(A) VCP protein structure. VCP assembles primarily as a homo-hexamer comprised of six monomers, each of which contains a globular N-domain and two ATPase domains (D1 and D2). Pathogenic mutations within each domain are indicated. (B) Directionality of substrate unfolding by Cdc48p. Cdc48p encounters a type I branch point when the initiator ubiquitin reaches the pore of the hexamer. Unfolding continues toward the C-terminus of the ubiquitin. At a type II branch point, Cdc48p can move toward the N- or C-terminus to eventually unfold the substrate. (C) Structure and substrate processing by VCP. VCP interacts with adaptor proteins to identify ubiquitylated substrates for degradation by the proteasome. Through interaction with p37, VCP can unfold substrates such as I3 independently of ubiquitylation. Phosphorylation of VCP by ULK1/2 increases the ATPase activity of VCP to enhance stress granule disassembly, while phosphorylation by PLK1 regulates VCP function in mitosis. Trimethylation of VCP monomers at Lys315 occurs by the action of METTL21D/C/E. The ASPL adaptor disassembles VCP hexamers to form ASPL–VCP hetero-tetramers (structure modeled in PyMOL using PDB 5IFS). Two hexamers that interact at the C-terminal tail provide an interface for the formation of dodecamers (structure modeled in PyMOL using PDB 7K57).