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영어

Misfolded and misassembled glycoproteins are retained in the endoplasmic reticulum (ER) where they are exposed to the protein folding machinery and protein quality control. The Unfolded Protein Response is activated in response to an accumulation of unfolded or misfolded proteins in the lumen of the ER. Eventually, misfolded and mis-assembled glycoproteins are targeted for degradation by a process called ER-associated protein degradation (ERAD). EDEM1 is an ERAD component that interacts with misfolded luminal glycoproteins and routes them for dislocation. This is followed by their ubiquitination and proteasomal degradation. Although EDEM1 was proposed to be a lectin–like protein and to react with Man8GlcNAc2 oligosaccharides of glycoproteins, still very little is known about the turnover and degradation mechanism of EDEM1 and how this relates to the fate of its substrates. Previously, we reported that EDEM1 exits the ER by a vesicular non-COPII-mediated mechanism and becomes rapidly degraded by basal autophagy. Here, we provide detailed insight into the mechanism by which EDEM1 becomes degraded. After its dislocation to the cytosol, EDEM1 is apparently making complexes with the selective autophagy receptors p62, NBR1 and Alfy. We observed co-distribution of EDEM1 and selective autophagy receptors by double and triple confocal laser scanning immunofluorescence. By quantifying the relationship of EDEM1 and the selective autophagy receptors as visualized by confocal laser scanning immunofluorescence and double immunogold electron microscopy, dramatical changes were observed in HepG2 cells. Following inhibition of autophagy by wortmannin, the number and size of cytoplasmic clusters composed of EDEM1 and the selective autophagy cargo receptors dramatically increased and this aggregate formation was independent of the activity of HDAC6. We observed that deglycosylation of EDEM1 occurred by the action of the cytosolic peptide N-glycanase since treatment with inhibitors resulted in a strong increase in the amount of glycosylated EDEM1. Inhibition of cytosolic peptide N-glycanase also inhibited wortmannin-induced aggregation of EDEM1 and its complex formation with p62. This indicates that deglycosylation of EDEM1 is a prerequisite for subsequent ubiquitination and interaction with selective autophagy receptors. This demonstrates that the ERAD component EDEM1 itself undergoes ERAD involving selective autophagy