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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 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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Figure 1: Expression of yeast base subcomplexes in E. coli and reconstitution of the 26S proteasome.
Figure 2: Proteasomes reconstituted with endogenous or heterologously expressed base exhibit similar degradation activities for a polyubiquitinated substrate.
Figure 3: Degradation activities for base variants with a Walker-B E→Q mutation in individual Rpt subunits correlate with the subunit's position in the spiral staircase arrangement of the base.
Figure 4: Degradation activities for base variants containing single-subunit pore-loop mutations.
Figure 5: Rpt C-terminal tails contribute to gate opening differentially and in a nucleotide-independent manner.
Figure 6: Affinities of core-peptidase binding for base variants lacking individual C-terminal tails.
Figure 7: Model for the contribution of individual ATPase subunits to substrate processing and core particle interaction.

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Acknowledgements

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.).

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R.B., E.E., E.J.W. and A.M. designed experiments, expressed and purified proteasome constructs, performed biochemical experiments and analyzed data. R.B. and A.M. prepared the manuscript.

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Correspondence to Andreas Martin.

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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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