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 a fluorogenic peptide substrate to characterize the composition of proteasome complexes in WT yeast, and the changes these complexes undergo upon the deletion of Pre9 (Δα3) or of Sem1 (ΔSem1). A comparison of whole-cell proteomic analysis to activity-guided proteasome profiling indicates that the amounts of proteasomal proteins and proteasome interacting proteins in the assembled active proteasomes differ significantly from their total amounts in the cell as a whole. Using this activity-guided approach, we characterized the changes in the abundance of subunits of various active proteasome species in different strains, quantified the relative abundance of active proteasomes across these strains, and charted the overall distribution of different proteasome species within each strain. The distributions obtained by our mass spectrometry-based quantification were markedly higher for some proteasome species than those obtained by activity-based quantification alone, suggesting that the activity of some of these species is impaired. The impaired activity appeared mostly among 20S Blm10 proteasome species which account for 20% of the active proteasomes in WT. To identify 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 novel evidence for the presence of non-mature and therefore inactive proteasome protease subunits β2 and β5 in the fully assembled proteasomes. Significance Statement Proteasomes, essential protein complexes in eukaryotic cells, degrade misfolded, damaged, or unstructured proteins. Here we present an activity-guided proteomic method to characterize the composition and abundance of proteasomes. When applied to yeast proteasomes, this method revealed discrepancies between proteasome distributions determined by mass spectrometry and peptidase activity. This implies that a substantial portion of the proteasomes may exhibit reduced activity. Our findings indicate that these changes in proteasome activity could be linked to proteasome inhibition by Fub1. Furthermore, we identified signature peptides that indicate incomplete maturation of some of the β2 and β5 proteolytic subunits in fully assembled proteasomes, suggesting that proteasome core particle assembly can proceed even without the complete maturation of all β subunits.