A thrombospondin-dependent pathway for a protective ER stress response.

Thrombospondin (Thbs) proteins are induced in sites of tissue damage or active remodeling. The endoplasmic reticulum (ER) stress response is also prominently induced with disease where it regulates protein production and resolution of misfolded proteins. Here we describe a function for Thbs as ER-resident effectors of an adaptive ER stress response. Thbs4 cardiac-specific transgenic mice were protected from myocardial injury, whereas Thbs4(-/-) mice were sensitized to cardiac maladaptation. Thbs induction produced a unique profile of adaptive ER stress response factors and expansion of the ER and downstream vesicles. Thbs bind the ER lumenal domain of activating transcription factor 6α (Atf6α) to promote its nuclear shuttling. Thbs4(-/-) mice showed blunted activation of Atf6α and other ER stress-response factors with injury, and Thbs4-mediated protection was lost upon Atf6α deletion. Hence, Thbs can function inside the cell during disease remodeling to augment ER function and protect through a mechanism involving regulation of Atf6α.

Proteomic analysis of fatty liver induced by starvation of medaka fish larvae.

When medaka fish (Oryzias latipes) larvae are grown in the absence of exogenous nutrition, the liver becomes dark and positive to Oil Red O staining from 7 days post-hatch (dph). We determined the mechanism of this starvation-induced development of fatty liver by proteomic analysis using livers obtained from larvae grown in the presence or absence of 2% glucose at 5 dph. Results showed that changes in the expression levels of enzymes involved in glycolysis or the tricarboxylic acid cycle were modest, whereas the expression levels of enzymes involved in amino acid catabolism or ß-oxidation of fatty acids were significantly elevated, suggesting that they become major energy sources under starvation conditions. Expression levels of enzymes for the uptake and ß-oxidation of fatty acids as well as synthesis of triacylglycerol were elevated, whereas those for the synthesis of cholesterol as well as export of cholesterol and triacylglycerol were decreased under starvation conditions, which explains the accumulation of triacylglycerol in the liver. Our results provide the basis for future research to understand how gene malfunction(s) affects the development of fatty liver, which can lead to nonalcoholic steatohepatitis and then to liver cirrhosis.Key words: amino acid catabolism, ß-oxidation, triacylglycerol, cholesterol, export.

Endoplasmic Reticulum-Associated Degradation Controls Virus Protein Homeostasis, Which Is Required for Flavivirus Propagation.

Many positive-stranded RNA viruses encode polyproteins from which viral proteins are generated by processing the polyproteins. This system produces an equal amount of each viral protein, though the required amounts for each protein are not the same. In this study, we found the extra membrane-anchored nonstructural (NS) proteins of Japanese encephalitis virus and dengue virus are rapidly and selectively degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway. Our gene targeting study revealed that ERAD involving Derlin2 and SEL1L, but not Derlin1, is required for the viral genome replication. Derlin2 is predominantly localized in the convoluted membrane (CM) of the viral replication organelle, and viral NS proteins are degraded in the CM. Hence, these results suggest that viral protein homeostasis is regulated by Derlin2-mediated ERAD in the CM, and this process is critical for the propagation of these viruses. IMPORTANCE The results of this study reveal the cellular ERAD system controls the amount of each viral protein in virus-infected cells and that this "viral protein homeostasis" is critical for viral propagation. Furthermore, we clarified that the "convoluted membrane (CM)," which was previously considered a structure with unknown function, serves as a kind of waste dump where viral protein degradation occurs. We also found that the Derlin2/SEL1L/HRD1-specific pathway is involved in this process, whereas the Derlin1-mediated pathway is not. This novel ERAD-mediated fine-tuning system for the stoichiometries of polyprotein-derived viral proteins may represent a common feature among polyprotein-encoding viruses.

Development of a Rapid in vivo Assay to Evaluate the Efficacy of IRE1-specific Inhibitors of the Unfolded Protein Response Using Medaka Fish.

Three types of transmembrane protein, IRE1α/IRE1ß, PERK, and ATF6α/ATF6ß, are expressed ubiquitously in vertebrates as transducers of the unfolded protein response (UPR), which maintains the homeostasis of the endoplasmic reticulum. IRE1 is highly conserved from yeast to mammals, and transmits a signal by a unique mechanism, namely splicing of mRNA encoding XBP1, the transcription factor downstream of IRE1 in metazoans. IRE1 contains a ribonuclease domain in its cytoplasmic region which initiates splicing reaction by direct cleavage of XBP1 mRNA at the two stem loop structures. As the UPR is considered to be involved in the development and progression of various diseases, as well as in the survival and growth of tumor cells, UPR inhibitors have been sought. To date, IRE1 inhibitors have been screened using cell-based reporter assays and fluorescent-based in vitro cleavage assays. Here, we used medaka fish to develop an in vivo assay for IRE1α inhibitors. IRE1α, IRE1ß, ATF6α and ATF6ß are ubiquitously expressed in medaka. We found that IRE1α/ATF6α-double knockout is lethal, similarly to IRE1α/IRE1ß- and ATF6α/ATF6ß-double knockout. Therefore, IRE1 inhibitors are expected to confer lethality to ATF6α-knockout medaka but not to wild-type medaka. One compound named K114 was obtained from 1,280 compounds using this phenotypic screening. K114 inhibited ER stress-induced splicing of XBP1 mRNA as well as reporter luciferase expression in HCT116 cells derived from human colorectal carcinoma, and inhibited ribonuclease activity of human IRE1α in vitro. Thus, this phenotypic assay can be used as a quick test for the efficacy of IRE1α inhibitors in vivo.Key words: endoplasmic reticulum, inhibitor screening, mRNA splicing, phenotypic assay, unfolded protein response.

Reinvestigation of Disulfide-bonded Oligomeric Forms of the Unfolded Protein Response Transducer ATF6.

ATF6α is an endoplasmic reticulum (ER)-embedded transcription factor which is rapidly activated by ER stress, and a major regulator of ER chaperone levels in vertebrates. We previously suggested that ATF6α occurs as a monomer, dimer and oligomer in the unstressed ER of Chinese hamster ovary cells due to the presence of two evolutionarily conserved cysteine residues in its luminal region (C467 and C618), and showed that ATF6α is reduced upon ER stress, such that only reduced monomer ATF6α is translocated to the Golgi apparatus for activation by proteolysis. However, mutagenesis analysis (C467A and C618A) revealed that the C618A mutant behaves in an unexpected manner (monomer and oligomer) during non-reducing SDS-PAGE, for reasons which remained unclear. Here, we used human colorectal carcinoma-derived HCT116 cells deficient in ATF6α and its relevant ATF6ß, and found that ATF6α dimer and oligomer are both dimers, which we designated C618-dimer and C467-dimer, respectively. We demonstrated that C467-dimer (previously considered an oligomer) behaved bigger than C618-dimer (previously considered a dimer) during non-reducing SDS-PAGE, based on their disulfide-bonded structures. Furthermore, ATF6α monomer physically associates with another ATF6α monomer in the absence of disulfide bonding, which renders two C467 residues in close proximity so that formation of C467-dimer is much easier than that of C618-dimer. In contrast, C618-dimer is more easily reduced upon ER stress. Thus, our analysis revealed that all forms of ATF6α, namely monomer, C618-dimer and C467-dimer, are activated by single reduction of a disulfide bond in response to ER stress, ensuring the rapidity of ATF6α activation.Key words: disulfide-bonded structure, endoplasmic reticulum, membrane-bound transcription factor, non-reducing SDS-PAGE, unfolded protein response.

SEL1L-dependent Substrates Require Derlin2/3 and Herp1/2 for Endoplasmic Reticulum-associated Degradation.

Accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). The ATF6 pathway is one of the three major pathways in vertebrates. Although ATF6, a transmembrane-type glycoprotein in the ER, functions as a UPR sensor/transducer, it is an unstable protein with a half-life of approximately 2 h and is constitutively subjected to the ER-associated degradation system with the location of the misfolded part in the ER lumen (ERAD-L). ERAD-L substrates are delivered to the cytosol through the retrotranslocon, which likely contains HRD1 (E3), gp78 (E3), SEL1L (a partner of HRD1), Derlin1/2/3 and Herp1/2. We previously showed that ATF6 represents a novel transmembrane-type ERAD-L substrate requiring both EDEM1/2/3-mediated mannose trimming and SEL1L. Here, by constructing and analyzing chicken DT40 cells deficient in various components of the retrotranslocon, we show that degradation of ATF6 requires Derlin2 or Derlin3 and that Derlin2 and Derlin3 are redundant for ERAD-L of ATF6. We further show that degradation of ATF6 requires Herp1 or Herp2 and that Herp1 and Herp2 are redundant for ERAD-L of ATF6. Furthermore, by investigating five more ERAD-L substrates, we show that SEL1L-dependent substrates require Derlin2/3 and Herp1/2 regardless of their soluble or transmembrane nature. Our results suggest that ERAD-L substrates take several routes to the cytosol. The HRD1-engaged route 1 requires SEL1L, Derlin2 or Derlin3, and Herp1 or Herp2, whereas the HRD1-engaged route 2 does not require them functionally. It remains to be determined whether the latter requires Derlin1 and whether these two routes are compositionally distinct.Key words: endoplasmic reticulum, proteasome, protein degradation, protein misfolding, ubiquitin.

An IFN-γ-stimulated ATF6-C/EBP-ß-signaling pathway critical for the expression of Death Associated Protein Kinase 1 and induction of autophagy.

The IFN family of cytokines operates a frontline defense against pathogens and neoplastic cells in vivo by controlling the expression of several genes. The death-associated protein kinase 1 (DAPK1), an IFN-γ-induced enzyme, controls cell cycle, apoptosis, autophagy, and tumor metastasis, and its expression is frequently down-regulated in a number of human tumors. Although the biochemical action of DAPK1 is well understood, mechanisms that regulate its expression are unclear. Previously, we have shown that transcription factor C/EBP-ß is required for the basal and IFN-γ-induced expression of DAPK1. Here, we show that ATF6, an ER stress-induced transcription factor, interacts with C/EBP-ß in an IFN-stimulated manner and is obligatory for Dapk1 expression. IFN-stimulated proteolytic processing of ATF6 and ERK1/2-mediated phosphorylation of C/EBP-ß are necessary for these interactions. More importantly, IFN-γ failed to activate autophagic response in cells lacking either ATF6 or C/EBP-ß. Consistent with these observations, the Atf6(-/-) mice were highly susceptible to lethal bacterial infections compared with the wild-type mice. These studies not only unravel an IFN signaling pathway that controls cell growth and antibacterial defense, but also expand the role of ATF6 beyond ER stress.

The unfolded protein response transducer ATF6 represents a novel transmembrane-type endoplasmic reticulum-associated degradation substrate requiring both mannose trimming and SEL1L protein.

Proteins misfolded in the endoplasmic reticulum (ER) are cleared by the ubiquitin-dependent proteasome system in the cytosol, a series of events collectively termed ER-associated degradation (ERAD). It was previously shown that SEL1L, a partner protein of the E3 ubiquitin ligase HRD1, is required for degradation of misfolded luminal proteins (ERAD-Ls substrates) but not misfolded transmembrane proteins (ERAD-Lm substrates) in both mammalian and chicken DT40 cells. Here, we analyzed ATF6, a type II transmembrane glycoprotein that serves as a sensor/transducer of the unfolded protein response, as a potential ERAD-Lm substrate in DT40 cells. Unexpectedly, degradation of endogenous ATF6 and exogenously expressed chicken and human ATF6 by the proteasome required SEL1L. Deletion analysis revealed that the luminal region of ATF6 is a determinant for SEL1L-dependent degradation. Chimeric analysis showed that the luminal region of ATF6 confers SEL1L dependence on type I transmembrane protein as well. In contrast, degradation of other known type I ERAD-Lm substrates (BACE457, T-cell receptor-α, CD3-δ, and CD147) did not require SEL1L. Thus, ATF6 represents a novel type of ERAD-Lm substrate requiring SEL1L for degradation despite its transmembrane nature. In addition, endogenous ATF6 was markedly stabilized in wild-type cells treated with kifunensine, an inhibitor of α1,2-mannosidase in the ER, indicating that degradation of ATF6 requires proper mannose trimming. Our further analyses revealed that the five ERAD-Lm substrates examined are classified into three subgroups based on their dependence on mannose trimming and SEL1L. Thus, ERAD-Lm substrates are degraded through much more diversified mechanisms in higher eukaryotes than previously thought.

The antipsychotic olanzapine induces apoptosis in insulin-secreting pancreatic ß cells by blocking PERK-mediated translational attenuation.

Patients with schizophrenia receive medication to alleviate various symptoms, but some efficacious second generation antipsychotics, particularly olanzapine, can cause obesity, dyslipidemia, and diabetes mellitus. It has been generally considered that olanzapine contributes to the development of diabetes by inducing obesity and subsequent insulin resistance. In this study, we examined the effect of olanzapine and risperidone, another second generation antipsychotic, on a hamster pancreatic ß cell line, and found that both evoked mild endoplasmic reticulum (ER) stress, as evidenced by mild activation of the ER stress sensor molecule PERK. Surprisingly, only olanzapine induced marked apoptosis. Phosphorylation of the α subunit of eukaryotic initiation factor 2, an event immediately downstream of PERK activation, was not observed in cells treated with olanzapine, protein synthesis continued despite PERK activation, and ER stress was thereby sustained. Secretion of insulin was markedly inhibited, and both proinsulin and insulin accumulated inside olanzapine-treated cells. Inhibition of protein synthesis and knockdown of insulin mRNA, which result in less unfolded protein burden, both attenuated subsequent olanzapine-induced apoptosis. Given clinical observations that some patients taking olanzapine exhibit hyperlipidemia and hyperglycemia without gaining weight, our observations suggest that damage to pancreatic ß cells may contribute to the undesirable metabolic consequences of olanzapine treatment in some cases.

Loss of ATF6α in a human carcinoma cell line is compensated not by its paralogue ATF6ß but by sustained activation of the IRE1 and PERK arms for tumor growth in nude mice.

To survive poor nutritional conditions, tumor cells activate the unfolded protein response, which is composed of the IRE1, PERK, and ATF6 arms, to maintain the homeostasis of the endoplasmic reticulum, where secretory and transmembrane proteins destined for the secretory pathway gain their correct three-dimensional structure. The requirement of the IRE1 and PERK arms for tumor growth in nude mice is established. Here we investigated the requirement for the ATF6 arm, which consists of ubiquitously expressed ATF6α and ATF6ß, by constructing ATF6α-knockout (KO), ATF6ß-KO, and ATF6α/ß-double KO (DKO) in HCT116 cells derived from human colorectal carcinoma. Results showed that these KO cells grew similarly to wild-type (WT) cells in nude mice, contrary to expectations from our analysis of ATF6α-KO, ATF6ß-KO, and ATF6α/ß-DKO mice. We then found that the loss of ATF6α in HCT116 cells resulted in sustained activation of the IRE1 and PERK arms in marked contrast to mouse embryonic fibroblasts, in which the loss of ATF6α is compensated for by ATF6ß. Although IRE1-KO in HCT116 cells unexpectedly did not affect tumor growth in nude mice, IRE1-KO HCT116 cells with ATF6α knockdown grew significantly more slowly than WT or IRE1-KO HCT116 cells. These results have unraveled the situation-dependent differential compensation strategies of ATF6α.

Activation of XBP1 but not ATF6α rescues heart failure induced by persistent ER stress in medaka fish.

The unfolded protein response is triggered in vertebrates by ubiquitously expressed IRE1α/ß (although IRE1ß is gut-specific in mice), PERK, and ATF6α/ß, transmembrane-type sensor proteins in the ER, to cope with ER stress, the accumulation of unfolded and misfolded proteins in the ER. Here, we burdened medaka fish, a vertebrate model organism, with ER stress persistently from fertilization by knocking out the AXER gene encoding an ATP/ADP exchanger in the ER membrane, leading to decreased ATP concentration-mediated impairment of the activity of Hsp70- and Hsp90-type molecular chaperones in the ER lumen. ER stress and apoptosis were evoked from 4 and 6 dpf, respectively, leading to the death of all AXER-KO medaka by 12 dpf because of heart failure (medaka hatch at 7 dpf). Importantly, constitutive activation of IRE1α signaling-but not ATF6α signaling-rescued this heart failure and allowed AXER-KO medaka to survive 3 d longer, likely because of XBP1-mediated transcriptional induction of ER-associated degradation components. Thus, activation of a specific pathway of the unfolded protein response can cure defects in a particular organ.

Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6alpha and XBP1.

Metazoans express three unfolded protein response transducers (IRE1, PERK, and ATF6) ubiquitously to cope with endoplasmic reticulum (ER) stress. ATF6 is an ER membrane-bound transcription factor activated by ER stress-induced proteolysis and has been duplicated in mammals. Here, we generated ATF6alpha- and ATF6beta-knockout mice, which developed normally, and then found that their double knockout caused embryonic lethality. Analysis of mouse embryonic fibroblasts (MEFs) deficient in ATF6alpha or ATF6beta revealed that ATF6alpha is solely responsible for transcriptional induction of ER chaperones and that ATF6alpha heterodimerizes with XBP1 for the induction of ER-associated degradation components. ATF6alpha(-/-) MEFs are sensitive to ER stress. Unaltered responses observed in ATF6beta(-/-) MEFs indicate that ATF6beta is not a negative regulator of ATF6alpha. These results demonstrate that ATF6alpha functions as a critical regulator of ER quality control proteins in mammalian cells, in marked contrast to worm and fly cells in which IRE1 is responsible.

Drop-Dry Deposition of Ni(OH)2 Precursors for Fabrication of NiO Thin Films.

Drop-dry deposition (DDD) is a method of depositing thin films by heating and drying the deposition solution dropped on a substrate. We prepared Ni(OH)2 precursor thin films by DDD and annealed them in air to prepare NiO thin films. The appropriate deposition conditions were found by changing the number of drop-dry cycles and the concentrations of chemicals in the solution, and the Ni(OH)2 precursor film with a thickness of 0.3 µm and optical transmittance of more than 95% was successfully deposited. Raman and X-ray diffraction measurements were performed, and it was found that the NiO film was successfully fabricated after annealing at 400 °C. The p-type conductivity of the annealed film was confirmed by photoelectrochemical measurements. In addition, we prepared n-type ZnO by electrochemical deposition on NiO thin films. The current-voltage measurement results show that the ZnO/NiO heterojunction had rectification properties.

A motor neuron disease-associated mutation produces non-glycosylated Seipin that induces ER stress and apoptosis by inactivating SERCA2b.

A causal relationship between endoplasmic reticulum (ER) stress and the development of neurodegenerative diseases remains controversial. Here, we focused on Seipinopathy, a dominant motor neuron disease, based on the finding that its causal gene product, Seipin, is a protein that spans the ER membrane twice. Gain-of-function mutations of Seipin produce non-glycosylated Seipin (ngSeipin), which was previously shown to induce ER stress and apoptosis at both cell and mouse levels albeit with no clarified mechanism. We found that aggregation-prone ngSeipin dominantly inactivated SERCA2b, the major calcium pump in the ER, and decreased the calcium concentration in the ER, leading to ER stress and apoptosis in human colorectal carcinoma-derived cells (HCT116). This inactivation required oligomerization of ngSeipin and direct interaction of the C-terminus of ngSeipin with SERCA2b, and was observed in Seipin-deficient neuroblastoma (SH-SY5Y) cells expressing ngSeipin at an endogenous protein level. Our results thus provide a new direction to the controversy noted above.

SEL1L is required for endoplasmic reticulum-associated degradation of misfolded luminal proteins but not transmembrane proteins in chicken DT40 cell line.

Proteins misfolded in the endoplasmic reticulum (ER) are degraded in the cytosol by a ubiquitin-dependent proteasome system, a process collectively termed ER-associated degradation (ERAD). Unraveling the molecular mechanisms of mammalian ERAD progresses more slowly than that of yeast ERAD due to the laborious procedures required for gene targeting and the redundancy of components. Here, we utilized the chicken B lymphocyte-derived DT40 cell line, which exhibits an extremely high homologous recombination frequency, to analyze ERAD mechanisms in higher eukaryotes. We disrupted the SEL1L gene, which encodes the sole homologue of yeast Hrd3p in both chickens and mammals; Hrd3p is a binding partner of yeast Hrd1p, an E3 ubiquitin ligase. SEL1L-knockout cells grew only slightly more slowly than the wild-type cells. Pulse chase experiments revealed that chicken SEL1L was required for ERAD of misfolded luminal proteins such as glycosylated NHK and unglycosylated NHK-QQQ but dispensable for that of misfolded transmembrane proteins such as NHK(BACE) and CD3-δ, as in mammals. The defect of SEL1L-knockout cells in NHK degradation was restored by introduction of not only chicken SEL1L but also mouse and human SEL1L. Deletion analysis showed the importance of Sel1-like tetratricopeptide repeats but not the fibronectin II domain in the function of SEL1L. Thus, our reverse genetic approach using the chicken DT40 cell line will provide highly useful information regarding ERAD mechanisms in higher eukaryotes which express ERAD components redundantly.

Luminal domain of ATF6 alone is sufficient for sensing endoplasmic reticulum stress and subsequent transport to the Golgi apparatus.

The transcription factor ATF6 is constitutively synthesized as a type II transmembrane protein embedded in the endoplasmic reticulum (ER). When unfolded proteins accumulate in the ER, ATF6 senses such ER stress via an as yet undetermined mechanism and relocates to the Golgi apparatus where it is cleaved by sequential action of Site-1 and Site-2 proteases, allowing liberated N-terminal fragments to translocate into the nucleus. This ATF6-mediated transcriptional induction of ER-localized molecular chaperones and folding enzymes together with components of ER-associated degradation leads to the maintenance of ER homeostasis in mammals. Here, we demonstrated that the luminal domain of ATF6 alone is sufficient for sensing ER stress and subsequent transportation to the Golgi apparatus. This domain of ATF6 was inserted between the N-terminal signal sequence and C-terminal tandem affinity purification tag. The resulting ATF6(C)-TAP translocated into the ER, where it was glycosylated and disulfide bonded. ATF6(C)-TAP occurred as monomer and dimer, and exhibited a relatively short half-life, similar to full-length ATF6. On application of dithiothreitol- or thapsigargin-induced ER stress, the ER chaperone BiP dissociated from ATF6(C)-TAP, and ATF6(C)-TAP was transported to the Golgi apparatus and then secreted into medium. Calnexin and protein disulfide isomerase were identified as cellular proteins capable of binding to ATF6(C)-TAP in addition to BiP, and subsequent analysis revealed that protein disulfide isomerase was bound to ATF6(C)-TAP with chaperone activity. These findings indicate that ATF6(C)-TAP can be used as a tool to isolate protein(s) that escort ATF6 from the ER to the Golgi apparatus in response to ER stress.

Vertebrate unfolded protein response: mammalian signaling pathways are conserved in Medaka fish.

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). The ER stress signal is sensed and transmitted by a transmembrane protein(s) in the ER. The number of these transducers has increased with evolution, one in yeast, three in worm and fly, and five in mammals. Here, we examined medaka fish, Oryzias latipes, as a vertebrate model organism, and found that the medaka genome encodes five UPR transducers. Analysis of a medaka embryonic cell line revealed that the mammalian UPR signaling mechanisms are very well conserved. Thus, XBP1 mRNA, which encodes the transcription factor XBP1 downstream of the IRE1 pathway, was spliced in response to ER stress, resulting in production of the active form of XBP1. Translation was generally attenuated in response to ER stress, which paradoxically induced the translation of ATF4, the transcription factor downstream of the PERK pathway. ATF6 was constitutively synthesized as a transmembrane protein and activated by ER stress-induced proteolysis. Results obtained with the overexpression of active ATF6α, ATF6ß, and XBP1 strongly suggested that ATF6α plays a major role in upregulating the major ER chaperone BiP, contrary to the case in non-vertebrates, in which the IRE1 pathway is essential to the induction of BiP. Physiological ER stress occurring during embryonic development was visualized using transgenic medaka carrying the enhanced green fluorescent protein gene under the control of the BiP promoter. Thus, analysis of the vertebrate UPR using medaka will help provide a more comprehensive understanding of the biology and physiology of the UPR.

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.

Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD.

Sequential mannose trimming of N-glycan, from M9 to M8B and then to oligosaccharides exposing the α1,6-linked mannosyl residue (M7A, M6, and M5), facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). We previously showed that EDEM2 stably disulfide-bonded to the thioredoxin domain-containing protein TXNDC11 is responsible for the first step (George et al., 2020). Here, we show that EDEM3 and EDEM1 are responsible for the second step. Incubation of pyridylamine-labeled M8B with purified EDEM3 alone produced M7 (M7A and M7C), M6, and M5. EDEM1 showed a similar tendency, although much lower amounts of M6 and M5 were produced. Thus, EDEM3 is a major α1,2-mannosidase for the second step from M8B. Both EDEM3 and EDEM1 trimmed M8B from a glycoprotein efficiently. Our confirmation of the Golgi localization of MAN1B indicates that no other α1,2-mannosidase is required for gpERAD. Accordingly, we have established the entire route of oligosaccharide processing and the enzymes responsible.