Specific lid-base contacts in the 26s proteasome control the conformational switching required for substrate degradation.

Eric R Greene,Andres H de la Peña,Andreas Martin,Gabriel C Lander,Ellen A Goodall,Mary E Matyskiela

doi:10.7554/elife.49806

Abstract

The 26S proteasome is essential for proteostasis and the regulation of vital processes through ATP-dependent degradation of ubiquitinated substrates. To accomplish the multi-step degradation process, the proteasome's regulatory particle, consisting of lid and base subcomplexes, undergoes major conformational changes whose origin is unknown. Investigating the Saccharomyces cerevisiae proteasome, we found that peripheral interactions between the lid subunit Rpn5 and the base AAA+ ATPase ring are important for stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states upon substrate engagement. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later processing steps remain unaffected. Similar defects in early degradation steps are observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control proteasome conformational transitions. These results provide important insight into interaction networks that coordinate conformational changes with various stages of degradation, and how modulators of conformational equilibria may influence substrate turnover.

Highlights

The 26S proteasome is the principal ATP-dependent protease in eukaryotic cells and responsible for the majority of targeted protein turnover, both through the degradation of short-lived regulatory proteins and the clearance of damaged or misfolded polypeptides for protein-quality control (Hershko and Ciechanover, 1998)
Structural rearrangements, the rotation of the lid relative to the base observed in response to substrate processing or binding of ATP analogs to the AAA+ motor, suggest that the lid may be directly involved in determining the proteasome conformational states (Matyskiela et al, 2013; Unverdorben et al, 2014; Wehmer et al, 2017; Eisele et al, 2018; Ding et al, 2017; de la Pena et al, 2018; Dong et al, 2019)
The conformational landscape of the proteasome can be biased by the nucleotide occupancy of the AAA+ motor (Sledzet al., 2013; Unverdorben et al, 2014; Wehmer et al, 2017; Eisele et al, 2018; Ding et al, 2017; Zhu et al, 2018) and the engagement of protein substrates (Matyskiela et al, 2013; de la Pena et al, 2018; Dong et al, 2019), but how the network of contacts within the regulatory particle, and in particular between the lid and base subcomplexes, affects conformational changes and equilibria remained unknown

Summary

Introduction

The 26S proteasome is the principal ATP-dependent protease in eukaryotic cells and responsible for the majority of targeted protein turnover, both through the degradation of short-lived regulatory proteins and the clearance of damaged or misfolded polypeptides for protein-quality control (Hershko and Ciechanover, 1998). Ubiquitin ligases mark obsolete proteins with poly-ubiquitin chains and thereby target them to ubiquitin receptors on the 26S proteasome, which represents the last component of the ubiquitin-proteasome system and mechanically unfolds, deubiquitinates, and translocates protein substrates into an internal chamber for proteolytic cleavage (Bard et al, 2018a). At the center of the 26S proteasome is a barrel-shaped core peptidase with sequestered proteolytic active sites (Groll et al, 1997) This core is capped on one or both ends by a regulatory particle that consists of two subcomplexes, referred to as the "lid" and the "base", and is responsible for the recognition, unfolding, and transfer of protein substrates into the core

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