Reconstitution of the 26S proteasome reveals functional asymmetries in its AAA+ unfoldase

Nat Struct Mol Biol. 2013 Oct;20(10):1164-72. doi: 10.1038/nsmb.2659. Epub 2013 Sep 8.


The 26S proteasome is the major eukaryotic ATP-dependent protease, yet the detailed mechanisms used by the proteasomal heterohexameric AAA+ unfoldase to drive substrate degradation remain poorly understood. To perform systematic mutational analyses of individual ATPase subunits, we heterologously expressed the unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro. Our studies demonstrate that the six ATPases have distinct roles in degradation, corresponding to their positions in the spiral staircases adopted by the AAA+ domains in the absence or presence of substrate. ATP hydrolysis in subunits at the top of the staircases is critical for substrate engagement and translocation. Whereas the unfoldase relies on this vertical asymmetry for substrate processing, interaction with the peptidase exhibits three-fold symmetry with contributions from alternate subunits. These diverse functional asymmetries highlight how the 26S proteasome deviates from simpler, homomeric AAA+ proteases.

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

  • Endopeptidase Clp / chemistry
  • Endopeptidase Clp / metabolism*
  • Escherichia coli / enzymology
  • Proteasome Endopeptidase Complex / chemistry
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Conformation
  • Saccharomyces cerevisiae / enzymology
  • Substrate Specificity


  • Endopeptidase Clp
  • Proteasome Endopeptidase Complex
  • ATP dependent 26S protease