EDEM1 Drives Misfolded Protein Degradation via ERAD and Exploits ER-Phagy as Back-Up Mechanism When ERAD Is Impaired

Abstract

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the main mechanism of targeting ER proteins for degradation to maintain homeostasis, and perturbations of ERAD lead to pathological conditions. ER-degradation enhancing α-mannosidase-like (EDEM1) was proposed to extract terminally misfolded proteins from the calnexin folding cycle and target them for degradation by ERAD. Here, using mass-spectrometry and biochemical methods, we show that EDEM1 is found in auto-regulatory complexes with ERAD components. Moreover, the N-terminal disordered region of EDEM1 mediates protein–protein interaction with misfolded proteins, whilst the absence of this domain significantly impairs their degradation. We also determined that overexpression of EDEM1 can induce degradation, even when proteasomal activity is severely impaired, by promoting the formation of aggregates, which can be further degraded by autophagy. Therefore, we propose that EDEM1 maintains ER homeostasis and mediates ERAD client degradation via autophagy when either dislocation or proteasomal degradation are impaired.

EDEM3 Domains Cooperate to Perform Its Overall Cell Functioning

“…We compared these clusters with those found for EDEM1 pulled–down in the same lysis detergent, displaying similar pull-down specificity towards polyclonal antibodies ( Figure 1 B) and comparable spectral counts with EDEM3 ( Appendix A Table A1 ). As shown in Figure 1 C,D and in a recent report [ 19 ], in contrast to EDEM3, the interactors of EDEM1 are abundantly occurring in both ERAD and folding linked networks of proteins. A complementary experiment using protein fractionation by ultracentrifugation ( Figure 1 E), placed EDEM3 in the lower range of the sucrose gradient fractions.…”

“…A complementary experiment using protein fractionation by ultracentrifugation ( Figure 1 E), placed EDEM3 in the lower range of the sucrose gradient fractions. This corresponded to an approximate mass distribution between 120 and 250 kDa, sustaining the diminished interaction profile of the molecule when compared to EDEM1 found in complexes spreading all over the gradient [ 19 ]. Importantly, this sedimentation equilibrium predicts the dimerization of the native EDEM3 molecule in the cell.…”

“…Though unexpectedly, the MS analysis of EDEM3 interactors showed infrequent stable interactions of EDEM3 with other ERAD components or with folding machinery proteins. This is in startling contrast with EDEM1, its closest related member of the EDEM family, which was shown to participate in various ER quality control complexes within the ER [ 19 ]. By contrast, ultracentrifugation experiments place EDEM3 at molecular masses varying between 120–250 kDa, indicating its functioning as a homo- or hetero-dimer within the ER.…”

“…This is interesting since, in the case of EDEM1, the inactivation of the active mannosidase center or the deletion of most of the domain, did not impair either the association with the ERAD substrate or the accelerated degradation effect [ 34 , 35 ]. EDEM1 has an intrinsically disordered domain shown to interact with the misfolded substrates but also with the ERAD components [ 17 , 19 ]. However, in the case of EDEM3, the mutant lacking the intrinsically disordered domain, ΔIDD, is not only enzymatically active, but also able to interact with and efficiently target both ST and NHK to degradation.…”

Endoplasmic Reticulum Associated Degradation of Spinocerebellar Ataxia-Related CD10 Cysteine Mutant

“…While exploring the factors in CD10 C143Y degradation, we found that cytoplasmic E3 ubiquitin ligase C-terminus of Hsp70-interacting protein (CHIP) could enhance the clearance of CD10 C143Y (Supplementary Figure S5). While this manuscript was in preparation, it was reported that EDEM1 is integrated into the functional ERAD complex, including SEL1L and Hrd1, to facilitate disposal of ER-misfolded proteins through the ERAD pathway or even autophagy [54]. Detailed characterization of the CD10 C143Y quality control and degradation pathway, and the biosynthetic pathway of WT CD10, would offer vital insights for application in SCA treatment, as well as for diseases associated with immature secretory and membrane proteins involved in ER quality control systems.…”