Cell
Volume 177, Issue 2, 4 April 2019, Pages 286-298.e15
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Article
The 26S Proteasome Utilizes a Kinetic Gateway to Prioritize Substrate Degradation

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Highlights

  • The rate-limiting step of protein degradation by the proteasome is unfolding

  • Substrate contact with the motor triggers a conformational switch of the proteasome

  • Substrates with poor initiation regions are quickly rejected

  • Supernumerary ubiquitin chains promote degradation of otherwise poor substrates

Summary

The 26S proteasome is the principal macromolecular machine responsible for protein degradation in eukaryotes. However, little is known about the detailed kinetics and coordination of the underlying substrate-processing steps of the proteasome, and their correlation with observed conformational states. Here, we used reconstituted 26S proteasomes with unnatural amino-acid-attached fluorophores in a series of FRET- and anisotropy-based assays to probe substrate-proteasome interactions, the individual steps of the processing pathway, and the conformational state of the proteasome itself. We develop a complete kinetic picture of proteasomal degradation, which reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation, and unfolding. Furthermore, we find that non-ideal substrates are rapidly rejected by the proteasome, which thus employs a kinetic proofreading mechanism to ensure degradation fidelity and substrate prioritization.

Keywords

26S proteasome
ubiquitin-proteasome system
AAA+ protease
ATP-dependent protein degradation
unnatural amino-acid incorporation

Cited by (0)

4

Present address: Department of Biochemistry & Molecular Biology, University of Chicago, Chicago, IL 60637, USA

5

Present address: Department of Early Discovery Biochemistry, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA

6

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