UC Berkeley

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, I heterologously expressed the unfoldase, also known as the base subcomplex, from Saccharomyces cerevisiae in Escherichia coli and reconstituted partially recombinant 26S proteasomes in vitro. My 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 of the base with the core peptidase exhibits three-fold symmetry with contributions from every other subunit. Preliminary data on establishing a bulk kinetic assay utilizing substrates of varying lengths to experimentally deconvolute the processes of substrate engagement and translocation is presented. The diverse structural and functional asymmetries explored in this body of work demonstrate how the mechanisms of substrate engagement and processing may differ between the eukaryotic 26S proteasome and other simpler, homomeric AAA+ proteases. My findings provide an initial framework for elucidating the mechanochemical operating principles of the heterohexameric proteasomal motor. Future studies will be required to determine whether emerging mechanistic models of ATP-dependent substrate translocation established for homomeric AAA+ proteases can be generalized to the 26S proteasome.