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 Man 9 GlcNAc 2 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.

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by a pathway termed ER-associated protein degradation (ERAD). Glycans are often removed from glycosylated ERAD substrates in the cytosol before substrate degradation, which maintains the efficiency of the proteasome. Png1, a deglycosylating enzyme, has long been suspected, but not proven, to be crucial in this process. We demonstrate that the efficient degradation of glycosylated ricin A chain requires the Png1–Rad23 complex, suggesting that this complex couples protein deglycosylation and degradation. Rad23 is a ubiquitin (Ub) binding protein involved in the transfer of ubiquitylated substrates to the proteasome. How Rad23 achieves its substrate specificity is unknown. We show that Rad23 binds various regulators of proteolysis to facilitate the degradation of distinct substrates. We propose that the substrate specificity of Rad23 and other Ub binding proteins is determined by their interactions with various cofactors involved in specific degradation pathways.

It has been proposed that cytoplasmic peptide: N -glycanase (PNGase) may be involved in the proteasome-dependent quality control machinery used to degrade newly synthesized glycoproteins that do not correctly fold in the ER. However, a lack of information about the structure of the enzyme has limited our ability to obtain insight into its precise biological function. A PNGase-defective mutant ( png1-1 ) was identified by screening a collection of mutagenized strains for the absence of PNGase activity in cell extracts. The PNG1 gene was mapped to the left arm of chromosome XVI by genetic approaches and its open reading frame was identified. PNG1 encodes a soluble protein that, when expressed in Escherichia coli , exhibited PNGase activity. PNG1 may be required for efficient proteasome-mediated degradation of a misfolded glycoprotein. Subcellular localization studies indicate that Png1p is present in the nucleus as well as the cytosol. Sequencing of expressed sequence tag clones revealed that Png1p is highly conserved in a wide variety of eukaryotes including mammals, suggesting that the enzyme has an important function.