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.
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We thank the members of the Martin lab for helpful discussions. E.J.W. acknowledges support from a National Science Foundation Graduate Research Fellowship. This research was funded in part by the Searle Scholars Program (A.M.), US National Institutes of Health grant R01-GM094497-01A1 (A.M.) and start-up funds from the Molecular and Cell Biology Department at University of California, Berkeley (A.M.).
The authors declare no competing financial interests.
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Beckwith, R., Estrin, E., Worden, E. et al. Reconstitution of the 26S proteasome reveals functional asymmetries in its AAA+ unfoldase. Nat Struct Mol Biol 20, 1164–1172 (2013). https://doi.org/10.1038/nsmb.2659
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