.U. & Finley, D. Chaperone rings in protein folding and degradation. Proc. Natl. Acad. Sci. USA 96, 11033–11040 (1999).
Conti, E., Muller, C.W. & Stewart, M. Karyopherin flexibility in nucleocytoplasmic transport. Curr. Opin. Struct. Biol. 16, 237–244 (2006).
Petosa, C. et al. Architecture of CRM1/Exportin1 suggests how cooperativity is achieved during formation of a nuclear export complex. Mol. Cell 16, 761–775 (2004).
Osmulski, P.A. & Gaczynska, M. Atomic force microscopy of the proteasome. Methods Enzymol. 398, 414–425 (2005).
Acknowledgements
We thank D. Cassel, O. Kleifeld and T. Rosenzweig for comments and critically reading the manuscript. A. Kajava is acknowledged for advice. N. Reis provided technical assistance. We thank D. Fass for assisting with AUC runs and analysis. We thank Y. Matiuhin (Technion) for Rad23 constructs. This work was funded by grants from the Israel Academy of Science/Israel Science Foundation (ISF), The USA-Israel Binational Science Foundation (BSF) and the Malat Family Foundation (via the Technion VP for research) to M.H.G., the NIH R01 grant (M.G.), and the Enhancement Research Grant and San Antonio Cancer Institute (SACI) support for P.A.O. R.R. was partially supported by an anonymous scholarship award (via the Technion Graduate School).
Author information
Authors and Affiliations
Contributions
R.R. performed biochemical experiments. M.G. and P.A.O. carried out AFM imaging. M.G. and M.H.G. designed and supervised experiments. All authors discussed the results and participated in writing the manuscript.
Corresponding authors
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–7 and Supplementary Methods (PDF 2832 kb)
Rights and permissions
About this article
Cite this article
Rosenzweig, R., Osmulski, P., Gaczynska, M. et al. The central unit within the 19S regulatory particle of the proteasome. Nat Struct Mol Biol 15, 573–580 (2008). https://doi.org/10.1038/nsmb.1427
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.1427
- Springer Nature America, Inc.
This article is cited by
-
RACK1 facilitates breast cancer progression by competitively inhibiting the binding of β-catenin to PSMD2 and enhancing the stability of β-catenin
Cell Death & Disease (2023)
-
The life cycle of the 26S proteasome: from birth, through regulation and function, and onto its death
Cell Research (2016)
-
The Arabidopsis MIEL1 E3 ligase negatively regulates ABA signalling by promoting protein turnover of MYB96
Nature Communications (2016)
-
C-terminal UBA domains protect ubiquitin receptors by preventing initiation of protein degradation
Nature Communications (2011)
-
The proteasome assembly line
Nature (2009)