Abstract
The ubiquitin-directed AAA-ATPase VCP/p97 facilitates degradation of damaged or misfolded proteins in diverse cellular stress response pathways. Resolving the complexity of its interactions with partner and substrate proteins and understanding its links to stress signaling is therefore a major challenge. Here, we used affinity-purification SWATH mass spectrometry (AP-SWATH) to identify proteins that specifically interact with the substrate-trapping mutant, p97-E578Q. AP-SWATH identified differential interactions over a large detection range from abundant p97 cofactors to pathway-specific partners and individual ligases such as RNF185 and MUL1 that were trapped in p97-E578Q complexes. In addition, we identified various substrate proteins and candidates including the PP1 regulator CReP/PPP1R15B that dephosphorylates eIF2α and thus counteracts attenuation of translation by stress-kinases. We provide evidence that p97 with its Ufd1-Npl4 adapter ensures rapid constitutive turnover and balanced levels of CReP in unperturbed cells. Moreover, we show that p97-mediated degradation, together with a reduction in CReP synthesis, is essential for timely stress-induced reduction of CReP levels and, consequently, for robust eIF2α phosphorylation to enforce the stress response. Thus, our results demonstrate that p97 not only facilitates bulk degradation of misfolded proteins upon stress, but also directly modulates the integrated stress response at the level of signaling.
Highlights
Key elements of the response to cellular stresses that threaten protein homeostasis include the induction of protein folding factors such as chaperones, the efficient clearance of terminally misfolded proteins and the attenuation of protein synthesis [1]
We aimed to “freeze” p97-mediated unfolding reactions and entangle substrates as well as associated cofactors and regulators, whose identification could reveal previously unidentified links of p97 to relevant signaling pathways. We combined this approach with SWATH-MS analysis [43], a data independent acquisition method that deterministically records fragment ion spectra for all detectable peptide precursors which are later analyzed in a targeted fashion with reference to spectral libraries where the mass spectrometric and chromatographic coordinates for peptides of interest have been established
SWATH-MS combines the advantages of data dependent acquisition with those of selected reaction monitoring and we have previously established the advantages of this method for quantitative analysis of affinity purification samples [30]
Summary
Key elements of the response to cellular stresses that threaten protein homeostasis include the induction of protein folding factors such as chaperones, the efficient clearance of terminally misfolded proteins and the attenuation of protein synthesis [1]. The AAAϩ-type ATPase Valosin-containing protein (VCP)/p97 ( called Cdc or Ter94) is a critical bottleneck in these processes [10, 11] It helps mobilize and unfold large cohorts of ubiquitin-modified substrates to facilitate their degradation in the proteasome in diverse pathways and compartments ranging from ER-associated degradation (ERAD), ribosomal quality control, mitochondrial stress response to macroautophagy and degradation of chromatin-associated proteins [11,12,13,14]. They include more than thirty cofactor proteins that directly interact with the regulatory N-domain or the C-terminal tail of p97 through dedicated interaction domains, and assist p97 as ubiquitin adapters, targeting factors or regulators [16, 22] It is often unclear if and how binding of accessory factors in diverse pathways is linked to substrate unfolding. P97 targets large cohorts of misfolded proteins in established stress pathways, only few regulatory degradation substrates are known so far, and it is unclear if and how p97 governs stress signaling by directly targeting stress regulators
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