Hexamerization of p97-VCP is promoted by ATP binding to the D1 domain and required for ATPase and biological activities

Abstract

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.

The AAA-ATPase p97-Ufd1-Npl4 is required for ERAD but not for spindle disassembly in Xenopus egg extracts

“…Thus, the E305Q mutation might impair hexamer formation and weaken the dominantnegative effect although dissociation-reassembly assays argue otherwise (Wang et al, 2003). We generated versions of Xenopus p97 with an intact D1 but inactivated D2 domain by introducing an E578Q point mutation and a double K524A-E578Q mutation (Kitami et al, 2006), resulting in the p97-Q and p97-AQ mutants, respectively.…”

The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes

“…S1 and S2) and is associated with diverse cellular activities involving ubiquitin-proteasome-mediated protein degradation (12,13). We found a G-to-A substitution mutation in a conserved Ser codon in the amino acid motif called D1, implicated in ATP binding and hexamerization (14) (Fig. 1B and SI Appendix, Fig.…”

HDAC6, at the crossroads between cytoskeleton and cell signaling by acetylation and ubiquitination

“…High-affinity binding of HDAC6 to ubiquitin was shown to hinder the recognition of cellular ubiquitinated proteins by other ubiquitin-binding factors and to subsequently delay their processing by the proteasome or USPs (Boyault et al, 2006). The HDAC6 partner p97/VCP is a chaperone involved in the control of a variety of cellular functions, many of them relying on its 'segregase' activity disassembling various complexes, including those containing ubiquitinated proteins (Wang et al, 2003;Romisch, 2005). p97/VCP, upon its binding to HDAC6, is able to extract HDAC6 bound to ubiquitinated proteins and therefore allows their further processing.…”