dictyNews
Electronic Edition
Volume 47, number 18
September 10, 2021

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Abstracts
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Domain organization of the UBX domain containing protein 9 (UBXD9) 
and analysis of its interactions with the homohexameric AAA+ ATPase 
p97 (VCP)

Jana Riehl, Ramesh Rijal, Leonie Nitz, Christoph S. Clemen, Andreas 
Hofmann, and Ludwig Eichinger


Frontiers in Cell and Developmental Biology, accepted

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 constructand 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 beta 0, alpha -1, 
alpha 0 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 UBXD9 (261-573). Results from 
Blue Native PAGE as well as structural model prediction suggest that 
hexamers of UBXD9 or UBXD9 (261-573) 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.


submitted by:  Ludwig Eichinger [[log in to unmask]]
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[End dictyNews, volume 47, number 18]