Bidirectional substrate shuttling between the 26S proteasome and the Cdc48 ATPase promotes protein degradation

Area of Science:

• Molecular Biology
• Protein Degradation
• Ubiquitin-Proteasome System

Background:

• Eukaryotic protein degradation primarily involves the 26S proteasome.
• Proteins lacking unstructured regions require unfolding by the Cdc48 ATPase (p97/VCP) complexed with Ufd1-Npl4 (UN) before proteasomal degradation.

Purpose of the Study:

• To reconstitute and analyze the Cdc48 ATPase-dependent degradation of well-folded model substrates by the 26S proteasome using purified yeast components.

Main Methods:

• Reconstitution of a minimal in vitro system for Cdc48-dependent proteasomal degradation.
• Utilized purified yeast 26S proteasome, Cdc48-UN complex, Rad23, Ubx5, and Shp1.
• Investigated the roles of cofactors in substrate binding and unfolding.

Main Results:

• A minimal system comprising the 26S proteasome, Cdc48-UN, Rad23, Ubx5, and Shp1 successfully mediated degradation of well-folded substrates.
• Rad23 and Ubx5 enhance polyubiquitin binding to the proteasome and Cdc48-UN, respectively, facilitating substrate competition.
• Shp1 was identified as a stimulator of protein unfolding by the Cdc48-UN complex.

Conclusions:

• Established a minimal reconstituted system for studying Cdc48-dependent protein degradation.
• Demonstrated the cooperative roles of Rad23, Ubx5, and Shp1 in facilitating the degradation of challenging protein substrates.
• Confirmed bidirectional substrate shuttling between the 26S proteasome and Cdc48 in vivo, highlighting a dynamic degradation pathway.