The Ufd1 cofactor determines the linkage specificity of polyubiquitin chain engagement by the AAA+ ATPase Cdc48

• Published in: Molecular cell 2023-03, Vol.83 (5), p.759-769.e7
• Main Authors: Williams, Cameron, Dong, Ken C., Arkinson, Connor, Martin, Andreas
• Format: Article
• Language: English
• Subjects: AAA+ ATPase; ATPases Associated with Diverse Cellular Activities - metabolism; Cdc48/p97; Cell Cycle Proteins - metabolism; linkage specificity; Nucleocytoplasmic Transport Proteins - metabolism; Polyubiquitin - metabolism; Saccharomyces cerevisiae Proteins - metabolism; Ubiquitin - metabolism; ubiquitin-dependent protein unfolding; ubiquitin-proteasome system; Ubiquitins - metabolism; Ufd1/Npl4; unfolding initiation; Valosin Containing Protein - metabolism; Vesicular Transport Proteins - metabolism
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2023.01.016

The AAA+ ATPase Cdc48 utilizes the cofactor Ufd1/Npl4 to bind and thread polyubiquitinated substrates for their extraction from complexes or membranes and often for subsequent proteasomal degradation. Previous studies indicated that Cdc48 engages polyubiquitin chains through the Npl4-mediated unfolding of an initiator ubiquitin; yet, the underlying principles remain largely unknown. Using FRET-based assays, we revealed the mechanisms and kinetics of ubiquitin unfolding, insertion into the ATPase, and unfolding of the ubiquitin-attached substrate. We found that Cdc48 uses Ufd1’s UT3 domain to bind a K48-linked ubiquitin on the initiator’s proximal side of the chain, thereby directing the initiator toward rapid unfolding by Npl4 and engagement by Cdc48. Ubiquitins on the initiator’s distal side increase substrate affinity and facilitate unfolding but impede substrate release from Cdc48-Ufd1/Npl4 in the absence of additional cofactors. Our findings explain how Cdc48-UN efficiently processes substrates with K48-linked chains of 4–6 ubiquitins, which represent most cellular polyubiquitinated proteins. [Display omitted] •Cdc48-Ufd1/Npl4 engages substrates through rapid unfolding of an initiator ubiquitin•Ufd1’s UT3 domain is a linkage-specific sensor for an initiator-proximal ubiquitin•Chains of 4–6 ubiquitins allow robust engagement through additional Npl4 interactions•Processing of initiator-distal ubiquitins is rate limiting for substrate release The AAA+ ATPase Cdc48/p97 mobilizes polyubiquitinated proteins from macromolecular assemblies or membranes for their proteasomal degradation. Using fluorescence-based assays, Williams et al. show how Cdc48’s Ufd1/Npl4 cofactor is utilized for engaging substrate-attached polyubiquitin chains in a length-, linkage-, and position-specific manner for mechanical substrate processing by the ATPase motor.