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Protein unfolding, one substrate at a time Ubiquitin marks proteins for degradation by the proteasome. However, many substrates cannot be directly degraded because they are well folded or are located in cell membranes or in multimeric complexes. These proteins are first unfolded by the Cdc48 adenosine triphosphatase (ATPase), which forms a hexameric assembly that pulls polypeptides through its central pore. Twomey et al. determined structures of Cdc48 at an initiation stage of substrate processing. Surprisingly, a ubiquitin molecule in the substrate-linked polyubiquitin chain could be unfolded simply by binding to the Cdc48 complex. A segment of the unfolded ubiquitin inserts into the ATPase ring and initiates substrate unfolding. This explains why Cdc48 can deal with a broad range of substrates—even ones that are folded. Cooney et al. report the cryo–electron microscopy structure of Cdc48 in complex with an authentic substrate. In contrast to previously reported Cdc48 structures, an asymmetric spiraling assembly wraps around the extended substrate polypeptide. Thus, Cdc48 uses a hand-over-hand mechanism of translocation, which supports a common mechanism for protein substrate unfolding for AAA+ ATPases. Science, this issue p. eaax1033, p. 502 The Cdc48 ATPase processes polyubiquitinated substrates by unfolding and translocating an attached ubiquitin molecule. INTRODUCTION Many cellular proteins are polyubiquitinated but cannot be directly degraded by the proteasome because they are well folded, associated with binding partners, or located in membranes. These proteins are first unfolded by the Cdc48 adenosine triphosphatase (ATPase) in yeast or its orthologs p97 or valosin-containing protein in higher organisms. Mutations in p97 can cause neurodegenerative diseases. Cdc48/p97 belongs to the AAA+ (ATPases associated with diverse cellular activities) family. It contains an N-terminal domain and two tandem ATPase domains (D1 and D2). Six monomers form a double-ring structure, with a central pore. Cdc48 often collaborates with the heterodimeric cofactor Ufd1/Npl4. Substrate initially binds through the attached polyubiquitin chain to Ufd1/Npl4 and then moves through the pore of the ATPase rings and is thereby unfolded. This translocation process requires adenosine 5′-triphosphate (ATP) hydrolysis by the D2 domains and involves their pore loop residues. RATIONALE The mechanism of substrate processing by Cdc48 is poorly understood. For example, it is unknown how Cdc48 can deal with a broad range of even well-folded substrates, the only requirement being an attached polyubiquitin chain. Specifically, it remains unclear how a segment of a folded substrate can pass through the D1 ring to contact the D2 pore loops that power translocation. How these D2 subunits then translocate the substrate is also not known. Structures of related hexameric ATPases indicate a spiral-staircase arrangement of pore loops around the substrate, but structures of the Cdc48 ATPase engaged with a polyubiquitinated substrate have not yet been determined. RESULTS We here report cryo–electron microscopy (cryo-EM) structures of the substrate-engaged Cdc48 ATPase complex. The complexes were assembled with purified Cdc48, Ufd1/Npl4 cofactor, and polyubiquitinated substrate. They represent initiation states of substrate processing, captured by the use of a slowly ATP-hydrolyzing Cdc48 mutant or by addition of adenosine diphosphate and beryllium fluoride (ADP-BeFx). The structures show two folded ubiquitin molecules bound to Npl4 located on top of Cdc48’s D1 ring. Hydrogen-deuterium exchange experiments indicate additional ubiquitin-binding sites on Ufd1. Surprisingly, one ubiquitin molecule is unfolded and bound to a groove of Npl4, which contains conserved amino acids required for substrate unfolding. Unfolding of ubiquitin is remarkable, given that it is an extremely stable protein that can survive boiling. The unfolded ubiquitin molecule projects its N-terminal segment through the D1 ATPase ring and engages the pore loops of the D2 ATPases. These pore loops form a staircase that acts as a “conveyer belt” to move the polypeptide through the central pore. Photo–cross-linking experiments show that the insertion of ubiquitin’s N terminus across the ATPase rings can occur without ATP binding or hydrolysis. CONCLUSION Our results explain why the Cdc48 ATPase can act on a broad range of even well-folded proteins: It uses ubiquitin binding and unfolding to initiate substrate processing. Cdc48 first pulls on the N terminus of the unfolded ubiquitin molecule. The structure implies that if substrate is directly attached to the unfolded ubiquitin, it will next translocate through the central pore; otherwise, Cdc48 has to successively unfold the intervening ubiquitin molecules until it reaches and unfolds the substrate. Initiation of substrate processing by the Cdc48 ATPase complex. (A) Cryo-EM map of the substrate-engaged Cdc48/Ufd1/Npl4 complex. (B) The N terminus of an unfolded ubiquitin molecule engages pore loops of Cdc48’s second ATPase ring, arranged as a staircase. (C) Model of substrate processing. Some folded ubiquitins (purple) interact with Npl4 and Ufd1. Translocation starts with the N terminus of unfolded ubiquitin (red). Cdc48 pulls on ubiquitin molecules (pink), until it reaches and unfolds the substrate (green). The Cdc48 adenosine triphosphatase (ATPase) (p97 or valosin-containing protein in mammals) and its cofactor Ufd1/Npl4 extract polyubiquitinated proteins from membranes or macromolecular complexes for subsequent degradation by the proteasome. How Cdc48 processes its diverse and often well-folded substrates is unclear. Here, we report cryo–electron microscopy structures of the Cdc48 ATPase in complex with Ufd1/Npl4 and polyubiquitinated substrate. The structures show that the Cdc48 complex initiates substrate processing by unfolding a ubiquitin molecule. The unfolded ubiquitin molecule binds to Npl4 and projects its N-terminal segment through both hexameric ATPase rings. Pore loops of the second ring form a staircase that acts as a conveyer belt to move the polypeptide through the central pore. Inducing the unfolding of ubiquitin allows the Cdc48 ATPase complex to process a broad range of substrates.