The 26S Proteasome Utilizes a Kinetic Gateway to Prioritize Substrate Degradation

Cell. 2019 Apr 4;177(2):286-298.e15. doi: 10.1016/j.cell.2019.02.031. Epub 2019 Mar 28.


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; AAA(+) protease; ATP-dependent protein degradation; ubiquitin-proteasome system; unnatural amino-acid incorporation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Anisotropy
  • Binding Sites / physiology
  • Enzyme Activation
  • Kinetics
  • Models, Molecular
  • Proteasome Endopeptidase Complex / metabolism*
  • Proteasome Endopeptidase Complex / physiology*
  • Protein Binding
  • Protein Conformation
  • Protein Processing, Post-Translational / physiology
  • Proteolysis
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / metabolism
  • Substrate Specificity / physiology
  • Ubiquitin / metabolism


  • Saccharomyces cerevisiae Proteins
  • Ubiquitin
  • Proteasome Endopeptidase Complex
  • ATP dependent 26S protease