Deubiquitinase activity is required for the proteasomal degradation of misfolded cytosolic proteins upon heat-stress

Abstract

Elimination of misfolded proteins is crucial for proteostasis and to prevent proteinopathies. Nedd4/Rsp5 emerged as a major E3-ligase involved in multiple quality control pathways that target misfolded plasma membrane proteins, aggregated polypeptides and cytosolic heat-induced misfolded proteins for degradation. It remained unclear how in one case cytosolic heat-induced Rsp5 substrates are destined for proteasomal degradation, whereas other Rsp5 quality control substrates are otherwise directed to lysosomal degradation. Here we find that Ubp2 and Ubp3 deubiquitinases are required for the proteasomal degradation of cytosolic misfolded proteins targeted by Rsp5 after heat-shock (HS). The two deubiquitinases associate more with Rsp5 upon heat-stress to prevent the assembly of K63-linked ubiquitin on Rsp5 heat-induced substrates. This activity was required to promote the K48-mediated proteasomal degradation of Rsp5 HS-induced substrates. Our results indicate that ubiquitin chain editing is key to the cytosolic protein quality control under stress conditions.

The Degradation-promoting Roles of Deubiquitinases Ubp6 and Ubp3 in Cytosolic and ER Protein Quality Control

“…While Ubp3 had been known to support CytoQC under heat stress (Fang et al, 2016), our genetic screen further revealed that it supports both CytoQC and ERQC at the physiological temperature (30℃) ( Figure 1A and B). We proceeded to investigate Ubp3's functions in CytoQC, ERQC and the turnover of folded proteins.…”

“…Although DUbs function in a variety of cellular activities, little is known about the spectrum of DUbs involved in QC or the exact roles of a few DUbs implicated in QC pathways, such as Ubp3 and Ubp6. Ubp3 supports CytoQC under heat stress by suppressing the conjugation of lysine 63 (K63)-linked ubiquitin chains on misfolded proteins and facilitating K48linkage (Fang et al, 2014;Fang et al, 2016;Silva et al, 2015) but its function under the physiological temperature or in other QC pathways is unknown (Oling et al, 2014). Ubp6 was proposed to delay QC because deleting UBP6 reduced the steady-state abundance of some proteins (Boselli et al, 2017;Nielsen et al, 2014;Torres et al, 2010).…”

Precise replacement ofSaccharomyces cerevisiaeproteasome genes with human orthologs by an integrative targeting method

“…High temperature imposes proteotoxic stress on cells in the form of misfolded proteins and survival of this stress requires the ubiquitin-proteasome system [52][53][54][55][56] . As previously reported, pre9Δ yeast were very sensitive to high temperature 57 (Figure 5A).…”

“…High temperature has wide-ranging effects on cells, including changes in membrane composition 64,65 , and the induction of multiple transcriptional and metabolic pathways 66,67 . Ubiquitin ligases and deubiquitinases of the ubiquitin-proteasome system are required for the degradation of proteins that misfold as a result of high temperature 55,56 . With the exception of the non-essential α3 subunit Pre9 57 , the roles of the proteasome core subunits in surviving high temperature have not been deeply investigated, in part because they are essential.…”

Targeting HECT‐type E3 ligases – insights from catalysis, regulation and inhibitors

“…Alternatively, a deubiquitinating (DUB) enzyme may team up with the HECT enzyme to edit substrates, as was shown for the yeast orthologue of the NEDD4 family, Rsp5 that mediates cytosolic protein degradation under stress conditions . Upon heat stress, Ubp2 and Ubp3 deubiquitinases associate more with Rsp5 to prevent the assembly of K63‐linked ubiquitin chains on Rsp5 substrates . K63‐modified substrates might also be recognized by other E3 ligases – yet to be discovered – that subsequently modify the chains, like in the case of SUMO‐targeted ubiquitin ligase (STUbL) family proteins .…”