Cooperative assembly of p97 complexes involved in replication termination

Abstract

The p97 ATPase extracts polyubiquitylated proteins from diverse cellular structures in preparation for destruction by the proteasome. p97 functions with Ufd1-Npl4 and a variety of UBA-UBX co-factors, but how p97 complexes assemble on ubiquitylated substrates is unclear. To address this, we investigated how p97 disassembles the CMG helicase after it is ubiquitylated during replication termination. We show that p97Ufd1-Npl4 recruitment to CMG requires the UBA-UBX protein Ubxn7, and conversely, stable Ubxn7 binding to CMG requires p97Ufd1-Npl4. This cooperative assembly involves interactions between Ubxn7, p97, Ufd1-Npl4, and ubiquitin. Another p97 co-factor, Faf1, partially compensates for the loss of Ubxn7. Surprisingly, p97Ufd1-Npl4-Ubxn7 and p97Ufd1-Npl4-Faf1 also assemble cooperatively on unanchored ubiquitin chains. We propose that cooperative and substrate-independent recognition of ubiquitin chains allows p97 to recognize an unlimited number of polyubiquitylated proteins while avoiding the formation of partial, inactive complexes.

Bidirectional substrate shuttling between the 26S proteasome and the Cdc48 ATPase promotes protein degradation

“…Both bind to their respective partners through ubiquitin-like domains: Rad23 associates with the 26S proteasome through its UBL domain and Ubx5 with Cdc48 through its UBX domain. In both cases, the binding seems to be synergistic with polyubiquitinated substrate, which might be explained by avidity effects or by the relief of auto-inhibition caused by an intramolecular interaction between the UBA and ubiquitin-like domains (Kochenova et al, 2022; Ryu et al, 2003). Shp1 is not a recruitment factor, but seems to accelerate the rate of substrate unfolding, consistent with the mammalian homologs stimulating the ATPase activity of p97 (Zhang et al, 2015).…”

Expression and Localization of Fas-Associated Factor 1 in Testicular Tissues of Different Ages and Ovaries at Different Reproductive Cycle Phases of Bos grunniens

“…FAF1 contains several functional and structural domains, primarily N-terminal UBA, C-terminal UBX, death effector domain interacting domain(DEDID) and FAS-interacting domain(FID). UBA can recruit polyubiquitinated proteins essential for FAF1-mediated apoptosis and stress responses [6,11], thereby mediating apoptosis and stress responses.…”

“…UBX binds to the ubiquitin-proteasome system via a molecular chaperone-containing protein (valosin-containing protein, VCP) [11,12], the mammalian homolog of multifunctional Cdc48p in yeast and multifunctional p97 in Xenopus laevis [13], associated with various cellular activities. DEDID can be associated with Fas-associated protein with death domain (FADD) proteins and caspases [14].…”

Targeting of client proteins to the VCP/p97/Cdc48 unfolding machine

“…The third member, UBXN7 (Ubx5 in yeast), is localized exclusively in the nucleus and has been linked to various chromatin-associated functions of p97 ( Alexandru et al, 2008 ; Verma et al, 2011 ; Puumalainen et al, 2014 ; Chauhan et al, 2021 ). Both FAF1 and UBXN7 have been connected to the extraction of the replicative helicase from DNA ( Sonneville et al, 2017 ; Xia et al, 2021 ; Kochenova et al, 2022 ). The replicative helicase forms a ring consisting of the AAA proteins MCM2-7 and is assembled tightly around DNA during replication origin licensing.…”

“…Disassembly is triggered by ubiquitylation of MCM7 which is then targeted and extracted by p97 and Ufd1-Npl4 leading to destabilization of the whole complex. Crucially, UBXN7 assists p97-Ufd1-Npl4-mediated extraction of the helicase ( Sonneville et al, 2017 ; Xia et al, 2021 ; Kochenova et al, 2022 ). FAF1 can compensate for UBXN7, although this is controversial ( Fujisawa et al, 2022 ; Tarcan et al, 2022 ).…”