Direct observation of ATP-driven ubiquitin chain handling by Cdc48

Cdc48 (p97 or VCP in metazoans) targets polyubiquitin to selectively disassemble and degrade proteins. Cdc48 is believed to move along the ubiquitin chain towards linked proteins, but this has not been directly observed. By following single molecules in time, we find that the polypeptide branch points of ubiquitin chains are repeatedly inserted and rejected from the Cdc48 pore in ATP-driven manner, in bursts lasting up to seconds. This non-processive mode either ends by terminal substrate rejection, or advances to processive action, which drives ubiquitin unfolding in two steps, branch point translocation, and the extrusion of ubiquitin and linked protein as polypeptide loops in trans. Final retrograde movement can bring polypeptide segments back to cis. Our results establish the dynamics of ubiquitin chain handling by Cdc48, reveal key hallmarks of kinetic proofreading. We speculate that Cdc48 translocation may play a role in ubiquitin chain selection.