Visualizing chaperone-mediated multistep assembly of the human 20S proteasome

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• The proteasome core particle (CP) is a large molecular machine essential for protein degradation.
• Assembly of the 28-subunit CP requires dedicated assembly factors and chaperones.
• Understanding the assembly pathway is crucial for comprehending proteasome function and dysfunction.

Purpose of the Study:

• To visualize the structural intermediates of human proteasome core particle assembly.
• To elucidate the molecular mechanisms by which assembly factors facilitate proteasome biogenesis.
• To reveal how proteasome subunits and assembly factors adapt during assembly.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to reconstruct seven human proteasome subcomplexes.
• Recombinant subcomplexes representing various stages of the assembly pathway were analyzed.
• Structural comparisons were made between chaperone-bound intermediates and the mature proteasome core particle.

Main Results:

• Visualized all five chaperones and three active site propeptides across the assembly pathway.
• Identified molecular mechanisms governing the order of subunit addition.
• Demonstrated structural adaptations of subcomplexes and assembly factors during progressive subunit incorporation.
• Revealed how intermediates stabilize, facilitate subsequent steps, and rearrange for activation and gated access.

Conclusions:

• Established a method for structural analysis of multiprotein complex assembly intermediates.
• Illuminated specific functions of proteasome assembly factors.
• Provided conceptual principles underlying human proteasome biogenesis.
• Offered explanations for previous biochemical and genetic observations in proteasome assembly.