Abstract
Protein substrates of the proteasome must apparently be unfolded and translocated through a narrow channel to gain access to the proteolytic active sites of the enzyme. Protein folding in vivo is mediated by molecular chaperones. Here, to test for chaperone activity of the proteasome, we assay the reactivation of denatured citrate synthase. Both human and yeast proteasomes stimulate the recovery of the native structure of citrate synthase. We map this chaperone-like activity to the base of the regulatory particle of the proteasome, that is, to the ATPase-containing assembly located at the substrate-entry ports of the channel. Denatured but not native citrate synthase is bound by the base complex. Ubiquitination of citrate synthase is not required for its binding or refolding by the base complex of the proteasome. These data suggest a model in which ubiquitin–protein conjugates are initially tethered to the proteasome by specific recognition of their ubiquitin chains; this step is followed by a nonspecific interaction between the base and the target protein, which promotes substrate unfolding and translocation.
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References
Baumeister, W., Walz, J., Zühl, F. & Seemüller, E. The proteasome: paradigm of a self-compartmentalizing protease. Cell 92, 367–380 (1998).
Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).
Tanaka, K. Molecular biology of the proteasome. Biochem. Biophys. Res. Commun. 247, 537–541 ( 1998).
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).
Löwe, J. et al. Crystal structure of the 20 S proteasome from the archaeon T. acidophilum at 3.4 Å resolution. Science 268, 533–539 (1995).
Groll, M. et al. Structure of 20S proteasome from yeast at 2.4 Å resolution . Nature 386, 463–471 (1997).
Walz, J. et al. 26S Proteasome structure revealed by three-dimensional electron microscopy. J. Struct. Biol. 121, 19– 29 (1998).
Larsen, C. N. & Finley, D. Protein translocation channels in the proteasome and other proteases. Cell 91, 431–434 (1997).
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).
Buckau, B. & Horwich, A. L. The Hsp70 and Hsp60 chaperone machines. Cell 92, 351– 366 (1998).
Wickner, S. et al. A molecular chaperone, ClpA, functions like DnaK and DnaJ . Proc. Natl Acad. Sci. USA 91, 12218– 12222 (1994).
Wawrzynow, A. et al. The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease is a novel molecular chaperone. EMBO J. 14, 1867–1877 (1995).
Leonhard, K., Stiegler, A., Neupert, W. & Langer, T. Chaperone-like activity of the AAA domain of the yeast Yme1 AAA protease. Nature 398, 348–351 ( 1999).
Murakami, Y. et al. Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination. Nature 360, 597– 599 (1992).
Buchner, J., Grallert, H. & Jakob, U. Analysis of chaperone function using citrate synthase as nonnative substrate protein. Methods Enzymol. 290 , 323–338 (1998).
Zhi, W., Srere, P. A. & Evans, C. T. Conformational stability of pig citrate synthase and some active-site mutants. Biochemistry 30, 9281–9286 (1991).
Buchner, J. et al. GroE facilitates refolding of citrate synthase by suppressing aggregation. Biochemistry 30, 1586– 1591 (1991).
Bose, S., Weikl, T., Buegl, H. & Buchner, J. Chaperone function of Hsp90-associated proteins. Science 274, 1715–1717 (1996).
Ehrnsperger, M., Graeber, S., Gaestel, M. & Buchner, J. Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation. EMBO J. 16, 221– 229 (1997).
Netzer, W. J. & Hartl, F. U. Protein folding in the cytosol: chaperonin-dependent and -independent mechanisms. Trends Biochem. Sci. 23, 68–73 ( 1998).
Xu, Z., Horwich, A. L. & Siegler, P. B. The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature 388, 741– 750 (1997).
Henke, W. et al. Comparison of human COP9 signalosome and 26S proteasome ‘lid’ . Mol. Biol. Rep. 26, 29– 34 (1999).
Pickart, C. M. Targeting of substrates to the 26S proteasome. FASEB J. 11, 1055–1066 (1997).
Akiyama, Y., Ehrmann, M., Kihara, A. & Ito, K. Polypeptide binding of Escherichia coli FtsH (HflB). Mol. Microbiol. 28, 803–812 (1998).
Ciechanover, A., Finley, D. & Varshavsky, A. Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell 37, 57–66 (1984).
Schmidtke, G. et al. Analysis of mammalian 20S proteasome biogenesis: the maturation of (β-subunits is an ordered two-step mechanism involving autocatalysis . EMBO J. 15, 6887–6898 (1996).
Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).
Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 ( 1976).
West, S. M., Kelly, S. M. & Price, N. C. The unfolding and attempted refolding of citrate synthase from pig heart. Biochim. Biophys. Acta 1037 , 332–336 (1990).
Acknowledgements
We thank R. Gali and C. Larsen for providing us with the base–20S particle of the yeast proteasome; D. Zantopf and G. Grelle for technical support; and J. Buchner and W. Dubiel for citrate-synthase-specific and S10a-specific antibodies. This work was supported by grants from the Deutsche Forschungsgemeinschaft (to M. S., Schm 884/2-1; and P.-M. K., Kl 427 8-2/8-3), from the NIH (to D. F., GM43601) and from the Medical Foundation Charles Kings Trust and the J. and A. Taub Research Fund (to M. G.).
Correspondence and requests for materials should be addressed to M.S.
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Braun, B., Glickman, M., Kraft, R. et al. The base of the proteasome regulatory particle exhibits chaperone-like activity. Nat Cell Biol 1, 221–226 (1999). https://doi.org/10.1038/12043
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DOI: https://doi.org/10.1038/12043
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