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

Abstract

The 26S proteasome is the major eukaryotic ATP-dependent protease, yet the detailed mechanisms utilized 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 unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro. Our studies demonstrate that the six ATPases play distinct roles in degradation, corresponding to their positions in spiral staircases adopted by the AAA+ domains in the absence and presence of substrate. ATP hydrolysis in subunits at the top of the staircases is critical for substrate engagement and translocation. While the unfoldase relies on this vertical asymmetry for substrate processing, interaction with the peptidase exhibits three-fold symmetry with contributions from every other subunit. These diverse functional asymmetries highlight how the 26S proteasome deviates from simpler, homomeric AAA+ proteases.