Structural studies elucidating the biogenesis pathway of 20S proteasomes in Saccharomyces cerevisiae

In eukaryotic cells, the ubiquitin-proteasome system (UPS) regulates the degradation of numerous proteins involved in essential cellular processes. Malfunctions in the UPS are associated with various human diseases, e.g. neurodegenerative disorders, cancer, and diabetes. Central to the UPS is the 26S proteasome, a ~2.5 MDa protease complex responsible for ubiquitin-dependent protein degradation. It comprises the 20S core particle (CP) and the 19S regulatory particle (RP). The 20S CP serves as the catalytic unit of the 26S proteasome and assembles from two identical half-proteasomes, each consisting of 14 distinct subunits. Its assembly needs to follow a tightly controlled, multi-step pathway involving five assembly chaperones. Biochemical and genetic studies shed light onto the eukaryotic 20S biogenesis pathway. However, the precise spatial and temporal order of events remains incompletely understood. This work provides a structural characterization of multiple 20S assembly intermediates from Saccharomyces cerevisiae via single-particle cryo-EM, focusing on the late stage of half-proteasome assembly and subsequent dimerization of two half-proteasomes. The obtained structures elucidate the temporal order of assembly steps and reveal roles for the chaperones Pba1, Pba2 and Ump1, as well as the β-subunits propeptides. During 20S biogenesis, Ump1 forms contacts with the incoming β-subunits, resulting in the gradual structuring of the chaperone. The β2-propeptide is indicated to function mainly during half-proteasome assembly and is cleaved off shortly afterwards. In contrast, the β1- and β5-propeptides act after dimerization. The β5-propeptide functions as an intramolecular chaperone, forming an interaction network with Ump1, β1, and β5-7. Together, Ump1 and the propeptides serve as assembly checkpoints by preventing the subunits from premature interlocking. Finally, the obtained data suggest a stepwise release mechanism for the Pba1-2 heterodimer upon 20S biogenesis, resulting in its subsequent release.