Structural transitions in the stepwise assembly of proteasome core particles.

Abstract

20S catalytic core particles (CP) of eukaryotic 26S proteasomes are composed of two identical halves comprising 14 distinct subunits. 15S precursor complexes (PC) represent detectable half-CPs assembly intermediates lacking the β7-subunit but containing assembly chaperones Ump1 and Pba1-Pba2. Incorporation of β7 drives 15S-PC dimerisation and further CP maturation. Our cryo-EM structures of the yeast 15S-PC and all 13S-PC-derived intermediates suggest that assembly in yeast is not restricted to a single trajectory, but instead involves alternative, and potentially simultaneous pathways. Comparison of the intermediates reveals how Ump1 and β-subunits become structured with each additionally incorporated β-subunit, and how this prepares peptidase sites for auto-activation. We identify two transient interactions of Pba1 with the α-ring, which are important for an ordered progression of maturation. Pba1 loop 81-117 intercalates between subunits α3 and α4 in 13S-15S-PCs and is displaced upon 15S-PC dimerisation. The second interaction involves the α1 N-terminus, deletion of which leads to a defect in Pba1-Pba2 release. These findings indicate how changes in α-ring subunit conformations coordinate CP maturation with Pba1‑Pba2 release.