Oligosaccharide modification in the early secretory pathway directs the selection of a misfolded glycoprotein for degradation by the proteasome

The role of conformation-based quality control in the early secretory pathway is to eliminate misfolded polypeptides and unassembled multimeric protein complexes from the endoplasmic reticulum, ensuring the deployment of only functional molecules to distal sites. The intracellular fate of terminally misfolded human alpha1-antitrypsin was examined in hepatoma cells to identify the functional role of asparagine-linked oligosaccharide modification in the selection of glycoproteins for degradation by the cytosolic proteasome. Proteasomal degradation required physical interaction with the molecular chaperone calnexin. Altered sedimentation of intracellular complexes following treatment with the specific proteasome inhibitor lactacystin, and in combination with mannosidase inhibition, revealed that the removal of mannose from attached oligosaccharides abrogates the release of misfolded alpha1-antitrypsin from calnexin prior to proteasomal degradation. Intracellular turnover was arrested with kifunensine, implicating the participation of endoplasmic reticulum mannosidase I in the disposal process. Accelerated degradation occurred in a mannosidase-independent manner and was arrested by lactacystin, in response to the posttranslational inhibition of glucosidase II, demonstrating that the attenuated removal of glucose from attached oligosaccharides functions as the underlying rate-limiting step in the proteasome-mediated pathway. A model is proposed in which the removal of mannose from multiple attached oligosaccharides directs calnexin in the selection of misfolded alpha1-antitrypsin for degradation by the proteasome.     

Processing by endoplasmic reticulum mannosidases partitions a secretion-impaired glycoprotein into distinct disposal pathways

In the early secretory pathway, a distinct set of processing enzymes and family of lectins facilitate the folding and quality control of newly synthesized glycoproteins. In this regard, we recently identified a mechanism in which processing by endoplasmic reticulum mannosidase I, which attenuates the removal of glucose from asparagine-linked oligosaccharides, sorts terminally misfolded alpha(1)-antitrypsin for proteasome-mediated degradation in response to its abrogated physical dissociation from calnexin (Liu, Y., Choudhury, P., Cabral, C., and Sifers, R. N. (1999) J. Biol. Chem. 274, 5861-5867). In the present study, we examined the quality control of genetic variant PI Z, which undergoes inappropriate polymerization following biosynthesis. Here we show that in stably transfected hepatoma cells the additional processing of asparagine-linked oligosaccharides by endoplasmic reticulum mannosidase II partitions variant PI Z away from the conventional disposal mechanism in response to an arrested posttranslational interaction with calnexin. Intracellular disposal is accomplished by a nonproteasomal system that functions independently of cytosolic components but is sensitive to tyrosine phosphatase inhibition. The functional role of ER mannosidase II in glycoprotein quality control is discussed.     

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.     

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.     

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.     

A complex of Pdi1p and the mannosidase Htm1p initiates clearance of unfolded glycoproteins from the endoplasmic reticulum

Endoplasmic reticulum (ER)-resident mannosidases generate asparagine-linked oligosaccharide signals that trigger ER-associated protein degradation (ERAD) of unfolded glycoproteins. In this study, we provide in vitro evidence that a complex of the yeast protein disulfide isomerase Pdi1p and the mannosidase Htm1p processes Man(8)GlcNAc(2) carbohydrates bound to unfolded proteins, yielding Man(7)GlcNAc(2). This glycan serves as a signal for HRD ligase-mediated glycoprotein disposal. We identified a point mutation in PDI1 that prevents complex formation of the oxidoreductase with Htm1p, diminishes mannosidase activity, and delays degradation of unfolded glycoproteins in vivo. Our results show that Pdi1p is engaged in both recognition and glycan signal processing of ERAD substrates and suggest that protein folding and breakdown are not separated but interconnected processes. We propose a stochastic model for how a given glycoprotein is partitioned into folding or degradation pathways and how the flux through these pathways is adjusted to stress conditions.     

Membrane traffic in animal cells: cellular glycoproteins return to the site of Golgi mannosidase I

The recycling of cellular glycoproteins to the site of Golgi mannosidase I, an enzyme of asparagine-linked oligosaccharide synthesis, was studied in K562 human erythroleukemia cells. Cells were metabolically labeled in the presence of deoxymannojirimycin, a reversible inhibitor of Golgi mannosidase I. This generates glycoproteins with immature oligosaccharides in their normal locations. Transport to the mannosidase I compartment was then assessed by testing for the conversion of oligosaccharides into mature forms during reculture without deoxymannojirimycin. Transferrin receptor (TfR) was acted on by mannosidase I during reculture, suggesting that it returned to the region of the Golgi complex where this enzyme resides. The slow rate of this transport (t1/2 greater than 6 h) implies that it is probably different than TfR movement during transferrin internalization (t1/2 = 10-20 min) and TfR transport to the sialyltransferase compartment in the Golgi complex (t1/2 = 2-3 h) (Snider, M. D., and O. C. Rogers, 1985, J. Cell Biol., 100:826-834). The total cell glycoprotein pool was also transported to the mannosidase I compartment with a half-time of 4 h. Because this transport is 5-10 times faster than the rate of de novo glycoprotein synthesis in these cells, it is likely that most of the glycoprotein traffic through the Golgi complex is composed of recycling molecules.     

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.     

In vitro mannose trimming property of human ER α-1,2 mannosidase I

Endoplasmic reticulum α-1,2 mannosidase I (ERManI) is an enzyme, which removes α(1-2) linked mannoses from asparagine-linked oligosaccharides on glycoproteins in the endoplasmic reticulum (ER). ERManI preferentially removes one α(1-2) linked mannose from B-chain of Man₉GlcNAc₂. When glycoproteins fail to achieve properly folding, increased removal of α(1-2) linked mannoses on their oligosaccharides is induced and leads them to be disposed and degraded by ER-associated degradation pathway. However, it is still inconclusive whether accelerated removal of α(1-2) linked mannoses on those glycoproteins is catalyzed by the α-1,2 mannosidase I, proteins similar to mannosidase I [e.g. ER degradation-enhancing α-1,2 mannosidase-like protein (EDEM)], or both of them. Therefore, to approach this issue, we have investigated its in vitro activities using various oligosaccharides and glycoproteins as substrates. A recombinant form of human ERManI (hERManI) was prepared by using Escherichia coli. First, the enzyme generated Man₆GlcNAc₂-PA and Man₅GlcNAc₂-PA from 100 μM Man₉GlcNAc₂-PA after a one-hour reaction. Second, we have exposed bovine thyroglobulin and soybean agglutinin to denaturing conditions, e.g. 8 M urea, and used those glycoproteins as substrates. Sugar moieties were released from the reactant by PNGase F and their structures and amounts were elucidated by HPLC analysis. Intriguingly, the enzyme was shown to remove mannoses from bovine thyroglobulin and soybean agglutinin to larger extents when they were exposed to a denaturant. Therefore, our results suggested that hERManI could recognize tertiary and/or quaternary structures of glycoproteins and remove more α-1,2 linked mannoses from misfolded glycoproteins in living cells.     

Protein quality control in the ER: The recognition of misfolded proteins

The mechanism, in molecular terms of protein quality control, specifically of how the cell recognizes and discriminates misfolded proteins, remains a challenge. In the secretory pathway the folding status of glycoproteins passing through the endoplasmic reticulum is marked by the composition of the N-glycan. The different glycoforms are recognized by specialized lectins. The folding sensor UGGT acts as an unusual molecular chaperone and covalently modifies the Man9 N-glycan of a misfolded protein by adding a glucose moiety and converts it to Glc1Man9 that rebinds the lectin calnexin. However, further links between the folding status of a glycoprotein and the composition of the N-glycan are unclear. There is little unequivocal evidence for other proteins in the ER recognizing the N-glycan and also acting as molecular chaperones. Nevertheless, based upon a few examples, we suggest that this function is carried out by individual proteins in several different complexes. Thus, calnexin binds the protein disulfide isomerase ERp57, that acts upon Glc1Man9 glycoproteins. In another example the protein disulfide isomerase ERdj5 binds specifically to EDEM (which is probably a mannosidase) and a lectin OS9, and reduces the disulfide bonds of bound glycoproteins destined for ERAD. Thus the glycan recognition is performed by a lectin and the chaperone function performed by a specific partner protein that can recognize misfolded proteins. We predict that this will be a common arrangement of proteins in the ER and that members of protein foldase families such as PDI and PPI will bind specifically to lectins in the ER. Molecular chaperones BiP and GRp94 will assist in the folding of proteins bound in these complexes as well as in the folding of non-glycoproteins.     

Mammalian ER mannosidase I resides in quality control vesicles,where it encounters its glycoprotein substrates

Endoplasmic reticulum α1,2 mannosidase I (ERManI), a central component of ER quality control and ER-associated degradation (ERAD), acts as a timer enzyme, modifying N-linked sugar chains of glycoproteins with time. This process halts glycoprotein folding attempts when necessary and targets terminally misfolded glycoproteins to ERAD. Despite the importance of ERManI in maintenance of glycoprotein quality control, fundamental questions regarding this enzyme remain controversial. One such question is the subcellular localization of ERManI, which has been suggested to localize to the ER membrane, the ER-derived quality control compartment (ERQC), and, surprisingly, recently to the Golgi apparatus. To try to clarify this controversy, we applied a series of approaches that indicate that ERManI is located, at the steady state, in quality control vesicles (QCVs) to which ERAD substrates are transported and in which they interact with the enzyme. Both endogenous and exogenously expressed ERManI migrate at an ER-like density on iodixanol gradients, suggesting that the QCVs are derived from the ER. The QCVs are highly mobile, displaying dynamics that are dependent on microtubules and COP-II but not on COP-I vesicle machinery. Under ER stress conditions, the QCVs converge in a juxtanuclear region, at the ERQC, as previously reported. Our results also suggest that ERManI is turned over by an active autophagic process. Of importance, we found that membrane disturbance, as is common in immunofluorescence methods, leads to an artificial appearance of ERManI in a Golgi pattern.     

Discrimination between lumenal and cytosolic sites of deglycosylation in endoplasmic reticulum-associated degradation of glycoproteins by using benzyl mannose in CHO cell lines

Recent studies demonstrated that deglycosylation step is a prerequisite for endoplasmic reticulum (ER)-associated degradation of misfolded glycoproteins. Here, we report the advantages of using benzyl mannose during pulse-chase experiments to study the subcellular location of the deglycosylation step in Chinese hamster ovary (CHO) cell lines. Benzyl mannose inhibited both the ER-to-cytosol transport of oligomannosides and the trimming of cytosolic-labeled oligomannosides by the cytosolic mannosidase in vivo. We pointed out the occurrence of two subcellular sites of deglycosylation. The first one is located in the ER lumen, and led to the formation of Man8GlcNAc2 (isomer B) in wild-type CHO cell line and Man4GlcNAc2 in Man-P-Dol-deficient cell line. The second one was revealed in CHO mutant cell lines for which a high rate of glycoprotein degradation was required. It occurred in the cytosol and led to the liberation of oligosaccharides species with one GlcNAc residue and with a pattern similar to the one bound onto glycoproteins. The cytosolic deglycosylation site was not specific for CHO mutant cell lines, since we demonstrated the occurrence of cytosolic pathway when the formation of truncated glycans was induced in wild-type cells.     

The ER glycoprotein quality control system

The endoplasmic reticulum (ER) is the major site for folding and sorting of newly synthesized secretory cargo proteins. One central regulator of this process is the quality control machinery, which retains and ultimately disposes of misfolded secretory proteins before they can exit the ER. The ER quality control process is highly effective and mutations in cargo molecules are linked to a variety of diseases. In mammalian cells, a large number of secretory proteins, whether membrane bound or soluble, are asparagine (N)-glycosylated. Recent attention has focused on a sugar transferase, UDP-Glucose: glycoprotein glucosyl transferase (UGGT), which is now recognized as a constituent of the ER quality control machinery. UGGT is capable of sensing the folding state of glycoproteins and attaches a single glucose residue to the Man9GlcNAc2 glycan of incompletely folded or misfolded glycoproteins. This enables misfolded glycoproteins to rebind calnexin and reenter productive folding cycles. Prolonging the time of glucose addition on misfolded glycoproteins ultimately results in either the proper folding of the glycoprotein or its presentation to an ER associated degradation machinery.     

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.     

Glucosidase and mannosidase inhibitors mediate increased secretion of mutant alpha1 antitrypsin Z

It is now well known that the addition and trimming of oligosaccharide side chains during post-translational modification play an important role in determining the fate of secretory, membrane, and lysosomal glycoproteins. Recent studies have suggested that trimming of oligosaccharide side chains also plays a role in the degradation of misfolded glycoproteins as a part of the quality control mechanism of the endoplasmic reticulum (ER). In this study, we examined the effect of several inhibitors of carbohydrate processing on the fate of the misfolded secretory protein alpha1 antitrypsin Z. Retention of this misfolded glycoprotein in the ER of liver cells in the classical form of alpha1 antitrypsin (alpha1-AT) deficiency is associated with severe liver injury and hepatocellular carcinoma and lack of its secretion is associated with destructive lung disease/emphysema. The results show marked alterations in the fate of alpha1 antitrypsin Z (alpha1-ATZ). Indeed, one glucosidase inhibitor, castanospermine (CST), and two mannosidase inhibitors, kifunensine (KIF) and deoxymannojirimycin (DMJ), mediate marked increases in secretion of alpha1-ATZ by distinct mechanisms. The effects of these inhibitors on secretion have interesting implications for our understanding of the quality control apparatus of the ER. These inhibitors may also constitute models for development of additional drugs for chemoprophylaxis of liver injury and emphysema in patients with alpha1-AT deficiency.     

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.     

The retrotranslocation protein Derlin-1 binds peptide:N-glycanase to the endoplasmic reticulum

The deglycosylating enzyme, peptide:N-glycanase, acts on misfolded N-linked glycoproteins dislocated from the endoplasmic reticulum (ER) to the cytosol. Deglycosylation has been demonstrated to occur at the ER membrane and in the cytosol. However, the mechanism of PNGase association with the ER membrane was unclear, because PNGase lacked the necessary signal to facilitate its incorporation in the ER membrane, nor was it known to bind to an integral ER protein. Using HeLa cells, we have identified a membrane protein that associates with PNGase, thereby bringing it in close proximity to the ER and providing accessibility to dislocating glycoproteins. This protein, Derlin-1, has recently been shown to mediate retrotranslocation of misfolded glycoproteins. In this study we demonstrate that Derlin-1 interacts with the N-terminal domain of PNGase via its cytosolic C-terminus. Moreover, we find PNGase distributed in two populations; ER-associated and free in the cytosol, which suggests the deglycosylation process can proceed at either site depending on the glycoprotein substrate.     

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.