Visualizing chaperone-mediated multistep assembly of the human 20S proteasome

Abstract

Dedicated assembly factors orchestrate the stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here we report cryo-electron microscopy reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, as well as how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors and reveals conceptual principles underlying human proteasome biogenesis, thus providing an explanation for many previous biochemical and genetic observations.

ProEnd: A Comprehensive Database for Identifying HbYX Motif-Containing Proteins Across the Tree of Life

“…The most well-known and studied function of the HbYX motifs is its ability to bind to and activate the 20S proteasome. Known regulators such as PA200, PI31, and PACs, are capable of activating, inhibiting, and assembling the proteasome, respectively 22,59,60 . To determine additional functional diversity associated with the HbYX motif identified by our approach we performed a gene enrichment analysis 61 in model gnathostomata organisms.…”