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An unstructured initiation site is required for efficient proteasome-mediated degradation

Nature Structural & Molecular Biology volume 11, pages 830–837 (2004)Cite this article

Abstract

The proteasome is the main ATP-dependent protease in eukaryotic cells and controls the concentration of many regulatory proteins in the cytosol and nucleus. Proteins are targeted to the proteasome by the covalent attachment of polyubiquitin chains. The ubiquitin modification serves as the proteasome recognition element but by itself is not sufficient for efficient degradation of folded proteins. We report that proteolysis of tightly folded proteins is accelerated greatly when an unstructured region is attached to the substrate. The unstructured region serves as the initiation site for degradation and is hydrolyzed first, after which the rest of the protein is digested sequentially. These results identify the initiation site as a novel component of the targeting signal, which is required to engage the proteasome unfolding machinery efficiently. The proteasome degrades a substrate by first binding to its ubiquitin modification and then initiating unfolding at an unstructured region.

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Figure 1: Sequential degradation of two-domain proteins by the proteasome in two directions.
Figure 2: Proteasome degradation is accelerated by an unstructured region in the substrate protein.
Figure 3: Proteasome degradation initiates at an unstructured region in the substrate protein.
Figure 4: Tightly folded substrates must contain an unstructured region for efficient proteasome degradation.
Figure 5: Characterization of degradation initiation sites.
Figure 6: Schematic representation of the proposed proteasome degradation cycle.

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Acknowledgements

We thank C. Pickart, L. Hicke, J. Widom, C. Holmberg and O. Uhlenbeck for advice and helpful comments on the manuscript. We also thank P. Bellare, J. Schnell, N. Jaffe and A. Wilcox for carefully reading and correcting the manuscript. M. Fisher (University of Kansas Medical Center) provided purified GroEL and advice. We acknowledge the use of the instruments at the Keck Biophysics Facility of the Robert H. Lurie Comprehensive Cancer Center at Northwestern University. The work was supported by US National Institutes of Health grant R01GM63004, by a Scholar Award to A.M. from the Leukemia and Lymphoma Society and by a Gramm Travel Fellowship Award to S.P. from the Robert H. Lurie Comprehensive Cancer Center at Northwestern University.

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Authors and Affiliations

  1. Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, 60208, Illinois, USA

    Sumit Prakash, Lin Tian, Kevin S Ratliff, Rebecca E Lehotzky & Andreas Matouschek

  2. Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, 60611, Illinois, USA

    Sumit Prakash, Lin Tian, Kevin S Ratliff, Rebecca E Lehotzky & Andreas Matouschek

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Correspondence to Andreas Matouschek.

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Prakash, S., Tian, L., Ratliff, K. et al. An unstructured initiation site is required for efficient proteasome-mediated degradation. Nat Struct Mol Biol 11, 830–837 (2004). https://doi.org/10.1038/nsmb814

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