Proteasome Assembly and the Formation of Non‐canonical Complexes in Vivo

Abstract

The 26S proteasome is the heart of the ubiquitin‐proteasome system (UPS) in eukaryotes with a central role in the degradation of proteins and the maintenance of protein homeostasis. The 26S proteasome is comprised of a compartmentalized protease, the 20S proteasome or core particle (CP), whose activity is modulated by its interaction with one or more copies of the 19S regulatory particle (RP). The quaternary structure of the CP, found in all domains of life, is absolutely conserved and consists of four heptameric rings stacked coaxially. The rings are made of structurally related α and β subunits. In eukaryotes, assembly factors chaperone the seven different α and seven different β subunits to form a structure with α(1‐7)β(1‐7) β(1‐7) α(1‐7) stoichiometry. Here we demonstrate that CP subunits can assemble into structures other than the canonical CP in vivo. Specifically, the yeast α4 subunit forms high molecular weight complexes (α4 HMWCs) whose abundance increases when proteasome function is compromised. Results from a disulfide crosslinking approach are consistent with these complexes being ring‐shaped. Though several eukaryotic α subunits can form rings when expressed recombinantly in bacteria, this is the first evidence that such non‐canonical complexes exist in vivo. Our results place the α4 subunit at an important crossroads in the assembly of the CP. In addition to being present in the canonical CP, and these newly discovered α4 HMWCs, the α4 subunit also assembles into an alternate CP isoform called an α4–α4 proteasome, whose formation is evolutionarily conserved from yeast to humans. How α4 is distributed among these 3 possible complexes in vivo is not known. We present evidence suggesting that relative levels of the Rpn4 transcription factor might determine the extent to which α4 populates assembly pathways that lead to the formation of α4 HMWCs and α4–α4 proteasomes.Support or Funding InformationThis work was supported in part by a Research Support Funds Grant from Indiana University‐Purdue University Indianapolis (to A.R.K.).