The quality control mechanism in the endoplasmic reticulum (ER) discriminates correctly folded proteins from misfolded polypeptides and determines their fate. Terminally misfolded proteins are retrotranslocated from the ER and degraded by cytoplasmic proteasomes, a mechanism known as ER-associated degradation (ERAD). We report the cDNA cloning of Edem, a mouse gene encoding a putative type II ER transmembrane protein. Expression of Edem mRNA was induced by various types of ER stress. Although the luminal region of ER degradation enhancing α-mannosidase-like protein (EDEM) is similar to class I α1,2-mannosidases involved in N-glycan processing, EDEM did not have enzymatic activity. Overexpression of EDEM in human embryonic kidney 293 cells accelerated the degradation of misfolded α1-antitrypsin, and EDEM bound to this misfolded glycoprotein. The results suggest that EDEM is directly involved in ERAD, and targets misfolded glycoproteins for degradation in an N-glycan dependent manner.

Many proteins in the yeast Saccharomyces cerevisiae are modified by the attachment of N-linked saccharides to asparagine, of O-linked mannose glycans to serine or threonine, and of glycosylphosphoinositol membrane anchors. The biosynthetic events leading to these modifications are coupled to the secretory pathway. Early stages of N-linked glycosylation and the formation of glycosylphosphoinositol anchors have been conserved through evolution of eukaryotes. Studies of yeast offer a variety of genetic and molecular biological approaches, which have led to the isolation of different glycosylation mutants and of genes for enzymes involved in glycosylation. Yeast mutants are useful to identify biosynthetic intermediates, to establish whether a given enzyme is essential for viability, and to determine how cellular functions are affected when glycosylation is perturbed. Yeast glycosylation mutants and genes can be used to identify their counterparts in other eukaryotes.

Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing ␣-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I ␣-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded ␣1-antitrypsin variant (null (Hong Kong)) and of TCR␣. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded ␣1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent ␣1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of ␣1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has ␣1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.ER 3 quality control is an elaborate mechanism conserved from yeast to mammals, ensuring that newly synthesized proteins in the ER fold and assemble correctly and that only proteins that acquire their correct conformations are sorted further into the secretory pathway (1-4). During this process, proteins that fail to attain their native conformation due to mutations of the polypeptides or to ER stress conditions adverse for protein folding as well as orphan subunits are degraded in a process known as ER-associated degradation (ERAD) (3, 5-7). The recognition of misfolded proteins for ERAD is still poorly understood, but there is increasing evidence for a role of mannose trimming in the targeting of glycoproteins for ERAD (8, 9). In mammalian cells, overexpression of ER ␣-mannosidase I stimulates ERAD of misfolded glycoproteins (10, 11), whereas the ␣1,2-mannosidase inhibitors kifunensine and 1-deoxymannojirimycin stabilize misfolded glycoproteins (12-16). These observations suggested that Man 8 GlcNAc 2 isomer B, the major product of the ER ␣1,2-mannosidase, is a recognition marker for ERAD of glycoproteins, but this view is being challenged, since there is increasing evidence that trimming to smaller oligosaccharides occurs on ERAD substrates (10,(17)(18)(19). We previously cloned mouse EDEM (ER degradation enhancing ␣-mannosidase-like protein) as a cDNA whose expression is up-regulated by ER stress and showed that EDEM accelerates glycoprotein ERAD (20). EDEM is an integral ER membrane protein that has all the signature motifs of Class I ...

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Endoplasmic reticulum (ER) class I ␣1,2-mannosidase (also known as ER ␣-mannosidase I) is a critical enzyme in the maturation of N-linked oligosaccharides and ERassociated degradation. Trimming of a single mannose residue acts as a signal to target misfolded glycoproteins for degradation by the proteasome. Crystal structures of the catalytic domain of human ER class I ␣1,2-mannosidase have been determined both in the presence and absence of the potent inhibitors kifunensine and 1-deoxymannojirimycin. Both inhibitors bind to the protein at the bottom of the active-site cavity, with the essential calcium ion coordinating the O-2 and O-3 hydroxyls and stabilizing the six-membered rings of both inhibitors in a 1 C 4 conformation. This is the first direct evidence of the role of the calcium ion. The lack of major conformational changes upon inhibitor binding and structural comparisons with the yeast ␣1,2-mannosidase enzyme-product complex suggest that this class of inverting enzymes has a novel catalytic mechanism. The structures also provide insight into the specificity of this class of enzymes and provide a blueprint for the future design of novel inhibitors that prevent degradation of misfolded proteins in genetic diseases. Endoplasmic reticulum (ER)1 class I ␣1,2-mannosidase (also known as ER ␣-mannosidase I and Man 9 GlcNAc 2 -specific processing ␣-mannosidase, EC 3.2.1.113) is a key enzyme in the maturation of N-linked oligosaccharides in mammalian cells (for reviews, see Refs.

Misfolded glycoproteins synthesized in the endoplasmic reticulum (ER) are degraded by cytoplasmic proteasomes, a mechanism known as ERAD (ER-associated degradation

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We had previously shown that endoplasmic reticulum (ER)-associated degradation (ERAD) of glycoproteins in mammalian cells involves trimming of three to four mannose residues from the N-linked oligosaccharide Man(9)GlcNAc(2). A possible candidate for this activity, ER mannosidase I (ERManI), accelerates the degradation of ERAD substrates when overexpressed. Although in vitro, at low concentrations, ERManI removes only one specific mannose residue, at very high concentrations it can excise up to four alpha1,2-linked mannose residues. Using small interfering RNA knockdown of ERManI, we show that this enzyme is required for trimming to Man(5-6)GlcNAc(2) and for ERAD in cells in vivo, leading to the accumulation of Man(9)GlcNAc(2) and Glc(1)Man(9)GlcNAc(2) on a model substrate. Thus, trimming by ERManI to the smaller oligosaccharides would remove the glycoprotein from reglucosylation and calnexin binding cycles. ERManI is strikingly concentrated together with the ERAD substrate in the pericentriolar ER-derived quality control compartment (ERQC) that we had described previously. ERManI knockdown prevents substrate accumulation in the ERQC. We suggest that the ERQC provides a high local concentration of ERManI, and passage through this compartment would allow timing of ERAD, possibly through a cycling mechanism. When newly made glycoproteins cannot fold properly, transport through the ERQC leads to trimming of a critical number of mannose residues, triggering a signal for degradation.