Abstract
Many eukaryotic proteins are modified with a polyubiquitin chain and then recruited to either the Cdc48 ATPase (p97 or VCP in mammals) or the 26S proteasome by conserved cofactors. They can then shuttle between the Cdc48 ATPase and the 26S proteasome before being degraded. How substrates avoid being trapped on the Cdc48 ATPase complex is incompletely understood, as they can undergo repeated cycles of translocation through the ATPase pore. Here, we show that the deubiquitinating enzyme (DUB) Otu1 (Yod1 in mammals) can break this futile cycle. Otu1 trims the ubiquitin chain of the substrate before its translocation through the Cdc48 pore is initiated, allowing transfer to the proteasome and subsequent degradation. A cryo-EM structure shows that the mammalian homolog Yod1 binds to p97 simultaneously with other Cdc48/p97 cofactors. As in the yeast system, polypeptide translocation through the ATPase pore is initiated by the unfolding of a ubiquitin molecule, suggesting that the mechanism of substrate processing is conserved in all eukaryotes.
Data availability
Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB, https://www.ebi.ac.uk/pdbe/emdb/) under the accession codes EMD-73365 (composite map), EMD- 73375 (globally refined map), and EMD-73376 (map locally refined on p97 D1 and Npl4). The atomic model has been deposited in the Protein Data Bank under the accession codes PDB 9YRC.
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Acknowledgements
We thank R. Yan at the HHMI Janelia Research Campus cryo-EM facility for assistance with microscope operation and data collection, Z. Li, R. Walsh, C. Leistner, M. Mayer, R. Nair, and S. Rawson at the Harvard cryo-EM Center for Structural Biology for assistance with sample preparation, microscope operation, data collection, and image processing, the SBGrid team for software and workstation support. This work was supported by a NIGMS grant (R01 GM052586) to T.A.R. T.A.R. is a Howard Hughes Medical Institute Investigator.
Funding
This work was supported by a NIGMS grant (R01 GM052586) to T.A.R. T.A.R. is a Howard Hughes Medical Institute Investigator. This manuscript is the result of funding in whole or in part by the National Institutes of Health (NIH). It is subject to the NIH Public Access Policy. Through acceptance of this federal funding, NIH has been given a right to make this manuscript publicly available in PubMed Central upon the Official Date of Publication, as defined by NIH.
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H.L. performed all experiments, H.G. helped with cryo-EM sample preparation and data processing, T.A.R. supervised the project. H.L. and T.A.R. wrote the manuscript.
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Li, H., Guan, H. & Rapoport, T.A. The deubiquitinating enzyme Otu1 releases substrates from the conserved initiation complex of the Cdc48/p97 ATPase for proteasomal degradation. Sci Rep (2026). https://doi.org/10.1038/s41598-026-42811-6
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DOI: https://doi.org/10.1038/s41598-026-42811-6
