Mannose 6-phosphate Receptor Homology Domain Research Articles

The endoplasmic reticulum‐associated protein, OS‐9, behaves as a lectin in targeting the immature calcium‐sensing receptor

The mechanisms responsible for the processing and quality control of the calcium-sensing receptor (CaSR) in the endoplasmic reticulum (ER) are largely unknown. In a yeast two-hybrid screen of the CaSR C-terminal tail (residues 865-1078), we identified osteosarcoma-9 (OS-9) protein as a binding partner. OS-9 is an ER-resident lectin that targets misfolded glycoproteins to the ER-associated degradation (ERAD) pathway through recognition of specific N-glycans by its mannose-6-phosphate receptor homology (MRH) domain. We show by confocal microscopy that the CaSR and OS-9 co-localize in the ER in COS-1 cells. In immunoprecipitation studies with co-expressed OS-9 and CaSR, OS-9 specifically bound the immature form of wild-type CaSR in the ER. OS-9 also bound the immature forms of a CaSR C-terminal deletion mutant and a C677A mutant that remains trapped in the ER, although binding to neither mutant was favored over wild-type receptor. OS-9 binding to immature CaSR required the MRH domain of OS-9 indicating that OS-9 acts as a lectin most likely to target misfolded CaSR to ERAD. Our results also identify two distinct binding interactions between OS-9 and the CaSR, one involving both C-terminal domains of the two proteins and the other involving both N-terminal domains. This suggests the possibility of more than one functional interaction between OS-9 and the CaSR. When we investigated the functional consequences of altered OS-9 expression, neither knockdown nor overexpression of OS-9 was found to have a significant effect on CaSR cell surface expression or CaSR-mediated ERK1/2 phosphorylation.

Multiple Domains of GlcNAc-1-phosphotransferase Mediate Recognition of Lysosomal Enzymes

The Golgi enzyme UDP-GlcNAc:lysosomal enzymeN-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), an α2β2γ2hexamer, mediates the initial step in the addition of the mannose 6-phosphate targeting signal on newly synthesized lysosomal enzymes. This tag serves to direct the lysosomal enzymes to lysosomes. A key property of GlcNAc-1-phosphotransferase is its unique ability to distinguish the 60 or so lysosomal enzymes from the numerous non-lysosomal glycoproteins with identical Asn-linked glycans. In this study, we demonstrate that the two Notch repeat modules and the DNA methyltransferase-associated protein interaction domain of the α subunit are key components of this recognition process. Importantly, different combinations of these domains are involved in binding to individual lysosomal enzymes. This study also identifies the γ-binding site on the α subunit and demonstrates that in the majority of instances the mannose 6-phosphate receptor homology domain of the γ subunit is required for optimal phosphorylation. These findings serve to explain how GlcNAc-1-phosphotransferase recognizes a large number of proteins that lack a common structural motif.

OS9 Protein Interacts with Na-K-2Cl Co-transporter (NKCC2) and Targets Its Immature Form for the Endoplasmic Reticulum-associated Degradation Pathway

Mutations in the renal specific Na-K-2Cl co-transporter (NKCC2) lead to type I Bartter syndrome, a life-threatening kidney disease featuring arterial hypotension along with electrolyte abnormalities. We have previously shown that NKCC2 and its disease-causing mutants are subject to regulation by endoplasmic reticulum-associated degradation (ERAD). The aim of the present study was to identify the protein partners specifically involved in ERAD of NKCC2. To this end, we screened a kidney cDNA library through a yeast two-hybrid assay using NKCC2 C terminus as bait. We identified OS9 (amplified in osteosarcomas) as a novel and specific binding partner of NKCC2. Co-immunoprecipitation assays in renal cells revealed that OS9 association involves mainly the immature form of NKCC2. Accordingly, immunocytochemistry analysis showed that NKCC2 and OS9 co-localize at the endoplasmic reticulum. In cells overexpressing OS9, total cellular NKCC2 protein levels were markedly decreased, an effect blocked by the proteasome inhibitor MG132. Pulse-chase and cycloheximide-chase assays demonstrated that the marked reduction in the co-transporter protein levels was essentially due to increased protein degradation of the immature form of NKCC2. Conversely, knockdown of OS9 by small interfering RNA increased NKCC2 expression by increasing the co-transporter stability. Inactivation of the mannose 6-phosphate receptor homology domain of OS9 had no effect on its action on NKCC2. In contrast, mutations of NKCC2 N-glycosylation sites abolished the effects of OS9, indicating that OS9-induced protein degradation is N-glycan-dependent. In summary, our results demonstrate the presence of an OS9-mediated ERAD pathway in renal cells that degrades immature NKCC2 proteins. The identification and selective modulation of ERAD components specific to NKCC2 and its disease-causing mutants might provide novel therapeutic strategies for the treatment of type I Bartter syndrome.

Subunit interactions of the disease-related hexameric GlcNAc-1-phosphotransferase complex.

The multimeric GlcNAc-1-phosphotransferase complex catalyzes the formation of mannose 6-phosphate recognition marker on lysosomal enzymes required for receptor-mediated targeting to lysosomes. GNPTAB and GNPTG encode the α/β-subunit precursor membrane proteins and the soluble γ-subunits, respectively. Performing extensive mutational analysis, we identified the binding regions of γ-subunits in a previously uncharacterized domain of α-subunits comprising residues 535-698, named GNPTG binding (GB) domain. Both the deletion of GB preventing γ-subunit binding and targeted deletion of GNPTG led to significant reduction in GlcNAc-1-phosphotransferase activity. We also identified cysteine 70 in α-subunits to be involved in covalent homodimerization of α-subunits which is, however, required neither for interaction with γ-subunits nor for catalytic activity of the enzyme complex. Finally, binding assays using various γ-subunit mutants revealed that residues 130-238 interact with glycosylated α-subunits suggesting a role for the mannose 6-phosphate receptor homology domain in α-subunit binding. These studies provide new insight into the assembly of the GlcNAc-1-phosphotransferase complex, and the functions of distinct domains of the α- and γ-subunits.

Expression of α-subunit of α-glucosidase II in adult mouse brain regions and selected organs.

α-Glucosidase II (GII), a resident of endoplasmic reticulum (ER) and an important enzyme in the folding of nascent glycoproteins, is heterodimeric, consisting of α (GIIα) and β (GIIβ) subunits. The catalytic GIIα subunit, with the help of mannose 6-phosphate receptor homology domain of GIIβ, sequentially hydrolyzes two α1-3-linked glucose residues in the second step of N-linked oligosaccharide-mediated protein folding. The soluble GIIα subunit is retained in the ER through its interaction with the HDEL-containing GIIβ subunit. N-glycosylation and correct protein folding are crucial for protein stability and trafficking and cell surface expression of several proteins in the brain. Alterations in N-glycosylation lead to abnormalities in neuronal migration and mental retardation, various neurodegenerative diseases, and invasion of malignant gliomas. Inhibitors of GII are used to inhibit cell proliferation and migration in a variety of different pathologies, such as viral infection, cancer, and diabetes. Despite the widespread use of GIIα inhibitory drugs and the role of GIIα in brain function, little is known about its expression in brain and other tissues. Here, we report generation of a highly specific chicken antibody to the GIIα subunit and its characterization by Western blotting and immunoprecipitation using cerebral cortical extracts. By using this antibody, we showed that the GIIα protein is highly expressed in testis, kidney, and lung, with the lowest amount in heart. GIIα polypeptide levels in whole brain were comparable to those in spleen. However, a higher expression of GIIα protein was detected in the cerebral cortex, reflecting its continuous requirement in correct folding of cell surface proteins.

Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II, an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum

Here we report for the first time the three-dimensional structure of a mannose 6-phosphate receptor homology (MRH) domain present in a protein with enzymatic activity, glucosidase II (GII). GII is involved in glycoprotein folding in the endoplasmic reticulum. GII removes the two innermost glucose residues from the Glc3Man9GlcNAc2 transferred to nascent proteins and the glucose added by UDP-Glc:glycoprotein glucosyltransferase. GII is composed of a catalytic GIIα subunit and a regulatory GIIβ subunit. GIIβ participates in the endoplasmic reticulum localization of GIIα and mediates in vivo enhancement of N-glycan trimming by GII through its C-terminal MRH domain. We determined the structure of a functional GIIβ MRH domain by NMR spectroscopy. It adopts a β-barrel fold similar to that of other MRH domains, but its binding pocket is the most shallow known to date as it accommodates a single mannose residue. In addition, we identified a conserved residue outside the binding pocket (Trp-409) present in GIIβ but not in other MRHs that influences GII glucose trimming activity.

Hepatocystin/80K-H inhibits replication of hepatitis B virus through interaction with HBx protein in hepatoma cell

Hepatitis B virus (HBV) X protein (HBx) is a key player in HBV replication as well as HBV-induced hepatocellular carcinoma (HCC). However, the pathogenesis of HBV infection and the mechanisms of host–virus interactions are still elusive. In this study, a combination of affinity purification and mass spectrometry was applied to identify the host factors interacting with HBx in hepatoma cells. Thirteen proteins were identified as HBx binding partners. Among them, we first focused on determining the functional significance of the interaction between HBx and hepatocystin. A physical interaction between HBx and hepatocystin was confirmed by co-immunoprecipitation and Western blotting. Immunocytochemistry demonstrated that HBx and hepatocystin colocalized in the hepatoma cells. Domain mapping of both proteins revealed that the HBx C-terminus (amino acids 110–154) was responsible for binding to the mannose 6-phosphate receptor homology domain (amino acids, 419–525) of hepatocystin. Using translation and proteasome inhibitors, we found that hepatocystin overexpression accelerated HBx degradation via a ubiquitin-independent proteasome pathway. We demonstrated that this effect was mediated by an interaction between both proteins using a HBx deletion mutant. Hepatocystin overexpression significantly inhibited HBV DNA replication and expression of HBs antigen concomitant with HBx degradation. Using the hepatocystin mutant constructs that bind HBx, we also confirmed that hepatocystin inhibited HBx-dependent HBV replication. In conclusion, we demonstrated for the first time that hepatocystin functions as a chaperon-like molecule by accelerating HBx degradation, and thereby inhibits HBV replication. Our results suggest that inducing hepatocystin may provide a novel therapeutic approach to control HBV infection.

Endoplasmic reticulum lectin XTP3‐B inhibits endoplasmic reticulum‐associated degradation of a misfolded α1‐antitrypsin variant

The endoplasmic reticulum (ER) is an organelle that synthesizes many secretory and membrane proteins. However, proteins often fold incorrectly. Terminally misfolded polypeptides in the ER are retro-translocated to the cytosol, where they are ultimately degraded by the ubiquitin-proteasome system, a process termed ER-associated degradation (ERAD). By recognizing the specific structures of N-linked oligosaccharides attached to polypeptides, lectins play an important role in the quality control of glycoproteins in the ER. Mammalian OS-9 and XTP3-B are ER-resident lectins that contain mannose 6-phosphate receptor homology (MRH) domains, which recognize sugar moieties; OS-9 has one MRH domain and XTP3-B has two. Both are involved in ERAD, but the functional differences between the two are poorly understood. The present study analyzed the function of human XTP3-B, and found, by frontal affinity chromatography analysis, that its C-terminal MRH domain specifically recognized the Man9 GlcNAc2 (M9) glycan invitro and M9 glycans on an ERAD substrate NHK, a terminally misfolded α1-antitrypsin variant, invivo. Furthermore, endogenous XTP3-B was a component of the HRD1-SEL1L membrane-embedded ubiquitin ligase complex, an association that was stabilized by a direct interaction with SEL1L. The lectin activity of XTP3-B was required for its binding to NHK, but not for its association with SEL1L. Unlike OS-9, XTP3-B did not enhance the degradation of misfolded glycoproteins, but instead inhibited the degradation of NHK bearing M9 oligosaccharides. Therefore, we propose that XTP3-B recognizes M9 glycans on unfolded polypeptides, thereby acting as a negative regulator of ERAD, and also protects newly synthesized immature polypeptides from premature degradation.

213 Structures and interactions of proteins involved in ER-associated protein degradation

Proteins are translocated into the endoplasmic reticulum (ER) of cells in an unfolded state, and acquire their native conformation in the ER lumen after signal peptide cleavage. ER-associated degradation (ERAD) of folding-incompetent protein chains is mediated by the protein complexes residing in the ER membrane. We study the architecture and function of one of these, the HRD complex assembled around the E3 ubiquitin ligase Hrd1. The recognition of ERAD substrates is linked to the maturation of their carbohydrate structures. The HRD complex-associated lectin Yos9 is involved in ERAD substrate recognition by binding carbohydrates through its mannose-6-phosphate receptor homology (MRH) domain. We have determined the crystal structure of a central domain of Yos9, adjacent to the MRH domain, which was previously annotated as interaction region with the HRD subunit Hrd3 (Hanna et al., 2012). We find that this domain does not support Hrd3 association which we map to the N-terminal half of Yos9 instead. In contrast, the domain has a function in Yos9 dimerization as seen in the crystal structure, in various solution experiments and as supported by mutagenesis of dimer interface residues. The dimerization of the ER-luminal Yos9, in conjunction with studies of the cytosolic domain of the HRD component Usa1 (Horn et al., 2009) and other biochemical data thus supports a model of a HRD complex that exists and functions as a dimer or a higher multimer. The delivery of ubiquitinated ERAD substrates to the proteasome is mediated by the cytosolic AAA ATPase Cdc48 (p97 in mammalian cells). The p97 (VCP) serves a wide variety of cellular functions in addition to its role in ERAD, including organelle membrane fusion, mitosis, DNA repair, and apoptosis. These different functions are linked to the binding of adaptor proteins to p97, many of which contain ubiquitin regulatory X (UBX) domains. One of these adaptors, ASPL (alveolar soft part sarcoma locus), uses a substantially extended UBX domain for binding to the N domain of p97 where a lariat-like, mostly α-helical extension wraps around one subunit of p97. By this binding ASPL triggers the dissociation of functional p97 hexamers leading to partial inactivation of the AAA ATPase. To the best of our knowledge, this is the first time that the structural basis for adaptor protein-induced inactivation by hexamer dissociation of p97 and, indeed, any AAA ATPase has been demonstrated. This observation has far reaching implications for AAA ATPase-regulated processes.

Structural and Biochemical Basis of Yos9 Protein Dimerization and Possible Contribution to Self-association of 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase Degradation Ubiquitin-Ligase Complex

In yeast, the membrane-bound HMG-CoA reductase degradation (HRD) ubiquitin-ligase complex is a key player of the ER-associated protein degradation pathway that targets misfolded proteins for proteolysis. Yos9, a component of the luminal submodule of the ligase, scans proteins for specific oligosaccharide modifications, which constitute a critical determinant of the degradation signal. Here, we report the crystal structure of the Yos9 domain that was previously suggested to confer binding to Hrd3, another component of the HRD complex. We observe an αβ-roll domain architecture and a dimeric assembly which are confirmed by analytical ultracentrifugation of both the crystallized domain and full-length Yos9. Our binding studies indicate that, instead of this domain, the N-terminal part of Yos9 including the mannose 6-phosphate receptor homology domain mediates the association with Hrd3 in vitro. Our results support the model of a dimeric state of the HRD complex and provide first-time evidence of self-association on its luminal side.

Structural Basis for Oligosaccharide Recognition of Misfolded Glycoproteins by OS-9 in ER-Associated Degradation

Misfolded glycoproteins are translocated from endoplasmic reticulum (ER) into the cytosol for proteasome-mediated degradation. A mannose-6-phosphate receptor homology (MRH) domain is commonly identified in a variety of proteins and, in the case of OS-9 and XTP3-B, is involved in glycoprotein ER-associated degradation (ERAD). Trimming of outermost α1,2-linked mannose on C-arm of high-mannose-type glycan and binding of processed α1,6-linked mannosyl residues by the MRH domain are critical steps in guiding misfolded glycoproteins to enter ERAD. Here we report the crystal structure of a human OS-9 MRH domain (OS-9(MRH)) complexed with α3,α6-mannopentaose. The OS-9(MRH) has a flattened β-barrel structure with a characteristic P-type lectin fold and possesses distinctive double tryptophan residues in the oligosaccharide-binding site. Our crystallographic result in conjunction with nuclear magnetic resonance (NMR) spectroscopic and biochemical results provides structural insights into the mechanism whereby OS-9 specifically recognizes Manα1,6Manα1,6Man residues on the processed C-arm through the continuous double tryptophan (WW) motif.

Functions of the α, β, and γ Subunits of UDP-GlcNAc:Lysosomal Enzyme N-Acetylglucosamine-1-phosphotransferase

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an alpha(2)beta(2)gamma(2) hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the gamma subunit along with kinetic studies of recombinant alpha(2)beta(2)gamma(2) and alpha(2)beta(2) forms of the transferase, we have explored the function of the alpha/beta and gamma subunits. The findings demonstrate that the alpha/beta subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the alpha/beta subunits phosphorylate about one-third of the acid hydrolases at close to wild-type levels but require the gamma subunit for optimal phosphorylation of the rest of the acid hydrolases. In addition to enhancing the activity of the alpha/beta subunits toward a subset of the acid hydrolases, the gamma subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the gamma subunit binds and presents the high mannose glycans of the acceptor to the alpha/beta catalytic site in a favorable manner.

Human XTP3-B binds to 1-antitrypsin variant nullHong Kong via the C-terminal MRH domain in a glycan-dependent manner

XTP3-B is a soluble endoplasmic reticulum (ER)-resident protein containing two mannose-6-phosphate receptor homology (MRH) domains in its sequence. XTP3-B interacts with a membrane-associated ubiquitin ligase complex, and, therefore, is thought to participate in ER-associated degradation (ERAD). In this study, the recombinant human XTP3-B fused with IgG-Fc (XTP3-B-Fc), XTP3-B without an N-terminal MRH domain fused with IgG-Fc (XTP3-BDelta1-Fc), or XTP3-B without a C-terminal MRH domain fused with IgG-Fc (XTP3-BDelta2-Fc) were prepared. XTP3-B-Fc and XTP3-BDelta1-Fc bound to Lec1 cells but not to CHO, Lec2, or Lec8 cells, while XTP3-BDelta2-Fc did not bind to any of these cells. The binding of XTP3-B-Fc and XTP3-BDelta1-Fc to Lec1 cells was abrogated by treatment of the cells with endo-beta-N-acetylglucosaminidase H, Manalpha1,6Man or Manalpha1,6(Manalpha1,3)Manalpha1,6(Manalpha1,3)Man, or by substitution of Arg428 or Tyr457 in the C-terminal MRH domain with alanine. Arg428 and Tyr457 are homologous to amino acids that mediate glycan binding by the cation-dependent mannose-6-phosphate receptor. An immunoprecipitation experiment using lysates of cells co-expressing wild-type alpha1-antitrypsin (AT), alpha1-antitrypsin variant null(Hong Kong) (AT(NHK)), and FLAG-tagged XTP3-B, or its mutants, demonstrated that AT(NHK), but not AT, specifically co-precipitated with XTP3-B and XTP3-BDelta1. The glycan-binding-deficient XTP3-BDelta2 did not bind either AT or AT(NHK). These results suggest that XTP3-B specifically binds to AT(NHK), which is a well-known substrate of ERAD, via a C-terminal MRH domain in a glycan-dependent manner.

Sugar-binding activity of the MRH domain in the ER -glucosidase II subunit is important for efficient glucose trimming

Glucosidase II (GII) is a glycan-processing enzyme that trims two alpha1,3-linked glucose residues from N-glycan on newly synthesized glycoproteins. Trimming of the first alpha1,3-linked glucose from Glc(2)Man(9)GlcNAc(2) (G2M9) is important for a glycoprotein to interact with calnexin/calreticulin (CNX/CRT), and cleavage of the innermost glucose from Glc(1)Man(9)GlcNAc(2) (G1M9) sets glycoproteins free from the CNX/CRT cycle and allows them to proceed to the Golgi apparatus. GII is a heterodimeric complex consisting of a catalytic alpha subunit (GIIalpha) and a tightly associated beta subunit (GIIbeta) that contains a mannose 6-phosphate receptor homology (MRH) domain. A recent study has suggested a possible involvement of the MRH domain of GIIbeta (GIIbeta-MRH) in the glucose trimming process via its putative sugar-binding activity. However, it remains unknown whether GIIbeta-MRH possesses sugar-binding activity and, if so, what role this activity plays in the function of GII. Here, we demonstrate that human GIIbeta-MRH binds to high-mannose-type glycans. Frontal affinity chromatography revealed that GIIbeta-MRH binds most strongly to the glycans with the alpha1,2-linked mannobiose structure. GII with the mutant GIIbeta that lost the sugar-binding activity of GIIbeta-MRH hydrolyzes p-nitrophenyl-alpha-glucopyranoside, but the capacity to remove glucose residues from G1M9 and G2M9 is significantly decreased. Our results clearly demonstrate the capacity of the GIIbeta-MRH to bind high-mannose-type glycans and its importance in efficient glucose trimming of N-glycans.

Human OS-9, a Lectin Required for Glycoprotein Endoplasmic Reticulum-associated Degradation, Recognizes Mannose-trimmed N-Glycans

In the endoplasmic reticulum (ER), lectins and processing enzymes are involved in quality control of newly synthesized proteins for productive folding as well as in the ER-associated degradation (ERAD) of misfolded proteins. ER quality control requires the recognition and modification of the N-linked oligosaccharides attached to glycoproteins. Mannose trimming from the N-glycans plays an important role in targeting of misfolded glycoproteins for ERAD. Recently, two mammalian lectins, OS-9 and XTP3-B, which contain mannose 6-phosphate receptor homology domains, were reported to be involved in ER quality control. Here, we examined the requirement for human OS-9 (hOS-9) lectin activity in degradation of the glycosylated ERAD substrate NHK, a genetic variant of alpha1-antitrypsin. Using frontal affinity chromatography, we demonstrated that the recombinant hOS-9 mannose 6-phosphate receptor homology domain specifically binds N-glycans lacking the terminal mannose from the C branch in vitro. To examine the specificity of OS-9 recognition of N-glycans in vivo, we modified the oligosaccharide structures on NHK by overexpressing ER alpha1,2-mannosidase I or EDEM3 and examined the effect of these modifications on NHK degradation in combination with small interfering RNA-mediated knockdown of hOS-9. The ability of hOS-9 to enhance glycoprotein ERAD depended on the N-glycan structures on NHK, consistent with the frontal affinity chromatography results. Thus, we propose a model for mannose trimming and the requirement for hOS-9 lectin activity in glycoprotein ERAD in which N-glycans lacking the terminal mannose from the C branch are recognized by hOS-9 and targeted for degradation.

Human XTP3-B Forms an Endoplasmic Reticulum Quality Control Scaffold with the HRD1-SEL1L Ubiquitin Ligase Complex and BiP

The recognition of terminally misfolded proteins in the endoplasmic reticulum (ER) and the extraction of these proteins to the cytoplasm for proteasomal degradation are determined by a quality control mechanism in the ER. In yeast, Yos9p, an ER lectin containing a mannose 6-phosphate receptor homology (MRH) domain, enhances ER-associated degradation (ERAD) of glycoproteins. We show here that human XTP3-B (hXTP3-B), an ER lectin containing two MRH domains, has two transcriptional variants, and both isoforms retard ERAD of the human alpha(1)-antitrypsin variant null Hong Kong (NHK), a terminally misfolded glycoprotein. The hXTP3-B long isoform strongly inhibited ERAD of NHK-QQQ, which lacks all of the N-glycosylation sites of NHK, but the short transcriptional variant of hXTP3-B had almost no effect. Examination of complex formation by immunoprecipitation and by fractionation using sucrose density gradient centrifugation revealed that the hXTP3-B long isoform associates with the HRD1-SEL1L membrane-anchored ubiquitin ligase complex and BiP, forming a 27 S ER quality control scaffold complex. The hXTP3-B short isoform, however, is excluded from scaffold formation. Another MRH domain-containing ER lectin, hOS-9, is incorporated into this large complex, but gp78, another mammalian homolog of the yeast ubiquitin ligase Hrd1p, is not. Based on these results, we propose that this large ER quality control scaffold complex, containing ER lectins, a chaperone, and a ubiquitin ligase, provides a platform for the recognition and sorting of misfolded glycoproteins as well as nonglycosylated proteins prior to retrotranslocation into the cytoplasm for degradation.