The 97-kDa valosin-containing protein (p97 or VCP) is a type-II AAA ( ATPases associated with a variety of activities) ATPases, which are characterized by possessing two conserved ATPase domains. VCP forms a stable homo-hexameric structure, and this two-tier ring-shaped complex acts as a molecular chaperone that mediates many seemingly unrelated cellular activities. The involvement of VCP in the ubiquitin-proteasome degradation pathway and the identification of VCP cofactors provided us important clues to the understanding of how this molecular chaperone works. In this review, we summarize the reported biological functions of VCP and explore the molecular mechanisms underlying the diverse cellular functions. We discuss the structural and biochemical studies, and elucidate how this sophisticated enzymatic machine converts chemical energy into the mechanical forces required for the chaperone activity.

The 97-kDa valosin-containing protein (p97-VCP) plays a role in a wide variety of cellular activities, many of which are regulated by the ubiquitin-proteasome (Ub-Pr)-mediated degradation pathway. We previously demonstrated that VCP binds to multi-ubiquitin chains and may act as a molecular chaperone that targets the ubiquitinated substrates to the proteasome for degradation. In this report, we show that although the ubiquitin chain-binding activity, carried out by the N-terminal 200 residues (N domain), is necessary for the degradation of proteasome substrates, it is not sufficient. Using in vitro degradation assays, we demonstrated that the entire VCP molecule, consisting of the N domain and two ATPase domains D1 and D2, is required for mediating the Ub-Pr degradation. The ATPase activity of VCP requires Mg 2؉ , and is stimulated by high temperature. Under optimal conditions, VCP hydrolyzes ATP with a K m of ϳ0.33 mM and a V max of ϳ0.52 nmol P i min ؊1 g ؊1 . At a physiological temperature, mutation in D2 significantly inhibits the ATPase activity, while that in D1 has little effect. Interestingly, mutations in D1, but not D2, abolish the heat-stimulated ATPase activity. Thus, we provide the first demonstration that the ATPase activity of VCP is required for mediating the Ub-Pr degradation, that D2 accounts for the major ATPase activity, and that D1 contributes to the heat-induced activity.

The 97-kDa valosin-containing protein (p97-VCP or VCP), a hexameric AAA ATPase, plays an important role in diverse cell activities, including ubiquitin-proteasome mediated protein degradation. In this report, we studied dissociation-reassembly kinetics to analyze the structure-function relationship in VCP. Urea-dissociated VCP can reassemble by itself, but addition of ATP, ADP, or ATP-gamma S accelerates the reassembly. Mutation in the ATP-binding site of D1, but not D2, domain abolishes the ATP acceleration effect and further delays the reassembly. Using hybrid hexamers of the wild type and ATP-binding site mutant, we show that hexameric structure and proper communication among the subunits are required for the ATPase activity and ubiquitin-proteasome mediated degradation. Thus, ATP-binding site in D1 plays a major role in VCP hexamerization, of which proper inter-subunit interaction is essential for the activities.