1. The Ufd1 cofactor determines the linkage specificity of polyubiquitin chain engagement by the AAA+ ATPase Cdc48
- Author
-
Williams, Cameron, Dong, Ken C, Arkinson, Connor, and Martin, Andreas
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Polyubiquitin ,Saccharomyces cerevisiae Proteins ,ATPases Associated with Diverse Cellular Activities ,Valosin Containing Protein ,Vesicular Transport Proteins ,Nucleocytoplasmic Transport Proteins ,Ubiquitin ,Ubiquitins ,Cell Cycle Proteins ,initiator unfolding ,Initiator pore insertion ,Proximal Ub processing ,Distal Ub processing ,L and substrate release ,AAA+ ATPase ,Cdc48/p97 ,Ufd1/Npl4 ,linkage specificity ,ubiquitin-dependent protein unfolding ,ubiquitin-proteasome system ,unfolding initiation ,Medical and Health Sciences ,Developmental Biology ,Biological sciences ,Biomedical and clinical sciences ,Health sciences - Abstract
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.
- Published
- 2023