Molecular Mechanism of Substrate Processing by the Cdc48 ATPase Complex

Area of Science:

• Protein biochemistry
• Molecular cell biology
• Enzymology

Background:

• Cdc48 ATPase and Ufd1/Npl4 (UN) are crucial for extracting polyubiquitinated proteins from cellular structures.
• The precise mechanism of substrate processing by the Cdc48 complex remained largely undefined.

Purpose of the Study:

• To elucidate the step-by-step mechanism by which the Cdc48-UN complex processes polyubiquitinated protein substrates.
• To understand the distinct roles of the D1 and D2 ATPase rings in substrate unfolding and release.

Main Methods:

• Biochemical assays using purified Cdc48, Ufd1/Npl4 (UN) cofactors, and polyubiquitinated protein substrates.
• Analysis of ATP hydrolysis-dependent substrate translocation and unfolding within the Cdc48 double-ring structure.

Main Results:

• The D2 ring's ATP hydrolysis drives substrate polypeptide translocation through the central pore, inducing unfolding.
• The D1 ring's ATP hydrolysis is essential for the subsequent release of the substrate from the complex.
• Substrate release necessitates collaboration with a deubiquitinase to trim polyubiquitin chains for translocation.

Conclusions:

• A novel model for Cdc48-mediated protein extraction is proposed, involving sequential ATP hydrolysis events in distinct rings.
• The findings highlight the coordinated action of Cdc48, its cofactors, and deubiquitinases in protein processing and degradation pathways.
• This mechanism provides insights into the function of Cdc48 and its mammalian homolog, p97/VCP.