Abstact

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 beta7-subunit but containing assembly chaperones Ump1 and Pba1-Pba2. Incorporation of beta7 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 beta-subunits become structured with each additionally incorporated beta-subunit, and how this prepares peptidase sites for auto-activation. We identify two transient interactions of Pba1 with the alpha-ring, which are important for an ordered progression of maturation. Pba1 loop 81-117 intercalates between subunits alpha3 and alpha4 in 13S-15S-PCs and is displaced upon 15S-PC dimerisation. The second interaction involves the alpha1 N-terminus, deletion of which leads to a defect in Pba1-Pba2 release. These findings indicate how changes in alpha-ring subunit conformations coordinate CP maturation with Pba1‑Pba2 release.