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bioRxiv · 10.64898/2026.03.07.710261

ATP-independent unfolding of ubiquitin by Ufd1 initiates Cdc48/p97-mediated substrate processing

Wang, Y.·Zhang, Z.·He, W.·Wang, P.·Ji, Z.
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Abstract

The Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactors Ufd1 and Npl4 to extract polyubiquitinated proteins from membranes or multi-subunit complexes, promoting their proteasomal degradation. A ubiquitin molecule in the chain is unfolded in an ATP-independent manner and initiates substrate translocation through the central pore of the ATPase. How ubiquitin is unfolded remains unclear. Here, we demonstrate that the UT3 domain of Ufd1 interacts simultaneously with two Lys48-linked ubiquitins and unfolds one of the ubiquitins by binding its C-termianl {beta}-strand into a cleft. This unfolded ubiquitin is subsequently captured by Npl4 and Cdc48/p97. Experiments in vitro and in cells show that unfolding-defective mutants of Ufd1 indeed compromise Cdc48/p97 function. Our results explain how simple protein-protein interactions cause the unfolding of the remarkably stable ubiquitin. We also establish a linkage-specific recognition that involves ubiquitin unfolding.

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Wang, Y., Zhang, Z., He, W., Wang, P., Ji, Z.. 2026-03-07. ATP-independent unfolding of ubiquitin by Ufd1 initiates Cdc48/p97-mediated substrate processing. https://doi.org/10.64898/2026.03.07.710261

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