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