Abstract
p97/Valosin-containing protein (VCP) is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD). It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell.
Highlights
Despite several structural studies, it remains unclear how the recruitment of the increasing number of various adaptor proteins is regulated [40]. This is exemplified by the two best characterized adaptor proteins, p47 and Ufd1/ Npl4, with the latter being a heterodimer of ubiquitin-fusion degradation protein 1 (Ufd1) and nuclear localization protein 4 (Npl4), and commonly referred to as UN
We focused on the effects of nucleotides, at physiological concentrations, on the binding of p47 and UN to the N-domain and the contribution of the proximal D1 domain to regulating the interactions of these adaptor proteins with p97/valosin-containing protein (VCP)
Together with the effect of ATP that enhanced binding to p97/ VCP of UN but not of p47, we propose that ATP binding to the D1 domain plays a role in regulating the exclusive recruitment of either UN or p47 to the p97/VCP proximal N-domain and in directing p97/VCP to either endoplasmic reticulum-associated degradation (ERAD) or homotypic fusion, respectively
Summary
P97, known as valosin-containing protein (VCP), is a member of the AAA (ATPase associated with various cellular activities) ATPase family. Being an AAA-ATPase, p97/VCP is implicated in multiple cellular processes, including molecular segregation, gene regulation, cell cycle regulation and spindle disassembly, homotypic membrane fusion, intracellular trafficking and protein quality control via ubiquitin-proteasome-mediated degradation [18,19,20,21,22] Of the multiple functions of p97/VCP, its involvement in the endoplasmic reticulum-associated protein degradation (ERAD) pathway is the best characterized [23,24,25]. Despite several structural studies, it remains unclear how the recruitment of the increasing number of various adaptor proteins is regulated [40] This is exemplified by the two best characterized adaptor proteins, p47 and Ufd1/ Npl, with the latter being a heterodimer of ubiquitin-fusion degradation protein 1 (Ufd1) and nuclear localization protein 4 (Npl4), and commonly referred to as UN. Our results assign a novel role to the D1 AAA-ATPase domain and link p97/VCP function to the ATP level and the metabolic status of the cell
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