Abstract:

The proteasome mediates most selective protein degradation. Proteolysis occurs within the 20S core particle (CP), a barrel-shaped chamber with an $\alpha$7$\beta$7$\beta$7$\alpha$7 configuration. CP biogenesis proceeds through an ordered multistep pathway requiring five chaperones, Pba1–4 and Ump1. Using Saccharomyces cerevisiae, we report high-resolution structures of CP assembly intermediates by cryogenic-electron microscopy. The first structure corresponds to the 13S particle, which consists of a complete $\alpha$-ring, partial $\beta$-ring ($\beta$2–4), Ump1 and Pba1/2. The second structure contains two additional subunits ($\beta$5–6) and represents a later pre-15S intermediate. These structures reveal the architecture and positions of Ump1 and $\beta$2/$\beta$5 propeptides, with important implications for their functions. Unexpectedly, Pba1's N terminus extends through an open CP pore, accessing the CP interior to contact Ump1 and the $\beta$5 propeptide. These results reveal how the coordinated activity of Ump1, Pba1 and the active site propeptides orchestrate key aspects of CP assembly.