Reconstitution of the 26S Proteasome Reveals Functional Asymmetries in its Heterohexameric AAA+ Unfoldase

Abstract

The 26S proteasome is the major protease in eukaryotic cells responsible for selective protein degradation to mediate protein quality control and regulation. However, the detailed mechanisms by which the proteasomal heterohexameric AAA+ unfoldase drives ATP-dependent protein degradation remain poorly understood. Delineating the roles of the six distinct ATPase subunits in substrate processing has been hindered by limitations in working with endogenous proteasomes due to misassembly or lethal degradation defects. We therefore developed a heterologous expression system to produce the unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro to perform systematic mutational analyses of the individual ATPase subunits. Our studies demonstrate that the six ATPases have distinct functions in degradation, corresponding to their positions in the spiral staircases adopted by their large 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 a pronounced three-fold symmetry. Only three ATPase subunits, arranged in alternate positions within the unfoldase ring, contain a conserved C-terminal hydrophobic/aromatic/unspecified (HbYX) motif that is critical for both peptidase binding and gate-opening, whereas the C-terminal tails of the interjacent ATPase subunits are dispensable. Our study provides an initial glimpse into the potential importance of the spiral staircase configurations of proteasomal ATPase subunits in substrate processing and highlights how the 26S proteasome may deviate from simpler, homomeric AAA+ proteases.