Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway

Soluble or transmembrane proteins in the ER possessing unfolded or misfolded parts in their luminal regions are retrotranslocated back to the cytoplasm and degraded by the proteasome, a series of events termed ER-associated degradation (ERAD)-L (Xie and Ng, 2010; Smith et al., 2011; Brodsky, 2012). ERAD-L deals with both glycoproteins (gpERAD) and non-glycoproteins (non-gpERAD). Misfolded parts of non-glycoproteins are most likely recognized by molecular chaperones represented by BiP. In contrast, glycoprotein degradation relies on the structure of N-glycans present on the surface of ERAD-L substrates. Glc3Man9GlcNAc2 attached to the asparagine residue is trimmed by glucosidase I and II to Man9GlcNAc2, which is in turn trimmed to Man8GlcNAc2 and then to Man7GlcNAc2. Man7GlcNAc2 exposing α1,6-linked mannose is recognized by the lectin molecule for the delivery of ERAD-L substrates to the HRD1 E3 complex present in the ER membrane (Molinari, 2007; Hosokawa et al., 2010; Kamiya et al., 2012). We have recently conducted comprehensive gene knockout (KO) analyses to precisely identify the roles of the four α1,2-mannosidase candidates for the mannose trimming that occurs during ERAD-L, namely ER mannosidase I (ERmanI), EDEM1, EDEM2, and EDEM3. These experiments were performed in chicken DT40 cells using conventional homologous recombination and in human HCT116 cells using the transcription activator-like effector nuclease method. Our results were surprising (Ninagawa et al., 2014). Initial trimming from Man9GlcNAc2 to Man8GlcNAc2 was conducted by EDEM2, which had been thought to have no mannosidase activity (Mast et al., 2005), whereas subsequent trimming from Man8GlcNAc2 to Man7GlcNAc2 was performed mostly by EDEM3 and to a slight extent by EDEM1. ERmanI appeared to play only a minor role in gpERAD. Here, by constructing and analyzing DT40 and HCT116 cells simultaneously deficient in EDEM1/2/3, we show that different ERAD pathways are used depending on the severity of Glycoproteins and non-glycoproteins possessing unfolded/misfolded parts in their luminal regions are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L with distinct mechanisms. Two-step mannose trimming from Man9GlcNAc2 is crucial in the ERAD-L of glycoproteins. We recently showed that this process is initiated by EDEM2 and completed by EDEM3/EDEM1. Here, we constructed chicken and human cells simultaneously deficient in EDEM1/2/3 and analyzed the fates of four ERAD-L substrates containing three potential N-glycosylation sites. We found that native but unstable or somewhat unfolded glycoproteins, such as ATF6α, ATF6α(C), CD3-δ–ΔTM, and EMC1, were stabilized in EDEM1/2/3 triple knockout cells. In marked contrast, degradation of severely misfolded glycoproteins, such as null Hong Kong (NHK) and deletion or insertion mutants of ATF6α(C), CD3-δ–ΔTM, and EMC1, was delayed only at early chase periods, but they were eventually degraded as in wild-type cells. Thus, higher eukaryotes are able to extract severely misfolded glycoproteins from glycoprotein ERAD and target them to the non-glycoprotein ERAD pathway to maintain the homeostasis of the ER. Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway