Cooperative assembly of p97 complexes involved in replication termination

Area of Science:

• Molecular and Cellular Biology
• Protein Degradation Pathways
• Ubiquitin-Proteasome System

Background:

• The p97 ATPase is crucial for extracting ubiquitylated proteins for proteasomal degradation.
• p97 functions with co-factors like Ufd1-Npl4, but substrate complex assembly remains poorly understood.
• Understanding p97 complex assembly is key to elucidating its role in cellular protein homeostasis.

Purpose of the Study:

• To investigate the assembly mechanism of p97 complexes on ubiquitylated substrates.
• To determine the role of UBA-UBX co-factors in p97 recruitment and function.
• To understand how p97 disassembles the CMG helicase during replication termination.

Main Methods:

• Investigated p97 complex assembly on ubiquitylated CMG helicase.
• Utilized biochemical assays to study protein-protein interactions.
• Examined the roles of co-factors Ubxn7 and Faf1 in p97 complex formation.

Main Results:

• p97^Ufd1-Npl4 recruitment to CMG requires Ubxn7, and Ubxn7 binding requires p97^Ufd1-Npl4, indicating cooperative assembly.
• Cooperative assembly involves direct interactions between Ubxn7, p97, Ufd1-Npl4, and ubiquitin.
• Faf1 partially rescues Ubxn7 loss, and both Ubxn7- and Faf1-containing p97 complexes assemble cooperatively on ubiquitin chains.
• p97 complexes exhibit substrate-independent recognition of ubiquitin chains.

Conclusions:

• Cooperative assembly of p97 complexes with co-factors like Ubxn7 and Faf1 is essential for efficient substrate processing.
• Substrate-independent ubiquitin chain recognition enables p97 to target a broad range of ubiquitylated proteins.
• This mechanism prevents the formation of non-functional p97 complexes, ensuring efficient protein degradation.