The 26S proteasome is a 2.5-MDa multisubunit protease complex that degrades polyubiquitylated proteins. Although its functions and structure have been extensively characterized, little is known about its dynamics in living cells. Here, we investigate the absolute concentration, spatio-temporal dynamics and complex formation of the proteasome in living cells using fluorescence correlation spectroscopy. We find that the 26S proteasome complex is highly mobile, and that almost all proteasome subunits throughout the cell are stably incorporated into 26S proteasomes. The interaction between 19S and 20S particles is stable even in an importin-a mutant, suggesting that the 26S proteasome is assembled in the cytoplasm. Furthermore, a genetically stabilized 26S proteasome mutant is able to enter the nucleus. These results suggest that the 26S proteasome completes its assembly process in the cytoplasm and translocates into the nucleus through the nuclear pore complex as a holoenzyme.

Background: Hsm3 is a proteasome-dedicated chaperone that forms a base precursor, Hsm3-Rpt1-Rpt2-Rpn1. Results: Crystal structures of Hsm3 and Hsm3-Rpt1 were determined. Conclusion: The Hsm3-Rpt1 interface is formed by a hydrophobic core and complementary charged interactions and is important for the proteasome assembly. Significance: The study provides the structural basis for the assembly mechanism of the base subcomplex of the 26 S proteasome.