Specific lid-base contacts in the 26S proteasome control the conformational switching required for substrate engagement and degradation

Abstract

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

Site-specific ubiquitination affects protein energetics and proteasomal degradation

“…The proteasomal degradation rates observed for poly-ubiquitinated barstar variants are notably lower than for barstar or other substrates with fused flexible tails 15,16,40 , suggesting that engagement of a spontaneously unfolding region represents the rate-limiting step for degradation. To probe this further, we turned to a proteasome variant, Rpn5-VTENKIF, whose mutations in the regulatory particle affect the conformational equilibrium of the proteasome and thereby hinder the insertion of flexible segments into the AAA+ pore, making engagement rate-determining even for moderately stable substrates with permanently unstructured tails 40 . Using this proteasome mutant, we see a three-fold (Ubn-barstarK2) and two-fold (Ubn-barstarK60) decrease in degradation rate (Supplementary Fig.…”