Abstract
The abundant homohexameric AAA + ATPase p97 (also known as valosin-containing protein, VCP) is highly conserved from Dictyostelium discoideum to human and a pivotal factor of cellular protein homeostasis as it catalyzes the unfolding of proteins. Owing to its fundamental function in protein quality control pathways, it is regulated by more than 30 cofactors, including the UBXD protein family, whose members all carry an Ubiquitin Regulatory X (UBX) domain that enables binding to p97. One member of this latter protein family is the largely uncharacterized UBX domain containing protein 9 (UBXD9). Here, we analyzed protein-protein interactions of D. discoideum UBXD9 with p97 using a series of N- and C-terminal truncation constructs and probed the UBXD9 interactome in D. discoideum. Pull-down assays revealed that the UBX domain (amino acids 384–466) is necessary and sufficient for p97 interactions and that the N-terminal extension of the UBX domain, which folds into a β0-α–1-α0 lariat structure, is required for the dissociation of p97 hexamers. Functionally, this finding is reflected by strongly reduced ATPase activity of p97 upon addition of full length UBXD9 or UBXD9261–573. Results from Blue Native PAGE as well as structural model prediction suggest that hexamers of UBXD9 or UBXD9261–573 interact with p97 hexamers and disrupt the p97 subunit interactions via insertion of a helical lariat structure, presumably by destabilizing the p97 D1:D1’ intermolecular interface. We thus propose that UBXD9 regulates p97 activity in vivo by shifting the quaternary structure equilibrium from hexamers to monomers. Using three independent approaches, we further identified novel interaction partners of UBXD9, including glutamine synthetase type III as well as several actin-binding proteins. These findings suggest a role of UBXD9 in the organization of the actin cytoskeleton, and are in line with the hypothesized oligomerization-dependent mechanism of p97 regulation.
Highlights
The AAA + (ATPase Associated with diverse cellular Activities) ATPase p97, known as valosin-containing protein (VCP), is a very abundant protein and evolutionarily highly conserved
UBX domain containing protein 9 (UBXD9) is a member of the ubiquitin X domain (UBXD) protein family, which makes up the largest subgroup of p97 cofactors
The UBXD9 domain structure is highly conserved across different eukaryotes and is composed of the N-terminal ubiquitin-like (UBL1) and the low homology Ubiquitin Regulatory X (UBX) (LHU) domains followed by a coiled coil domain in the middle part
Summary
The AAA + (ATPase Associated with diverse cellular Activities) ATPase p97, known as valosin-containing protein (VCP), is a very abundant protein and evolutionarily highly conserved. It was first described in 1982 and has since emerged as a fundamental player in a plethora of cellular processes and signaling pathways (Moir et al, 1982; Madsen et al, 2009; van den Boom and Meyer, 2018). The protein has a tripartite structure comprising a globular N domain followed by the D1 and D2 domains that bind and hydrolyze ATP (Figure 1A; Wendler et al, 2012). Since cancer cells have a heightened dependence on mechanisms of protein homeostasis, p97 is a promising target for anti-cancer therapy (Deshaies, 2014)
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