Identification of an Htm1 (EDEM)-dependent,Mns1-independent Endoplasmic Reticulum-associated Degradation (ERAD) Pathway in Saccharomyces cerevisiae: APPLICATION OF A NOVEL ASSAY FOR GLYCOPROTEIN ERAD*

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control system for newly synthesized proteins in the ER; nonfunctional proteins, which fail to form their correct folding state, are then degraded. The cytoplasmic peptide:N-glycanase is a deglycosylating enzyme that is involved in the ERAD and releases N-glycans from misfolded glycoproteins/glycopeptides. We have previously identified a mutant plant toxin protein, RTA (ricin A-chain nontoxic mutant), as the first in vivo Png1 (the cytoplasmic peptide:N-glycanase in Saccharomyces cerevisiae)-dependent ERAD substrate. Here, we report a new genetic device to assay the Png1-dependent ERAD pathway using the new model protein designated RTL (RTA-transmembrane-Leu2). Our extensive studies using different yeast mutants identified various factors involved in RTL degradation. The degradation of RTA/RTL was independent of functional Sec61 but was dependent on Der1. Interestingly, ER-mannosidase Mns1 was not involved in RTA degradation, but it was dependent on Htm1 (ERAD-related α-mannosidase in yeast) and Yos9 (a putative degradation lectin), indicating that mannose trimming by Mns1 is not essential for efficient ERAD of RTA/RTL. The newly established RTL assay will allow us to gain further insight into the mechanisms involved in the Png1-dependent ERAD-L pathway.     

Endo-β-N-acetylglucosamidases (ENGases) in the fungus Trichoderma atroviride: Possible involvement of the filamentous fungi-specific cytosolic ENGase in the ERAD process

N-Glycosylation is an important post-translational modification of proteins, which mainly occurs in the endoplasmic reticulum (ER). Glycoproteins that are unable to fold properly are exported to the cytosol for degradation by a cellular system called ER-associated degradation (ERAD). Once misfolded glycoproteins are exported to the cytosol, they are subjected to deglycosylation by peptide:N-glycanase (PNGase) to facilitate the efficient degradation of misfolded proteins by the proteasome. Interestingly, the ortholog of PNGase in some filamentous fungi was found to be an inactive deglycosylating enzyme. On the other hand, it has been shown that in filamentous fungi genomes, usually two different fungi-specific endo-β-N-acetylglucosamidases (ENGases) can be found; one is predicted to be localized in the cytosol and the other to have a signal sequence, while the functional importance of these enzymes remains to be clarified. In this study the ENGases of the filamentous fungus Trichoderma atroviride was characterized. By heterologous expression of the ENGases Eng18A and Eng18B in Saccharomyces cerevisiae, it was found that both ENGases are active deglycosylating enzymes. Interestingly, only Eng18B was able to enhance the efficient degradation of the RTL protein, a PNGase-dependent ERAD substrate, implying the involvement of this enzyme in the ERAD process. These results indicate that T. atroviride Eng18B may deglycosylate misfolded glycoproteins, substituting the function of the cytoplasmic PNGase in the ERAD process.     

N-Glycan-dependent and -independent Quality Control of Human δ Opioid Receptor N-terminal Variants

Quality control (QC) in the endoplasmic reticulum (ER) scrutinizes newly synthesized proteins and directs them either to ER export or ER-associated degradation (ERAD). Here, we demonstrate that the human δ-opioid receptor (hδOR) is subjected to ERQC in both N-glycan-dependent and -independent manners. This was shown by investigating the biosynthesis and trafficking of wild-type and non-N-glycosylated F27C variants in metabolic pulse-chase assays coupled with flow cytometry and cell surface biotinylation. Both QC mechanisms distinguished the minute one-amino acid difference between the variants, targeting a large fraction of hδOR-Cys²⁷ to ERAD. However, the N-glycan-independent QC was unable to compensate the N-glycan-dependent pathway, and some incompletely folded non-N-glycosylated hδOR-Cys²⁷ reached the cell surface in conformation incompatible with ligand binding. The turnover of receptors associating with the molecular chaperone calnexin (CNX) was significantly slower for the hδOR-Cys²⁷, pointing to an important role of CNX in the hδOR N-glycan-dependent QC. This was further supported by the fact that inhibiting the co-translational interaction of hδOR-Cys²⁷ precursors with CNX led to their ERAD. Opioid receptor pharmacological chaperones released the CNX-bound receptors to ER export and, furthermore, were able to rescue the Cys²⁷ variant from polyubiquitination and retrotranslocation to the cytosol whether carrying N-glycans or not. Taken together, the hδOR appears to rely primarily on the CNX-mediated N-glycan-dependent QC that has the capacity to assist in folding, whereas the N-glycan-independent mechanism constitutes an alternative, although less accurate, system for directing misfolded/incompletely folded receptors to ERAD, possibly in altered cellular conditions.     

Subcellular localization of endo-β-N-acetylglucosaminidase and high-mannose type free N-glycans in plant cell

Subcellular distribution of plant endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase) and high-mannose type free N-glycans produced by the endoglycosidase has been analyzed using cotyledons of pumpkin seedlings as the model plant cells. Each organelle in the cotyledons was fractionated by ultracentrifugation with the sucrose density gradient system and the endo-β-GlcNAc-ase activity in each fraction was assayed with fluorescence labeled N-glycans as substrates. The endoglycosidase activity was exclusively recovered in the soluble fraction (cytosol fraction) but not in other specific organellar fractions, suggesting that the endoglycosidase would reside predominantly in the cytosol. The quantitative analysis of high-mannose type free N-glycans occurring in each fraction showed that more than 70% of the free N-glycans was recovered from the soluble fraction, suggesting the endoglycosidase would work in the cytosol and the resulting free N-glycans would accumulate in the same fraction. The pumpkin endo-β-GlcNAc-ase (endo-CM) partially purified from the cotyledons showed optimum activity around pH 6.5, supporting this enzyme would reside in the cytosol. Furthermore, the detailed analysis of substrate specificity of endo-CM using various high-mannose type N-glycans showed that the pumpkin enzyme, as well as other plant endo-β-N-acetylglucosaminidases, were highly active toward the high-mannose type glycans bearing the Manα1-2Manα1-3Manβ1-structural unit.     

Non-lysosomal degradation pathway for N-linked glycans and dolichol-linked oligosaccharides

There is growing evidence that asparagine (N)-linked glycans play pivotal roles in protein folding and intra- or intercellular trafficking of N-glycosylated proteins. During the N-glycosylation of proteins, significant amounts of free oligosaccharides (fOSs) and phosphorylated oligosaccharides (POSs) are generated at the endoplasmic reticulum (ER) membrane by unclarified mechanisms. fOSs are also formed in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins destined for proteasomal degradation. This article summarizes the current knowledge of the molecular and regulatory mechanisms underlying the formation of fOSs and POSs in mammalian cells and Saccharomyces cerevisiae.     

Free N-linked oligosaccharide chains: Formation and degradation

There is growing evidence that N-linked glycans play pivotal roles in protein folding and intra- and/or intercellular trafficking of N-glycosylated proteins. It has been shown that during the N-glycosylation of proteins, significant amounts of free oligosaccharides (free OSs) are generated in the lumen of the endoplasmic reticulum (ER) by a mechanism which remains to be clarified. Free OSs are also formed in the cytosol by enzymatic deglycosylation of misfolded glycoproteins, which are subjected to destruction by a cellular system called “ER-associated degradation (ERAD).” While the precise functions of free OSs remain obscure, biochemical studies have revealed that a novel cellular process enables them to be catabolized in a specialized manner, that involves pumping free OSs in the lumen of the ER into the cytosol where further processing occurs. This process is followed by entry into the lysosomes. In this review we summarize current knowledge about the formation, processing and degradation of free OSs in eukaryotes and also discuss the potential biological significance of this pathway. Other evidence for the occurrence of free OSs in various cellular processes is also presented.     

Endoplasmic-reticulum-associated protein degradation inside and outside of the endoplasmic reticulum

Summary Newly synthesized polypeptides that enter the endomembrane system encounter a folding environment in the lumen of the endoplasmic reticulum (ER) constituted by enzymes, lectinlike proteins, and molecular chaperones. The folding process is under scrutiny of this abundant catalytic machinery, and failure of the new arrivals to assume a stable and functional conformation is met with targeting to proteolytic destruction, a process which has been termed ER-associated degradation (ERAD). In recent years it became clear that, in most cases, proteolysis appears to take place in the cytosol after retro-translocation of the substrate proteins from the ER, and to depend on the ubiquitin-proteasome pathway. On the other hand, proteolytic activities within the ER that have been widely neglected so far may also contribute to the turnover of proteins delivered to ERAD. Thus, ERAD is being deciphered as a complex process that requires communication-dependent regulated proteolytic activities within both the ER lumen and the cytosol. Here we discuss some recent findings on ERAD and their implications on possible mechanisms involved.Abbreviations lAT alpha-1-antitrypsin - apoB apolipoprotein B - BiP immunoglobulin-heavy-chain-binding protein - CFTR cystic fibrosis transmembrane conductance regulator - CPY carboxypeptidase Y - ER endoplasmic reticulum - ERAD ER associated degradation - HMG-CoA 3-hydroxy-3-methylglutaryl coenzyme A - MHC major histocompatibility complex - PDI protein disulflde isomerase - TCR T cell antigen receptor     

Wild Type RTA and Less Toxic Variants Have Distinct Requirements for Png1 for Their Depurination Activity and Toxicity in Saccharomyces cerevisiae

Ricin A chain (RTA) undergoes retrograde trafficking and is postulated to use components of the endoplasmic reticulum (ER) associated degradation (ERAD) pathway to enter the cytosol to depurinate ribosomes. However, it is not known how RTA evades degradation by the proteasome after entry into the cytosol. We observed two distinct trafficking patterns among the precursor forms of wild type RTA and nontoxic variants tagged with enhanced green fluorescent protein (EGFP) at their C-termini in yeast. One group, which included wild type RTA, underwent ER-to-vacuole transport, while another group, which included the G83D variant, formed aggregates in the ER and was not transported to the vacuole. Peptide: N-glycanase (Png1), which catalyzes degradation of unfolded glycoproteins in the ERAD pathway affected depurination activity and toxicity of wild type RTA and G83D variant differently. PreG83D variant was deglycosylated by Png1 on the ER membrane, which reduced its depurination activity and toxicity by promoting its degradation. In contrast, wild type preRTA was deglycosylated by the free pool of Png1 in the cytosol, which increased its depurination activity, possibly by preventing its degradation. These results indicate that wild type RTA has a distinct requirement for Png1 compared to the G83D variant and is deglycosylated by Png1 in the cytosol as a possible strategy to avoid degradation by the ERAD pathway to reach the ribosome.     

Roles of molecular chaperones in endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD)

Secreted proteins are synthesized at the endoplasmic reticulum (ER), and a quality control mechanism in the ER is essential to maintain secretory pathway homeostasis. Newly synthesized soluble and integral membrane secreted proteins fold into their native conformations with the aid of ER molecular chaperones before they are transported to post-ER compartments. However, terminally mis-folded proteins may be retained in the ER and degraded by a process called ER-associated degradation (ERAD). Recent studies using yeast have shown that molecular chaperones both in the ER and in the cytosol play key roles during the ERAD of mis-folded proteins. One important role for chaperones during ERAD is to prevent substrate protein aggregation. Substrate selection is another important role for molecular chaperones during ERAD.     

Sec61p is part of the endoplasmic reticulum‐associated degradation machinery

Endoplasmic reticulum‐associated degradation (ERAD) is a cellular pathway for the disposal of misfolded secretory proteins. This process comprises recognition of the misfolded proteins followed by their retro‐translocation across the ER membrane into the cytosol in which polyubiquitination and proteasomal degradation occur. A variety of data imply that the protein import channel Sec61p has a function in the ERAD process. Until now, no physical interactions between Sec61p and other essential components of the ERAD pathway could be found. Here, we establish this link by showing that Hrd3p, which is part of the Hrd‐Der ubiquitin ligase complex, and other core components of the ERAD machinery physically interact with Sec61p. In addition, we study binding of misfolded CPY^* proteins to Sec61p during the process of degradation. We show that interaction with Sec61p is maintained until the misfolded proteins are ubiquitinated on the cytosolic side of the ER. Our observations suggest that Sec61p contacts an ERAD ligase complex for further elimination of ER lumenal misfolded proteins.     

PDI reductase acts on Akita mutant proinsulin to initiate retrotranslocation along the Hrd1/Sel1L-p97 axis

In mutant INS gene–induced diabetes of youth (MIDY), characterized by insulin deficiency, MIDY proinsulin mutants misfold and fail to exit the endoplasmic reticulum (ER). Moreover, these mutants bind and block ER exit of wild-type (WT) proinsulin, inhibiting insulin production. The ultimate fate of ER-entrapped MIDY mutants is unclear, but previous studies implicated ER-associated degradation (ERAD), a pathway that retrotranslocates misfolded ER proteins to the cytosol for proteasomal degradation. Here we establish key ERAD machinery components used to triage the Akita proinsulin mutant, including the Hrd1-Sel1L membrane complex, which conducts Akita proinsulin from the ER lumen to the cytosol, and the p97 ATPase, which couples the cytosolic arrival of proinsulin with its proteasomal degradation. Surprisingly, we find that protein disulfide isomerase (PDI), the major protein oxidase of the ER lumen, engages Akita proinsulin in a novel way, reducing proinsulin disulfide bonds and priming the Akita protein for ERAD. Efficient PDI engagement of Akita proinsulin appears linked to the availability of Hrd1, suggesting that retrotranslocation is coordinated on the lumenal side of the ER membrane. We believe that, in principle, this form of diabetes could be alleviated by enhancing the targeting of MIDY mutants for ERAD to restore WT insulin production.     

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 α1-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 α1,2-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.Recognition and sorting of improperly folded proteins is essential to cell survival, and hence, an elaborate quality control system is found in cells. ER⁴ quality control is well characterized with respect to the N-linked oligosaccharides regulating the folding and degradation of newly synthesized proteins in the ER (1). Immediately after polypeptides enter the ER, Glc₃Man₉GlcNAc₂ (G3M9) precursor oligosaccharides are covalently attached and subsequently processed. Terminally misfolded proteins are removed from the ER by the ERAD machinery (1–4). Aberrant conformers are recognized, retrotranslocated to the cytosol, and degraded by the ubiquitin-proteasome system (5, 6). Processing of mannose residues from the N-linked oligosaccharides acts as a timer for the recognition of misfolded glycoproteins in the ER lumen (1, 7). ER α1,2-mannosidase I (ER ManI) in mammals and ER α-mannosidase in yeast preferentially trim mannose residues from the middle branch of N-glycans, generating the Man₈GlcNAc₂ (M8) isomer B (M8B) (8). In mammals, further mannose processing is required as a signal for degradation (1, 9, 10), whereas the presence of M8B is sufficient to signal degradation in yeast (11). The postulated lectin EDEMs in mammals, their yeast homolog Htm1p/Mnl1p, and the yeast MRH domain-containing lectin Yos9p have all been proposed to recognize glycoproteins targeted for degradation (12).The role of Yos9p in glycoprotein ERAD was identified using a genetic screen in Saccharomyces cerevisiae (13). Yos9p, a homolog of hOS-9, contains an MRH domain (14) and functions as a lectin. Yos9p recognizes substrates of the ERAD-lumenal pathway (15–17), generating a large ER membrane complex containing the Hrd1p-Hrd3p ubiquitin ligase core complex (18–20). The M8B and Man₅GlcNAc₂ (M5) N-glycans are predicted to function as ligands for Yos9p (17). Bipartite recognition of both glycan and polypeptide by Yos9p has also been reported (15).Recent studies revealed that two mammalian MRH domain-containing lectins, OS-9 and XTP3-B, are ER luminal proteins involved in ER quality control and form a large complex containing the HRD1-SEL1L ubiquitin-ligase in the ER membrane (21–24). The components of the complex are similar to yeast, suggesting evolutionary conservation, although the molecular mechanisms underlying the role of OS-9 and XTP3-B remain elusive. Studies using lectin mutants have suggested that the MRH domains are required not for binding to ERAD substrates but for interactions with SEL1L (21), which has multiple N-glycans (25, 26). Additionally, lectin activity appears to be dispensable for hOS-9 binding to misfolded glycoproteins (21, 24). Thus, to understand the role of hOS-9 in the ER quality control pathway, the specific carbohydrate structures recognized by the hOS-9 MRH domain need to be identified, and the requirement of the lectin domain in substrate recognition needs to be determined.In the present study we demonstrate that the lectin activity of hOS-9 is required for enhancement of glycoprotein ERAD. We identified the N-glycan structures recognized by the recombinant hOS-9 MRH domain in vitro by frontal affinity chromatography (FAC). Using a model ERAD substrate, NHK (27), we show that the ability of hOS-9 to enhance ERAD in vivo depends on the oligosaccharides present on NHK, consistent with the FAC results.     

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.     

A new autophagy-related checkpoint in the degradation of an ERAD-M target

The endoplasmic reticulum (ER) harbors elaborate quality control mechanisms to ensure proper folding and post-translational modifications of polypeptides targeted to this organelle. Once an aberrant protein is detected, it is dislocated from the ER and routed to the proteasome for destruction. Autophagy has been recently implicated in the elevation of the ER stress response; however, the involvement of this pathway in selective removal of ER-associated degradation (ERAD) substrates has not been demonstrated. In the present study, we show that an ER membrane lesion, associated with the accumulation of the yeast ERAD-M substrate 6Myc-Hmg2p elicits the recruitment of Atg8 and elements of the cytosol to vacuole targeting (CVT) to the membrane, leading to attenuation in the degradation process. Deletion of peptide:N-glycanase (PNG1) stabilizes this association, a process accompanied by slowdown of 6Myc-Hmg2p degradation. Truncation of the unstructured C-terminal 23 amino acids of 6Myc-Hmg2p rendered its degradation PNG1-independent and allowed its partial delivery to the vacuole in an autophagy-dependent manner. These findings demonstrate a new conduit for the selective vacuolar/lysosomal removal of ERAD misfolded proteins by an autophagy-related machinery acting concomitantly with the proteasome.     

Roles of Protein-disulfide Isomerase-mediated Disulfide Bond Formation of Yeast Mnl1p in Endoplasmic Reticulum-associated Degradation

The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway.The endoplasmic reticulum (ER)² is the first organelle in the secretory pathway of eukaryotic cells and provides an optimum environment for maturation of newly synthesized secretory and membrane proteins. Protein folding/assembly in the ER is aided by molecular chaperones and folding enzymes. Molecular chaperones in the ER assist folding of newly synthesized proteins and prevent them from premature misfolding and/or aggregate formation (1, 2). Protein folding in the ER is often associated with formation of disulfide bonds, which contribute to stabilization of native, functional states of proteins. Disulfide bond formation could be a rate-limiting step of protein folding both in vitro and in vivo (3, 4), and the ER has a set of folding enzymes including protein-disulfide isomerase (PDI) and its homologs that catalyze disulfide bond formation (5, 6).In parallel, protein folding/assembly in the ER relies on the inherent failsafe mechanism, i.e. the ER quality control system, to ensure that only correctly folded and/or assembled proteins can exit the ER. Misfolded or aberrant proteins are retained in the ER for refolding by ER-resident chaperones, whereas terminally misfolded proteins are degraded by the mechanism known as ER-associated degradation (ERAD). The ERAD consists of recognition and processing of aberrant substrate proteins, retrotranslocation across the ER membrane, and subsequent proteasome-dependent degradation in the cytosol. More than 20 different components have been identified to be involved in this process in yeast and mammals (7).The majority of proteins synthesized in the ER are glycoproteins, in which N-linked glycans are not only important for folding but also crucial for their ERAD if they fail in folding. Specifically, trimming of one or more mannose residues of Man₉GlcNAc₂ oligosaccharide and recognition of the modified mannose moiety represent a key step for selection of terminally misfolded proteins for disposal (8). A mannosidase I-like protein, Mnl1p/Htm1p (yeast), and EDEM (mammals, ER degradation enhancing α-mannosidase-like protein) were identified as candidates for lectins that recognize ERAD substrates with modified mannose moieties (9–11). Both Mnl1p and EDEM contain an N-terminal mannosidase homology domain (MHD), which lacks cysteine residues conserved among α1,2-mannosidase family members and is proposed to function in recognition of mannose-trimmed carbohydrate chains (supplemental Fig. S1). However, whether Mnl1p or EDEM indeed functions as an ERAD-substrate-binding lectin or has a mannosidase activity is still in debate (11–15), and Yos9p was suggested to take the role of ERAD-substrate binding lectin (14, 16–18). Mnl1p, but not EDEM, has a large C-terminal extension, which does not show any homology to known functional domains and is conserved only among fungal Mnl1p homologs (supplemental Fig. S1).After recognition of the modified mannose signal for degradation, aberrant proteins are maintained or converted to be retrotranslocation competent by ER chaperones including BiP (19). PDI was also indicated to be involved in these steps in the ERAD by, for example, its possible chaperone-like functions (20–23). The yeast PDI, Pdi1p, contains four thioredoxin-like domains, two of which have a CGHC motif as active sites, followed by a C-terminal extension containing the ER retention signal. During its catalytic cycle, PDI transiently forms a mixed disulfide intermediate with its substrate through an intermolecular disulfide bond between the cysteine residues of the active site of PDI and the substrate molecule.Here we report identification of PDI as an Mnl1p-interacting protein. Stable interactions between the C-terminal domain of Mnl1p and PDI involve intermolecular disulfide bonds. Stably interacting PDI is required for formation of the functionally essential intramolecular disulfide bond in the MHD of Mnl1p, which in turn stabilizes and prolongs the Mnl1p-PDI interactions. Possible roles for those stable interactions between Mnl1p and PDI in the ERAD will be discussed.     

Usa1 Protein Facilitates Substrate Ubiquitylation through Two Separate Domains

Background

Defects in protein folding are recognized as the root of many neurodegenerative disorders. In the endoplasmic reticulum (ER), secretory proteins are subjected to a stringent quality control process to eliminate misfolded proteins by the ER-associated degradation (ERAD) pathway. A novel ERAD component Usa1 was recently identified. However, the specific role of Usa1 in ERAD remains obscure.

Methodology/Principal Findings

Here, we demonstrate that Usa1 is important for substrate ubiquitylation. Furthermore, we defined key cis-elements of Usa1 essential for its degradation function. Interestingly, a putative proteasome-binding motif is dispensable for the functioning of Usa1 in ERAD. We identify two separate cytosolic domains critical for Usa1 activity in ERAD, one of which is involved in binding to the Ub-protein ligase Hrd1/Hrd3. Usa1 may have another novel role in substrate ubiquitylation that is separate from the Hrd1 association.

Conclusions/Significance

We conclude that Usa1 has two important roles in ERAD substrate ubiquitylation.     

An in vitro assay for the selective endoplasmic reticulum associated degradation of an unglycosylated secreted protein

The endoplasmic reticulum (ER) represents the first compartment into which nascent secreted proteins traffic, and not coincidentally the ER lumen houses a high concentration of factors that facilitate protein folding, such as molecular chaperones. To off-set the potentially lethal consequences of mis-folded secreted protein accumulation, aberrant proteins may be selected for degradation via a process known as ER associated degradation (ERAD). After their selection ERAD substrates are retro-translocated back to the cytoplasm and then degraded by the 26S proteasome. Key features of the selection, retro-translocation, and degradation steps that constitute the ERAD pathway were elucidated through the development of an in vitro ERAD assay. In this assay the fates of two yeast proteins can be distinguished after their translocation, or import into ER-derived microsomes. Whereas a wild type, glycosylated protein ("Gp(alpha)F") is stable, a non-glycosylated version of the same protein ("p(alpha)F") is rapidly degraded when microsomes containing radiolabeled forms of these substrates are incubated in cytosol and ATP. The purpose of this chapter is first to discuss the experimental findings from the use of the in vitro assay, and then to describe the assay in detail. Finally, future potential uses of the in vitro system are illustrated.     

Definitive evidence that a single N-glycan among three glycans on inducible costimulator is required for proper protein trafficking and ligand binding

Glycosylation is a widespread post-translational modification found in glycoproteins. Glycans play key roles in protein folding, quality control in the endoplasmic reticulum (ER) and protein trafficking within cells. However, it remains unclear whether all positions of protein glycosylation are involved in glycan functions, or if specific positions have individual roles. Here we demonstrate the integral involvement of a specific N-glycan from amongst the three glycans present on inducible costimulator (ICOS), a T-cell costimulatory molecule, in proper protein folding and intracellular trafficking to the cell surface membrane. We found that glycosylation-defective mutant proteins lacking N-glycan at amino-acid position 89 (N89), but not proteins lacking either N23 or N110, were retained within the cell and were not detected on the cell surface membrane. Additional evidence suggested that N89 glycosylation was indirectly involved in ICOS ligand binding. These data suggest that amongst the three putative ICOS glycosylation sites, N89 is required for proper ICOS protein folding in the ER, intracellular trafficking and ligand binding activity. This study represents a substantial contribution to the current mechanistic understanding of the necessity and potential functions of a specific N-glycan among the multiple glycans of glycoproteins.     

Endoplasmic Reticulum Calcium Regulates the Retrotranslocation of Trypanosoma Cruzi Calreticulin to the Cytosol

For most secretory pathway proteins, crossing the endoplasmic reticulum (ER) membrane is an irreversible process. However, in some cases this flow can be reversed. For instance, misfolded proteins retained in the ER are retrotranslocated to the cytosol to be degraded by the proteasome. This mechanism, known as ER associated degradation (ERAD), is exploited by several bacterial toxins to gain access to the cytosol. Interestingly, some ER resident proteins can also be detected in the cytosol or nucleus, calreticulin (CRT) being the most studied. Here we show that in Trypanosoma cruzi a minor fraction of CRT localized to the cytosol. ER calcium depletion, but not increasing cytosolic calcium, triggered the retrotranslocation of CRT in a relatively short period of time. Cytosolic CRT was subsequently degraded by the proteasome. Interestingly, the single disulfide bridge of CRT is reduced when the protein is located in the cytosol. The effect exerted by ER calcium was strictly dependent on the C-terminal domain (CRT-C), since a CRT lacking it was totally retained in the ER, whereas the localization of an unrelated protein fused to CRT-C mirrored that of endogenous CRT. This finding expands the regulatory mechanisms of protein sorting and may represent a new crossroad between diverse physiological processes.     

Quality control: another player joins the ERAD cast

The quality control system known as ERAD removes misfolded proteins from the ER to the cytosol for degradation. The AAA ATPase Cdc48p and ubiquitin ligases play crucial roles; their relationship has been unclear, but recent work has shown that the membrane protein Ubx2p links their functions in yeast.