Mannose trimming is required for delivery of a glycoprotein from EDEM1 to XTP3-B and to late endoplasmic reticulum-associated degradation steps

Although the trimming of α1,2-mannose residues from precursor N-linked oligosaccharides is an essential step in the delivery of misfolded glycoproteins to endoplasmic reticulum (ER)-associated degradation (ERAD), the exact role of this trimming is unclear. EDEM1 was initially suggested to bind N-glycans after mannose trimming, a role presently ascribed to the lectins OS9 and XTP3-B, because of their in vitro affinities for trimmed oligosaccharides. We have shown before that ER mannosidase I (ERManI) is required for the trimming and concentrates together with the ERAD substrate and ERAD machinery in the pericentriolar ER-derived quality control compartment (ERQC). Inhibition of mannose trimming prevents substrate accumulation in the ERQC. Here, we show that the mannosidase inhibitor kifunensine or ERManI knockdown do not affect binding of an ERAD substrate glycoprotein to EDEM1. In contrast, substrate association with XTP3-B and with the E3 ubiquitin ligases HRD1 and SCF(Fbs2) was inhibited. Consistently, whereas the ERAD substrate partially colocalized upon proteasomal inhibition with EDEM1, HRD1, and Fbs2 at the ERQC, colocalization was repressed by mannosidase inhibition in the case of the E3 ligases but not for EDEM1. Interestingly, association and colocalization of the substrate with Derlin-1 was independent of mannose trimming. The HRD1 adaptor protein SEL1L had been suggested to play a role in N-glycan-dependent substrate delivery to OS9 and XTP3-B. However, substrate association with XTP3-B was still dependent on mannose trimming upon SEL1L knockdown. Our results suggest that mannose trimming enables delivery of a substrate glycoprotein from EDEM1 to late ERAD steps through association with XTP3-B.     

EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming

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 alpha-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 alpha-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 alpha1-antitrypsin variant (null (Hong Kong)) and of TCRalpha. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded alpha1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent alpha1,2-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 alpha1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has alpha1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.     

Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ER-associated degradation

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.     

Endoplasmic reticulum (ER)-associated degradation of misfolded N-linked glycoproteins is suppressed upon inhibition of ER mannosidase I

To examine the role of early carbohydrate recognition/trimming reactions in targeting endoplasmic reticulum (ER)-retained, misfolded glycoproteins for ER-associated degradation (ERAD), we have stably expressed the cog thyroglobulin (Tg) mutant cDNA in Chinese hamster ovary cells. We found that inhibitors of ER mannosidase I (but not other glycosidases) acutely suppressed Cog Tg degradation and also perturbed the ERAD process for Tg reduced with dithiothreitol as well as for gamma-carboxylation-deficient protein C expressed in warfarin-treated baby hamster kidney cells. Kifunensine inhibition of ER mannosidase I also suppressed ERAD in castanospermine-treated cells; thus, suppression of ERAD does not require lectin-like binding of ER chaperones calnexin and calreticulin to monoglucosylated oligosaccharides. Notably, the undegraded protein fraction remained completely microsome-associated. In pulse-chase studies, kifunensine-sensitive degradation was still inhibitable even 1 h after Tg synthesis. Intriguingly, chronic treatment with kifunensine caused a 3-fold accumulation of Cog Tg in Chinese hamster ovary cells and did not lead to significant induction of the ER unfolded protein response. We hypothesize that, in a manner not requiring lectin-like activity of calnexin/calreticulin, the recognition or processing of a specific branched N-linked mannose structure enhances the efficiency of glycoprotein retrotranslocation from the ER lumen.     

N-linked oligosaccharide processing,but not association with calnexin/calreticulin is highly correlated with endoplasmic reticulum-associated degradation of antithrombin Glu313-deleted mutant

Previously we showed that two antithrombin mutants were degraded through an endoplasmic reticulum (ER)-associated degradation (ERAD) pathway [F. Tokunaga et al., FEBS Lett. 412 (1997) 65]. Here, we examined the combined effects of inhibitors of glycosidases, protein synthesis, proteasome, and tyrosine phosphatase on ERAD of a Glu313-deleted (DeltaGlu) mutant of antithrombin. We found that kifunensine, an ER mannosidase I inhibitor, suppressed ERAD, indicating that specific mannose trimming plays a critical role. Cycloheximide and puromycin, inhibitors of protein synthesis, also suppressed ERAD, the effects being cancelled by pretreatment with castanospermine. In contrast, kifunensine suppressed ERAD even in castanospermine-treated cells, suggesting that suppression of ERAD does not always require the binding of lectin-like ER chaperones-like calnexin and/or calreticulin. These results indicate that, besides proteasome inhibitors, inhibitors of ER mannosidase I and protein synthesis suppress ERAD of the antithrombin deltaGlu mutant at different stages, and processing of N-linked oligosaccharides highly correlated with the efficiency of ERAD.     

Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I

Misfolded glycoproteins synthesized in the endoplasmic reticulum (ER) are degraded by cytoplasmic proteasomes, a mechanism known as ERAD (ER-associated degradation). In the present study, we demonstrate that ERAD of the misfolded genetic variant-null Hong Kong alpha1-antitrypsin is enhanced by overexpression of the ER processing alpha1,2-mannosidase (ER ManI) in HEK 293 cells, indicating the importance of ER ManI in glycoprotein quality control. We showed previously that EDEM, an enzymatically inactive mannosidase homolog, interacts with misfolded alpha1-antitrypsin and accelerates its degradation (Hosokawa, N., Wada, I., Hasegawa, K., Yorihuzi, T., Tremblay, L. O., Herscovics, A., and Nagata, K. (2001) EMBO Rep. 2, 415-422). Herein we demonstrate a combined effect of ER ManI and EDEM on ERAD of misfolded alpha1-antitrypsin. We also show that misfolded alpha1-antitrypsin NHK contains labeled Glc1Man9GlcNAc and Man5-9GlcNAc released by endo-beta-N-acetylglucosaminidase H in pulse-chase experiments with [2-3H]mannose. Overexpression of ER ManI greatly increases the formation of Man8GlcNAc, induces the formation of Glc1Man8GlcNAc and increases trimming to Man5-7GlcNAc. We propose a model whereby the misfolded glycoprotein interacts with ER ManI and with EDEM, before being recognized by downstream ERAD components. This detailed characterization of oligosaccharides associated with a misfolded glycoprotein raises the possibility that the carbohydrate recognition determinant triggering ERAD may not be restricted to Man8GlcNAc2 isomer B as previous studies have suggested.     

Multiple endoplasmic reticulum-associated pathways degrade mutant yeast carboxypeptidase Y in mammalian cells

The degradation of misfolded and unassembled proteins by the endoplasmic reticulum (ER)-associated degradation (ERAD) has been shown to occur mainly through the ubiquitin-proteasome pathway after transport of the protein to the cytosol. Recent work has revealed a role for N-linked glycans in targeting aberrant glycoproteins to ERAD. To further characterize the molecular basis of substrate recognition and sorting during ERAD in mammalian cells, we expressed a mutant yeast carboxypeptidase Y (CPY*) in CHO cells. CPY* was retained in the ER in un-aggregated form, and degraded after a 45-min lag period. Degradation was predominantly by a proteasome-independent, non-lysosomal pathway. The inhibitor of ER mannosidase I, kifunensine, blocked the degradation by the alternate pathway but did not affect the proteasomal fraction of degradation. Upon inhibition of glucose trimming, the initial lag period was eliminated and degradation thus accelerated. Our results indicated that, although the proteasome is a major player in ERAD, alternative routes are present in mammalian cells and can play an important role in the disposal of both glycoproteins and non-glycoproteins.     

Degradation of trafficking-defective long QT syndrome type II mutant channels by the ubiquitin-proteasome pathway

Mutations in the human ether-a-go-go-related gene (hERG) cause chromosome 7-linked long QT syndrome type II (LQT2). We have shown previously that LQT2 mutations lead to endoplasmic reticulum (ER) retention and rapid degradation of mutant hERG proteins. In this study we examined the role of the ubiquitin-proteasome pathway in the degradation of the LQT2 mutation Y611H. We showed that proteasome inhibitors N-acetyl-L-leucyl-L-leucyl-L-norleucinal and lactacystin but not lysosome inhibitor leupeptin inhibited the degradation of Y611H mutant channels. In addition, ER mannosidase I inhibitor kifunensine and down-regulation of EDEM (ER degradation-enhancing alpha-mannosidase-like protein) also suppressed the degradation of Y611H mutant channels. Proteasome inhibition but not mannosidase inhibition led to the accumulation of full-length hERG protein in the cytosol. The hERG protein accumulated in the cytosol was deglycosylated. Proteasome inhibition also resulted in the accumulation of polyubiquitinated hERG channels. These results suggest that the degradation of LQT2 mutant channels is mediated by the cytosolic proteasome in a process that involves mannose trimming, polyubiquitination, and deglycosylation of mutant channels.     

Stimulation of ERAD of misfolded null Hong Kong alpha1-antitrypsin by Golgi alpha1,2-mannosidases

Terminally misfolded or unassembled proteins are degraded by the cytoplasmic ubiquitin-proteasome pathway in a process known as ERAD (endoplasmic reticulum-associated protein degradation). Overexpression of ER alpha1,2-mannosidase I and EDEMs target misfolded glycoproteins for ERAD, most likely due to trimming of N-glycans. Here we demonstrate that overexpression of Golgi alpha1,2-mannosidase IA, IB, and IC also accelerates ERAD of terminally misfolded human alpha1-antitrypsin variant null (Hong Kong) (NHK), and mannose trimming from the N-glycans on NHK in 293 cells. Although transfected NHK is primarily localized in the ER, some NHK also co-localizes with Golgi markers, suggesting that mannose trimming by Golgi alpha1,2-mannosidases can also contribute to NHK degradation.     

A novel ER alpha-mannosidase-like protein accelerates ER-associated degradation

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 alpha-mannosidase-like protein (EDEM) is similar to class I alpha1,2-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 alpha1-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.     

Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation

Proteins that are unfolded or misfolded in the endoplasmic reticulum (ER) must be refolded or degraded to maintain the homeostasis of the ER. Components of both productive folding and ER-associated degradation (ERAD) mechanisms are known to be up-regulated by the unfolded protein response (UPR). We describe two novel components of mammalian ERAD, Derlin-2 and -3, which show weak homology to Der1p, a transmembrane protein involved in yeast ERAD. Both Derlin-2 and -3 are up-regulated by the UPR, and at least Derlin-2 is a target of the IRE1 branch of the response, which is known to up-regulate ER degradation enhancing alpha-mannosidase-like protein (EDEM) and EDEM2, receptor-like molecules for misfolded glycoprotein. Overexpression of Derlin-2 or -3 accelerated degradation of misfolded glycoprotein, whereas their knockdown blocked degradation. Derlin-2 and -3 are associated with EDEM and p97, a cytosolic ATPase responsible for extraction of ERAD substrates. These findings indicate that Derlin-2 and -3 provide the missing link between EDEM and p97 in the process of degrading misfolded glycoproteins.     

N-glycan structure of a short-lived variant of ribophorin I expressed in the MadIA214 glycosylation-defective cell line reveals the role of a mannosidase that is not ER mannosidase I in the process of glycoprotein degradation.

A soluble form of ribophorin I (RI(332)) is rapidly degraded in Hela and Chinese hamster ovary (CHO) cells by a cytosolic proteasomal pathway, and the N-linked glycan present on the protein may play an important role in this process. Specifically, it has been suggested that endoplasmic reticulum (ER) mannosidase I could trigger the targeting of improperly folded glycoproteins to degradation. We used a CHO-derived glycosylation-defective cell line, MadIA214, for investigating the role of mannosidase(s) as a signal for glycoprotein degradation. Glycoproteins in MadIA214 cells carry truncated Glc(1)Man(5)GlcNAc(2) N-glycans. This oligomannoside structure interferes with protein maturation and folding, leading to an alteration of the ER morphology and the detection of high levels of soluble oligomannoside species caused by glycoprotein degradation. An HA-epitope-tagged soluble variant of ribophorin I (RI(332)-3HA) expressed in MadIA214 cells was rapidly degraded, comparable to control cells with the complete Glc(3)Man(9)GlcNAc(2) N-glycan. ER-associated degradation (ERAD) of RI(332)-3HA was also proteasome-mediated in MadIA214 cells, as demonstrated by inhibition of RI(332)-3HA degradation with agents specifically blocking proteasomal activities. Two inhibitors of alpha1,2-mannosidase activity also stabilized RI(332)-3HA in the glycosylation-defective cell line. This is striking, because the major mannosidase activity in the ER is the one of mannosidase I, specific for a mannose alpha1,2-linkage that is absent from the truncated Man(5) structure. Interestingly, though the Man(5) derivative was present in large amounts in the total protein pool, the two major species linked to RI(332)-3HA shortly after synthesis consisted of Glc(1)Man(5 )and Man(4), being replaced by Man(4 )and Man(3) when proteasomal degradation was inhibited. In contrast, the untrimmed intermediate of RI(332)-3HA was detected in mutant cells treated with mannosidase inhibitors. Our results unambiguously demonstrate that an alpha1,2-mannosidase that is not ER mannosidase I is involved in ERAD of RI(332-)3HA in the glycosylation-defective cell line, MadIA214.     

EDEM1 regulates ER-associated degradation by accelerating de-mannosylation of folding-defective polypeptides and by inhibiting their covalent aggregation

Proteins expressed in the endoplasmic reticulum (ER) are covalently modified by co-translational addition of pre-assembled core glycans (glucose(3)-mannose(9)-N-acetylglucosamine(2)) to asparagines in Asn-X-Ser/Thr motifs. N-Glycan processing is essential for protein quality control in the ER. Cleavages and re-additions of the innermost glucose residue prolong folding attempts in the calnexin cycle. Progressive loss of mannoses is a symptom of long retention in the ER and elicits preparation of terminally misfolded polypeptides for dislocation into the cytosol and proteasome-mediated degradation. The ER stress-induced protein EDEM1 regulates disposal of folding-defective glycoproteins and has been described as a mannose-binding lectin. Here we show that elevation of the intralumenal concentration of EDEM1 accelerates ER-associated degradation (ERAD) by accelerating de-mannosylation of terminally misfolded glycoproteins and by inhibiting formation of covalent aggregates upon release of terminally misfolded ERAD candidates from calnexin. Acceleration of Man(9) or Man(5)N-glycans dismantling upon overexpression was fully blocked by substitution in EDEM1 of one catalytic residue conserved amongst alpha1,2-mannosidases, thus suggesting that EDEM1 is an active mannosidase. This mutation did not affect the chaperone function of EDEM1.     

Human EDEM2, a novel homolog of family 47 glycosidases, is involved in ER-associated degradation of glycoproteins

In the endoplasmic reticulum (ER), misfolded proteins are retrotranslocated to the cytosol and degraded by the proteasome in a process known as ER-associated degradation (ERAD). Early in this pathway, a proposed lumenal ER lectin, EDEM, recognizes misfolded glycoproteins in the ER, disengages the nascent molecules from the folding pathway, and facilitates their targeting for disposal. In humans there are a total of three EDEM homologs. The amino acid sequences of these proteins are different from other lectins but are closely related to the Class I mannosidases (family 47 glycosidases). In this study, we characterize one of the EDEM homologs from Homo sapiens, which we have termed EDEM2 (C20orf31). Using recombinantly generated EDEM2, no alpha-1,2 mannosidase activity was observed. In HEK293 cells, recombinant EDEM2 is localized to the ER where it can associate with misfolded alpha1-antitrypsin. Overexpression of EDEM2 accelerates the degradation of misfolded alpha1-antitrypsin, indicating that the protein is involved in ERAD.     

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

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.     

Golgi localization of ERManI defines spatial separation of the mammalian glycoprotein quality control system

The Golgi complex has been implicated as a possible component of endoplasmic reticulum (ER) glycoprotein quality control, although the elucidation of its exact role is lacking. ERManI, a putative ER resident mannosidase, plays a rate-limiting role in generating a signal that targets misfolded N-linked glycoproteins for ER-associated degradation (ERAD). Herein we demonstrate that the endogenous human homologue predominantly resides in the Golgi complex, where it is subjected to O-glycosylation. To distinguish the intracellular site where the glycoprotein ERAD signal is generated, a COPI-binding motif was appended to the N terminus of the recombinant protein to facilitate its retrograde translocation back to the ER. Partial redistribution of the modified ERManI was observed along with an accelerated rate at which N-linked glycans of misfolded α1-antitrypsin variant NHK were trimmed. Despite these observations, the rate of NHK degradation was not accelerated, implicating the Golgi complex as the site for glycoprotein ERAD substrate tagging. Taken together, these data provide a potential mechanistic explanation for the spatial separation by which glycoprotein quality control components operate in mammalian cells.     

Characterization of Schizosaccharomyces pombe ER alpha-mannosidase: a reevaluation of the role of the enzyme on ER-associated degradation

It has been postulated that creation of Man8GlcNAc2 isomer B (M8B) by endoplasmic reticulum (ER) alpha-mannosidase I constitutes a signal for driving irreparably misfolded glycoproteins to proteasomal degradation. Contrary to a previous report, we were able to detect in vivo (but not in vitro) an extremely feeble ER alpha-mannosidase activity in Schizosaccharomyces pombe. The enzyme yielded M8B on degradation of Man9GlcNAc2 and was inhibited by kifunensin. Live S. pombe cells showed an extremely limited capacity to demannosylate Man9GlcNAc2 present in misfolded glycoproteins even after a long residence in the ER. In addition, no preferential degradation of M8B-bearing species was detected. Nevertheless, disruption of the alpha-mannosidase encoding gene almost totally prevented degradation of a misfolded glycoprotein. This and other conflicting reports may be best explained by assuming that the role of ER mannosidase on glycoprotein degradation is independent of its enzymatic activity. The enzyme, behaving as a lectin binding polymannose glycans of varied structures, would belong together with its enzymatically inactive homologue Htm1p/Mnl1p/EDEM, to a transport chain responsible for delivering irreparably misfolded glycoproteins to proteasomes. Kifunensin and 1-deoxymannojirimycin, being mannose homologues, would behave as inhibitors of the ER mannosidase or/and Htm1p/Mnl1p/EDEM putative lectin properties.     

Characterization of early EDEM1 protein maturation events and their functional implications

The endoplasmic reticulum (ER) quality control factor EDEM1 associates with a number of ER proteins and ER-associated degradation (ERAD) substrates; however, an understanding of its role in ERAD is unclear. The early maturation events for EDEM1 including signal sequence cleavage and glycosylation were analyzed, and their relationship to the function of EDEM1 was determined. EDEM1 has five N-linked glycosylation sites with the most C-terminal site recognized poorly cotranslationally, resulting in the accumulation of EDEM1 containing four or five glycans. The fifth site was modified post-translationally when bypassed cotranslationally. Signal sequence cleavage of EDEM1 was found to be a slow and inefficient process. Signal sequence cleavage produced a soluble form of EDEM1 that efficiently associated with the oxidoreductase ERdj5 and most effectively accelerated the turnover of a soluble ERAD substrate. In contrast, a type-II membrane form of EDEM1 was generated when the signal sequence was uncleaved, creating an N-terminal transmembrane segment. The membrane form of EDEM1 efficiently associated with the ER membrane protein SEL1L and accelerated the turnover of a membrane-associated ERAD substrate. Together, these results demonstrated that signal sequence cleavage functionally regulated the association of EDEM1-soluble and membrane-integrated isoforms with distinct ERAD machinery and substrates.     

Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD

The Htm1/EDEM protein has been proposed to act as a "degradation lectin" for endoplasmic reticulum-associated degradation (ERAD) of misfolded glycoproteins. In this study, we provide genetic and biochemical evidence that Yos9 protein in Saccharomyces cerevisiae is essential for efficient degradation of mutant glycoproteins. Yos9 is a member of the OS-9 protein family, which is conserved among eukaryotes and shows similarities with mannose-6-phosphate receptors (MPRs). We found that amino acids conserved among OS-9 family members and MPRs were essential for Yos9 protein function. Immunoprecipitation showed that Yos9 specifically associated with misfolded carboxypeptidase Y (CPY*), an ERAD substrate, but only when it carried Man8GlcNAc2 or Man5GlcNAc2 N-glycans. Our experiments further suggested that Yos9 acts in the same pathway as Htm1/EDEM. Yos9 protein is important for glycoprotein degradation and may act via its MRH domain as a degradation lectin-like protein in the glycoprotein degradation pathway.