Activity-Guided Proteomic Profiling of Proteasomes Uncovers a Variety of Active (And Inactive) Proteasome species

Proteasomes are multi-subunit, multi-catalytic protein complexes present in eukaryotic cells that degrade misfolded, damaged, or unstructured proteins. In this study, we used an activity-guided proteomic methodology based on fluorogenic peptide substrates to characterize the composition of proteasome complexes in WT yeast, and the changes they undergo upon deletion of Pre9 (Δα3; one of the 20S subunits) or of Sem1 (ΔSem1; one of the 19S subunits).A comparison of whole-cell proteomics to the activity-guided proteasome proteomics indicates that the amounts of proteasomal proteins and proteasome interacting proteins (PIPs) in the assembled active proteasomes differ significantly from their total amounts in the cell. Using this activity-guided approach, we characterized the changes in the abundance of subunits in various active proteasome species in the different strains, accurately quantified the relative abundance of active proteasomes across the strains, and charted the overall distribution of different proteasome species within each strain. The distributions obtained by our MS-based quantification were markedly higher for some proteasome species than those obtained just by activity-based quantification, suggesting that the activity of some of these species is impaired. The impaired activity appeared mostly among 20SBlm10proteasome species that account for 20% of the active proteasomes in WT.To define the factors behind this impaired activity, we mapped and quantified known proteasome-interacting proteins. Our results suggested that some of the reduced activity might be due to the association of the proteasome inhibitor Fub1. Additionally, we provide evidence for the presence of non-mature and therefore inactive proteasome protease subunits β2 and β5 in the fully assembled proteasomes.