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The EDEM and Yos9p families of lectin-like ERAD factors
Seminars in Cell & Developmental Biology 18 (2007) 743–750
Review
The EDEM and Yos9p families of lectin-like ERAD factors Kazue Kanehara, Shinichi Kawaguchi, Davis T.W. Ng ∗ Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore 117604, Singapore Available online 8 September 2007
Abstract Protein quality control pathways monitor the folding of newly synthesized proteins throughout the cell. Irreversibly misfolded proteins are sorted and degraded to neutralize their potential toxicity. In the secretory pathway, multiple strategies have evolved to test the wide diversity of molecules that traffic through the endoplasmic reticulum. The organelle has adapted the use of N-linked glycans to signal protein folding states. The signals are read by the EDEM and Yos9 protein families that take substrates out of folding cycles for degradation. © 2007 Elsevier Ltd. All rights reserved. Keywords: ER-associate degradation; ER quality control; ERAD; EDEM; Htm1p; Yos9p; Misfolded proteins; Lectin; N-linked glycosylation
Contents 1. 2. 3. 4. 5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-linked carbohydrates: active participants in ERQC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The ␣-mannosidase-like lectins of ERAD: EDEMs and Htm1p/Mnl1p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yos9p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Quality control systems that monitor folding operate everywhere that proteins are synthesized (for review, see [4,38,54]). Although the basic principle is simple—misfolded proteins are recognized and degraded—how cells accomplish the task remains mysterious. Protein synthesis in the endoplasmic reticulum (ER) is a particular challenge. The organelle’s dynamic nature and the sheer volume of cargo traversing it require that the flux be tightly regulated. ER localization of chaperones and modifying enzymes require unfolded proteins to be retained and the transport of folded proteins unfettered. The monitoring mechanisms must also contend with structurally diverse substrates. These include soluble luminal proteins, proteins linked to lipids, and integral membrane proteins. Membrane proteins can
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be intrinsically more difficult because they can have structures in the lumen, the lipid bilayer, and the cytosol. This review will focus on the terminal phase of ER quality control (ERQC) called ER-associated degradation (ERAD). Substrates of all ERAD pathways are degraded by the cytosolic 26S proteasome. Thus, misfolded proteins are recognized in the ER and transported to the cytosol for degradation. Although ERAD appears to be the major mechanism of disposal, recent studies have reported that aberrant glycoproteins can sometimes take alternative routes [33]. The current understanding of ERAD comes mostly from studies of the budding yeast and mammalian model systems [16,17,30]. Given the evolutionary expanse between fungi and mammals, it was not surprising to observe differences in their specific strategies and compositions. To monitor the diversity of substrates, each organism relies on multiple ERAD pathways. Usually, the distinct steps of recognition and degradation are linked by dedicated protein complexes that receive substrates, ubiquitinate them, and facilitate their transfer to the proteasome.
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Studies from budding yeast indicate that their repertoire of ERAD pathways represent a basic conserved standard set [4,37,40]. Higher eukaryotes differ by an expansion of components and pathways, presumably to satisfy the needs of more complex organisms. In budding yeast, three ERQC pathways have been described that can account for much of the organelle’s needs. These are the ERAD-C (ER-associated degradation-Cytosol), ERAD-M (-Membrane), and ERAD-L (Lumen) pathways that monitor folding of cytosolic, membrane, and luminal domains of client proteins, respectively (Fig. 1). Two E3 ubiquitin ligases, Hrd1p and Doa10p, organize factors involved in substrate recognition, extraction, and ubiquitination for these pathways [7,8,15]. Doa10p organizes a complex that includes the E2 ubiquitin conjugating enzyme Ubc7p (tethered to the membrane via Cue1p) and the AAA-ATPase Cdc48p with its cofactors Npl4p and Ufd1p (tethered to the membrane via Ubx2p) [7]. These factors are required to initiate the degradation of membrane proteins bearing cytosolic domain lesions (Fig. 1, ERAD-C). The Cdc48p complex provides the mechanical energy to extraction substrates from the ER membrane [2,26,53,61]. It should be noted that the
factors of the Doa10p complex are localized almost entirely or on the cytosolic face of the ER membrane (Fig. 1, Doa10p). Although this organization might reflect its role in monitoring cytosolic domains, the large transmembrane domain of Doa10p has been proposed to serve as a conduit for substrate extraction [32]. Hrd1p, like Doa10p, also assembles with the Ubc7p/Cue1p dimer and the Cdc48p complex (Fig. 1, ERAD-M and ERAD-L). However, additional partners function to detect and process substrates with misfolded luminal domains. These include a direct 1:1 interaction with Hrd3p, a transmembrane protein with a large luminal domain [13]. Bound to Hrd3p is the lectin-like factor Yos9p. Also part of this complex is the integral membrane protein Der1p. Usa1p, whose function is unknown, links Der1p to Hrd1p [7]. A notable factor that works through the Hrd1p complex but is not a stable member is the lectin-like factor Htm1p (also known as Mnl1p). Nearly all known ERAD factors in yeast have homologues in mammals. Among E3 ubiquitin ligases, mammalian HRD1 (also known as synoviolin) is implicated in the turnover of several substrates including the Pael receptor, misfolded insulin,
Fig. 1. ERAD pathways in yeast and mammals. In yeast (unshaded) substrates bearing cytosolic lesions follow the ERAD-C pathway (blue arrows; glycanindependent), membrane lesions follow the ERAD-M pathway (green arrows; glycan-independent), and luminal lesions follow the ERAD-L pathway (pink arrows; glycan-dependent) pathways in yeast. In mammals (shaded green), EDEM family proteins take the substrate out of the calnexin folding cycle (lower right) to the Hrd1 complex (upper right). Other established mammalian ERAD pathways are not shown for the sake of simplicity. After binding to E3 complexes, the substrates are ubiquitinated and extracted from the membranes via the Cdc48p/p97 complex. Substrates are then deglycosylated by protein N-glycanase (Png1) and escorted to the proteasome for degradation.
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and the unassembled T-cell receptor subunits TCR-␣ and CD3␦ [1,27,52]. The human TEB4 protein, on the other hand, is a homolog of yeast Doa10p but its involvement in ERAD is yet to be established [32]. Other mammalian ERAD E3 enzymes have no counterparts in yeast. These include gp78 (which bears some resemblance to Hrd1p), CHIP, and RMA1 [9,39,64]. The list, yet to be completed, illustrates the expanded repertoire of pathways that make up mammalian ERAD. The Cdc48p complex and Ubc7p, common to both yeast E3 complexes, have mammalian counterparts with established roles in ERAD [27,53,62]. Of exclusive factors of the Hrd1p complex, mammalian homologs of Hrd3p (SEL1), Der1p (Derlin-1, -2, -3), and Yos9p (OS-9 and XTP3-B) are already known [44,49,57]. The role of Der1p is not yet clear but one of its mammalian homologues Derlin-1 was proposed to function as the protein channel that mediates retrotranslocation of substrates [35,63]. The mammalian homolog for Htm1p is EDEM (ER degradation enhancing ␣-mannosidaselike protein) [23]. At its essence, ERQC encompasses a series of pathways that integrates folding, traffic control, sorting and disposal of misfolded proteins. Understanding the system requires the characterization of individual pathways, the identification of components and their organization, the precise dissection of the sequential events, and how individual factors fit into each step. Several articles in this volume bring together the current understanding of one major aspect of ERQC—how misfolded glycoproteins are recognized and degraded. This article will review the functions of the lectin-like factors Htm1p/EDEM and Yos9p that act at an early step of glycoprotein ERAD. 2. N-linked carbohydrates: active participants in ERQC The attachment of N-linked carbohydrates is essential for the maturation of many proteins [19]. Some depend on the calnexin chaperone cycle while others use glycans to prevent protein aggregation or to provide the correct chemical composition at those sites. Glycans can play a critical role in the recognition and disposal of misfolded proteins. In Saccharomyces cerevisiae, a mutant carboxypeptidase Y enzyme called CPY* misfolds, is retained in the ER, and rapidly degraded by ERAD [10]. A non-glycosylated version of CPY* is subject to ER retention by ERQC but fails to degrade [29]. This suggested a role for N-linked glycans for entry into the ERAD pathway. In both mammalian and yeast cells, the carbohydrate structure seems to be important for ERAD (See Caramilo and Parodi, this issue). N-linked carbohydrates in all eukaryotes share the same core structure when first attached to the protein (Fig. 2). A series of carbohydrate trimming events begins with the removal of the terminal glucose residue by glucosidase I. Glucosidase II removes the next two progressively. This process is rapid in budding yeast but can be staggered in other organisms for the calnexin folding cycle [19]. In yeast, the ER ␣1,2-mannosidase Mns1p cleaves the terminal mannose residue on the B branch of the glycan leaving the structure Man8 GlcNAc2 [6]. In mammalian cells, the ER ␣-mannosidase I carries out the same reaction and can sometimes remove an additional mannose residue [18].
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Fig. 2. Schematic representation of the conserved core N-linked glycan. NAcetylglucosamine (GlcNAc) is represented as blue squares, mannose as pink circles, and glucose as lavender triangles. The glycosidic bond linkages between each sugar moiety are indicated.
It was observed that the ␣-mannosidase inhibitors kifunensine and 1-deoxymannojirimycin or cells deficient in ␣-mannosidase activity disrupt the degradation of misfolded glycoproteins [56]. Furthermore, overexpression of mammalian ER mannosidase I accelerates glycan trimming and degradation of misfolded substrates [22]. Since ER ␣-mannosidases are slow acting compared to the glucosidases, it was proposed that the enzyme might act as a molecular timer for folding [18,25]. It was an appealing hypothesis since it would enable the quality control system to recognize a trimmed glycan as a marker for degradation. However, it is unlikely that it could serve as a signal alone because glycans from folded proteins also proceed to Man8 GlcNAc2 [6]. A modification of the hypothesis emerged recently that might overcome the limitation. Processing to forms smaller than Man8 GlcNAc2 has been reported for some ERAD substrates [11]. The Man7 GlcNAc2 , Man6 GlcNAc2 , and Man5 GlcNAc2 glycans have been proposed as favored for ERAD since they are poor substrates for reentry into the calnexin cycle. Alternatively, studies of the model substrates CPY* and PrA*, misfolded versions of yeast vacuolar proteases, revealed that each harbored a single, specific carbohydrate that is necessary and sufficient for ERAD [31,55]. Since all four glycans of CPY* have the Man8 GlcNAc2 structure only (with no further trimming [58]), the findings provided evidence that the signal for ERAD-L is likely bipartite and composed of the glycan and a protein determinant. This provides the ERAD machinery with a means of differentiating folding (Man9 , unfolded determinant), folded (Man8 , folded determinant), and misfolded (Man8 , unfolded determinant) proteins. The nature of a putative protein determinant remains uncharacterized.
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Recently, the mannosidase timer hypothesis was challenged. Parodi and coworkers reported that although the fission yeast Schizosaccharomyces pombe expresses a functional ER ␣1,2mannosidase (a Mns1p homolog called Spmns1p), misfolded glycoproteins primarily possess Man9 GlcNAc2 glycans due its poor activity [43]. Despite the deficiency, CPY* degrades as efficiently as it does in S. cerevisiae. The data support the idea that a Man8 GlcNAc2 glycan is not required for ERAD in fission yeast. Surprisingly, deletion of the gene encoding the Spmns1 or addition of kifunensine led to strong stabilization of CPY*. To explain the seemingly contradictory results, the authors proposed that Spmns1p’s ERAD function is distinct from its enzymatic activity. One possibility is that it acts like a lectin factor similar to that proposed for Htm1/EDEM. This might help explain how kifunensine, a molecule that inserts into the carbohydrate-binding pocket of Spmns1p, would also inhibit ERAD. In budding yeast, strains lacking glucosidase I or glucosidase II degrade CPY* poorly [21,25]. The Mns1p ␣-mannosidase is present in both cases and active in a gls2 mutant. Similarly, alg3, alg9, and alg12 mutants that result in the attachment of Man5 GlcNAc2 , Man6 GlcNAc2 , and Man7 GlcNAc2 glycans (which were proposed to stimulate ERAD in mammals [11]), respectively, also resulted in the stabilization of CPY* [25]. Thus, whether the conversion from Man9 GlcNAc2 to Man8 GlcNAc2 is more incidental than causal for substrate entry into ERAD, the attachment of N-linked carbohydrates and their proper structures remain an essential element even as the optimal structure might vary among organisms. 3. The ␣-mannosidase-like lectins of ERAD: EDEMs and Htm1p/Mnl1p The importance of glycans led to a proposal that a lectin receptor could be involved in ERAD [25]. The subsequent discovery of EDEM and Htm1p/Mnl1p in mammals and yeast, respectively, confirmed the notion [23,24,41,46]. EDEM1 encodes a 652 amino acid protein with sequence similarity to class I ␣1,2-mannosidases. Initially, EDEM1 appeared to be a type II integral protein localized to the ER [23] but recent data from the Molinari group indicate that EDEM1 is a soluble luminal protein in some cell types [51]. Functionally, its regulation by the unfolded protein response (UPR) suggested a role in ER stress tolerance [23]. By contrast, ER mannosidase I transcription was unchanged under ER stress. Consistent with its possible role as a lectin, ␣1,2-mannosidase activity was undetectable for this ER protein. Although EDEM1 loss-offunctions experiments were not available at the time, EDEM1 overexpression accelerated the turnover of the NHK variant of ␣1-antitrypsin, an established ERAD substrate [23,41]. Furthermore, EDEM1 was found associated with the NHK substrate in co-immunopreciptation experiments. These data supported the view that EDEM1 is an ERAD factor. In parallel, two reports describing the yeast EDEM homolog provided complementary evidence for a role in ERAD [24,46]. The lone EDEM homolog, called HTM1/MNL1, encodes a protein of 796 amino acids with an N-terminal cleaved signal
sequence. HTM1/MNL1 deleted cells are viable and exhibit no defects in glycan trimming. They do, however, exhibit strong defects in the turnover of misfolded glycoproteins. The non-glycosylated ERAD substrates Gp␣F (misfolded pro␣factor) and Sec61-2p (unstable translocon subunit), however, are degraded normally in its absence. Interestingly, unlike mammalian EDEM1, HTM1/MNL1 expression is unresponsive to ER stress [59]. The significance of this difference is unknown but enhancement of ERAD by the UPR is not dependent on its upregulation in yeast [59]. Taken together, these reports established that the EDEM/Htm1p/Mnl1p lectin-like orthologues perform a conserved ERAD function, specifically in the degradation of misfolded glycoproteins. How does EDEM/Htm1p/Mnl1p fit into established ERQC pathways? In yeast, Htm1p/Mnl1p functions in the ERAD-L pathway through the Hrd1p complex [60]. It is not a stable member of the complex, and as a soluble protein, likely acts at a step upstream [7,8,14]. The function of mammalian EDEM1 is better defined. In a series of elegant studies from the Nagata and Molinari groups, EDEM1 was shown to be a factor that connects the calnexin cycle to ERAD [41,48]. Calnexin (CNX) is an ER lectin that assists the folding of some glycoproteins. The calnexin cycle is reviewed separately in this volume and therefore only briefly described here (See Caramilo and Parodi, this issue). During or immediately following protein translocation and glycosylation, N-linked glycans are rapidly trimmed of their two terminal glucose residues by glucosidases I and II (Fig. 2). Monoglucosylated glycoproteins are then substrates of calnexin or calreticulin. After a round of folding attempts, the substrate is released, deglucosylated by glucosidase II, and scanned for unfolded structures by UDP-glucose:glycoprotein glucotransferase (UGT). If the molecule not fully folded, UGT will reglucosylate the substrate so it can reenter the calnexin cycle for another round (Fig. 1). Not all proteins that enter the calnexin cycle will fold. If a protein becomes irreversibly misfolded, EDEM1 interrupts otherwise futile folding cycles and diverts the substrate to ERAD. EDEM and calnexin interact directly to facilitate this step [48]. Elevation of EDEM1 to its equivalent level under ER stress reduces substrate intervals in the calnexin cycle and accelerates their degradation [41,48]. Under these conditions, it was observed that substrates shift more to the unglucosylated state suggesting a role of EDEM1 is to shield or divert molecules from UGT. Kifunensine treatment blocks these effects altogether suggesting that mannose trimming is required for EDEM1 function [41]. The very act of taking misfolded proteins out of a chaperone environment would risk aggregation, a physical state that is incompatible with ERAD. In yeast, the ER chaperone BiP maintains substrates in a soluble, ERAD-competent state [47]. Similarly, EDEM1 overexpression inhibits the formation of disulfide-linked higher order structures for clients of the calnexin cycle, which can be less dependent on BiP for their folding [42]. Therefore, EDEM1 appears to play a dual role. First, as an escort of substrates out of the calnexin cycle, second, as a lectin chaperone maintaining substrate solubility for ERAD.
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In budding yeast, the function of Htm1p/Mnl1p was placed into the Hrd1 pathway genetically [60]. However, a direct interaction with factors of the complex has not been reported. In mammals, EDEM1 can be co-immunoprecipitated with Derlin2 and Derlin-3, homologs of the yeast Der1p ERAD protein [49]. The Derlin family of integral membrane proteins was proposed to form all or part of the membrane channel for substrate retrotranslocation [35,63]. They form complexes with the Cdc48p homolog p97 and with the mammalian Hrd1/SEL1 complex [35,44,63]. These data indicate that EDEM1 provides a link between the calnexin folding pathway and the Hrd1 ERAD substrate-processing site in the ER membrane (Fig. 1). Although less well characterized, the other members of the EDEM family, EDEM2 and EDEM3, also participate in ERQC. The homologues share significant sequence identity within the ER mannosidase I homology domain and vary sharply in other segments (Fig. 3 and [36]). The human EDEM2 gene encodes a soluble protein of 578 amino acids while mouse EDEM3 encodes the largest member at 931 amino acids. EDEM3 carries a long C-terminal extension beyond the mannosidase homology domain that includes a protease-like domain and the KDEL retention signal [20]. EDEM1 and EDEM2, on the other hand, lack any obvious retention signals, suggesting that their localization may require association with other ER resident proteins. The mouse and human orthologues share 90% or greater sequence identity [20,36]. EDEM2 is ubiquitously expressed in all tissues examined and it localizes to the ER. Like the earlier experiments performed with EDEM1, EDEM2 lacks a detectable ␣1,2-mannosidase
activity when examined in vitro and in vivo [51]. Overexpression of EDEM2 accelerated the degradation of two variants of ␣1-antitrypsin, NHK and PI Z, but not a non-glycosylated variant. Thus, EDEM2 shares several functional similarities with EDEM1. Their substrate overlap also suggests that the homologues might perform semi-redundant functions. This notion is supported by the modest effect on BACE457 degradation when only EDEM1 expression is knocked-down [41]. At first glance, EDEM3 appears to be most enigmatic member with its prominent C-terminal extension. However, its characterization revealed that EDEM family members share some characteristics [20]. All mammalian members encode soluble ER proteins that are transcriptionally regulated by the UPR. Like the others, EDEM3 overexpression accelerates the degradation of the glycosylated substrates NHK and the unassembled ␣ subunit of the T-cell receptor. One difference is the extent of demannosylation of N-linked glycans. In addition to the Man8 GlcNAc2 glycan, EDEM3 overexpression stimulated the formation of Man7 GlcNAc2 , Man6 GlcNAc2 glycans and to a lesser extent, Man5 GlcNAc2 structures. The finding was interesting because of other reports showing accumulated misfolded proteins can be demannosylated to structures smaller than Man8 GlcNAc2 in mammalian cells [11]. Based on structural insights gleaned from ␣-mannosidases, the Molinari group reexamined the issue of whether EDEM1 can itself act as an ␣1,2-mannosidase. They found that EDEM1 overexpression enhanced demannosylation of ERAD substrates [50]. Although the study could not rule out indirect effects of EDEM overexpression on mannosidase activity (e.g., activating other ␣-mannosidases), it was argued that all the critical residues required for catalysis are intact in the EDEMs and that the missing cysteine residues conserved in most ␣-mannosidases may not be essential for enzymatic activity [50]. Indeed, inserting a mutation in one of EDEM1’s conserved catalytic residues abolished enhanced ␣-mannosidase activity while largely retaining the factor’s ability to accelerate degradation. From these data, the authors propose an interesting hypothesis. Here, the ER mannosidase I (not a UPR target) slowly converts Man9 GlcNAc2 to Man8 GlcNAc2 for typical ER quality control. Under ER stress, the UPR upregulates EDEM1 and EDEM3. Their activation causes the rapid and extensive demannosylation of N-glycans (the A branch in particular). This reduces reglucosylation of substrates by UGT and accelerates the clearance of aberrant proteins that would otherwise overload the ER.
Fig. 3. Schematic representation of EDEM and Yos9 protein families. (A) MRH domain containing proteins. The mannose 6-phosphate receptor homology (MRH) domain is shown in lavender, the predicted signal sequence in black and the predicted transmembrane region in green. The C-terminal HDEL ER retention signal is indicated. CD-MPR; cation-dependent mannose 6-phosphate receptor (bovine, P11456), OS-9 (mouse, NP 808282), Yos9p (Saccharomyces cerevisiae, YDR057w). (B) ER mannosidase I and the EDEM family. The mannosidase homology domain (MHD) is shown in blue, the predicted signal sequence in black and the predicted transmembrane domain in green. The C-terminal KDEL ER retention signal is indicated. ER Man I; ER mannosidase I (human, NP 057303), EDEM1 (human, CAC69370), EDEM2 (human, NP 060687), EDEM3 (human, NP 079467) and Htm1/Mnl1 (Saccharomyces cerevisiae, YHR204w).
4. Yos9p First characterized in budding yeast, Yos9p represents a second class of lectin-like ERAD factors. This class is evolutionarily distinct from the EDEM family because it shares its lectin-like domain with the mannose 6-phosphate receptor (MPR) family (Fig. 3). The YOS9 (yeast osteosarcoma 9) gene was given its curious moniker because of its similarity to OS-9, a gene found in amplified chromosomal DNA from human tumors [57]. YOS9 predicts a 542 amino acid protein with four N-linked glycosylation sites, an amino terminal cleaved signal sequence,
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and a C-terminal HDEL ER retention signal. Initial biochemical analysis suggested that Yos9p is peripherally associated with the ER membrane [12]. Sean Munro first suggested that Yos9p might function by binding N-glycans [45]. His work on Mrl1p, a yeast relative of the mammalian MPRs, helped reveal the conservation of the MPR glycan-binding module across multiple protein classes and organisms. Appearance of the modules, called mannose 6phosphate homology domains (MRH), in otherwise dissimilar proteins, suggested an evolutionary assimilation of the domain for diverse functions. It would be a few years later before Yos9p’s role in ERAD was reported [3,5,28,58]. Although conserved evolutionarily, functional analysis of the homologues OS-9 and XTP3-B has not yet been reported. Yos9p was discovered variously through genetic screens for mutants that disrupted ERAD. The large number of substrates examined among the studies revealed a clear pattern. Glycosylated substrates bearing lesions in the ER lumen (both soluble and membrane bound) requires Yos9p function while those bearing lesions in their transmembrane or cytosolic domains do not. Engineered YOS9 mutants that alter conserved residues in the carbohydrate-binding domain abolish ERAD function. This provided indirect evidence that Yos9 functions through the binding of glycans [3,58]. Despite the obvious functional similarity with the EDEM family, genetic analyses indicated that Yos9p and Htm1p perform distinct functions within the same pathway. Both are equally indispensable for ERAD-L and strains deleted of both genes are no worse off than individual knock-outs [3,5,28,58]. Yos9p exhibits the expected characteristics of a recognition factor for misfolded proteins. In co-immunoprecipitation and crosslinking experiments, Yos9p binds to misfolded proteins and not to folded ones [3,28,58]. Unfortunately, the role of the glycans was not clarified through these analyses. In one study, Yos9p prefers CPY* molecules bearing Man8 GlcNAc2 and Man5 GlcNAc2 glycans [58]. This result mirrors the carbohydrate structures that are optimal for efficient degradation [25]. By contrast, the second study reported that Yos9p could bind to CPY* lacking any glycans [3]. Although the results appear contradictory, it is important to note that the experiments were performed differently. In the first study, the pull-down experiment utilized a Yos9p-protein A fusion expressed at endogenous levels. In the second, crosslinking was applied to the HA-tagged Yos9p expressed from a multi-copy plasmid. The difference could reflect a greater stringency inherent to the Yos9p-protein A approach that revealed differences in binding affinity. Conversely, the combination of chemical crosslinking and Yos9p overexpression may have increased sensitivity for weaker interactions. With Yos9p firmly established as a critical ERAD factor, a second wave of studies provided the best view to date into the inner workings of ERAD. The Sommer, Weissman, and Rapoport groups used Yos9p and the ERAD E3s as points of reference for detailed biochemical analyses. Tandem affinity tagged (TAP) versions of the E3 ubiquitin ligases Hrd1p and Doa10p revealed that they organize two distinct ERAD-specific protein complexes [7]. On the luminal side, Yos9p is part of complex that includes the Hrd1/Hrd3p dimer and the chaperone Kar2p
[7,8,14]. Hrd1p directly interacts with its E2 Cue1p/Ubc7p, Der1p, Usa1p, and the Cdc48p complex (Fig. 1). Cells deleted of HRD1 do not disrupt the Kar2p/Yos9p/Hrd3p luminal complex but these proteins no longer interact with the Cdc48p complex [8,15]. Thus, Hrd1p forms the critical link between recognition events in the lumen and the ubiquitination and protein extraction activities in the cytosol. Surprisingly, Yos9p and Hrd3p can bind substrates whether they are glycosylated or not [8,15]. If substrates can be recruited efficiently to Hrd3p (and therefore, the Hrd1 complex), what purpose does Yos9p serve? Despite the binding data, it should be emphasized that ERAD-L glycoprotein substrates like CPY* degrade very poorly in the absence of Yos9p. Non-glycosylated variants are degraded poorly whether or not Yos9p is present. Thus, the Hrd1 complex lectin and substrate glycans remain critical elements of ERAD-L even as binding experiments suggest that neither are required for substrate recruitment. To explain these data, it was proposed that Yos9p performs a “proofreading” or “gating function” [8,15]. In this scenario, Hrd3p can recruit misfolded proteins but Yos9p scans the substrate for Man8 GlcNAc2 glycans. Only if this requirement is satisfied will the substrate be passed through a pore complex (which remains unidentified) for ubiquitination and degradation. This extra level of screening would ensure that folding intermediates would not be degraded. Lending support for this idea, the Weissman group showed that overexpression of Hrd1p itself can partially overcome the loss of Hrd3p and Yos9p in degrading CPY*. More intriguingly, the non-glycosylated variant is now degraded equally to CPY*. These data suggest that while the Hrd1p complex can be made functional in the absence of these factors, the fidelity of substrate selection is impaired [8]. 5. Concluding remarks Early observations that the structures of N-linked glycans can be determinants in ERQC brought forth the hypothesis that glycan trimming could serve as a molecular timer for folding. The search for mediators uncovered the EDEM and Yos9 families of ER lectin-like ERAD factors. The studies presented in this review highlight the importance of glycans in ERQC. However, it should be emphasized that this system represents but a small part of the cellular strategies deployed for protein quality control. By definition, ERAD-M, ERAD-C, and quality control anywhere outside the ER lumen would employ entirely different mechanisms due to the absence of N-linked glycans. Indeed, even within the ER lumen exists a pathway defined by the nonglycosylated variant of the yeast pro␣-factor that depends on the proteasomal degradation but not ubiquitination [34]. Although the understanding of how lectin-like factors function in ER quality control has advanced dramatically in recent years, many questions remained unanswered. Perhaps most conspicuous is whether the EDEM and Yos9 proteins are, in fact, lectins. Although the accumulated data strongly suggest that they recognize glycans, direct biochemical evidence is needed to understand precisely what structures they recognize. Secondly, how the EDEM family functions in ERAD remains incomplete. Although it is clear that EDEM1 interacts with the calnexin
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cycle to take misfolded proteins out of futile folding cycles in mammalian cells, whether EDEMs work as functional ␣mannosidases is unclear. Its function in yeast remains a mystery since the canonical calnexin cycle does not appear to be used in budding yeast. The elegant models of luminal folding surveillance formulated by the Sommer and Weissman groups do not include a clear role for Htm1p. Yet, no doubt remains that it is an essential member of the pathway. Thus, uncovering Htm1p function should be considered a priority to understand the pathway. Since only a subset glycoproteins use the calnexin cycle for folding in mammals, principles learned from its role in yeast will likely be generally applicable. Beyond glycan recognition, there is ample evidence that sorting determinants are bipartite, composed of glycan and protein. What is the nature of this determinant? The view that they are simply glycans linked to a misfolded protein is insufficient, as there are ample examples of misfolded glycoproteins retained by ERQC but not subject to ERAD-L [31,55]. The use of a specifically positioned glycan in CPY*, even as the other glycans share the same Man8 GlcNAc2 structure, suggests that the protein determinant is distinctive. Finally, perhaps a reevaluation of the mannose timer hypothesis that has guided the field for years is in order. ER mannose trimming does not appear to be needed for efficient and accurate ERAD in fission yeast [43]. In budding yeast, deletion of the sole ER ␣-mannosidase in yeast leaves N-glycans entirely in Man9 GlcNAc2 form yet ERAD is only modestly affected [24,46]. The Parodi hypothesis that the ER ␣-mannosidase functions in ERAD, not as a glycosidase, but as an alternative lectin to Htm1p, could explain the curious phenotype. The field of glycoprotein quality control has experienced its share of twists and turns. However, with the tremendous strides witnessed over the past few years, it is well on its way to becoming one of the better-understood biological mechanisms. With its discoveries bearing implications for human conformational protein diseases, there is no doubt that the investigators of this field will bring this promise to fruition. Acknowledgements Research support was provided by the Temasek Life Sciences Laboratory and in part by the Japan Society for the Promotion of Science Postdoctoral Fellowships for Research Abroad (to KK). References [1] Allen JR, Nguyen LX, Sargent KE, Lipson KL, Hackett A, Urano F. High ER stress in beta-cells stimulates intracellular degradation of misfolded insulin. Biochem Biophys Res Commun 2004;324(1):166–70. [2] Bays NW, Wilhovsky SK, Goradia A, Hodgkiss-Harlow K, Hampton RY. HRD4/NPL4 is required for the proteasomal processing of ubiquitinated ER proteins. Mol Biol Cell 2001;12(12):4114–28. [3] Bhamidipati A, Denic V, Quan EM, Weissman JS. Exploration of the topological requirements of ERAD identifies Yos9p as a lectin sensor of misfolded glycoproteins in the ER lumen. Mol Cell 2005;19(6):741–51. [4] Bukau B, Weissman J, Horwich A. Molecular chaperones and protein quality control. Cell 2006;125(3):443–51. [5] Buschhorn BA, Kostova Z, Medicherla B, Wolf DH. A genome-wide screen identifies Yos9p as essential for ER-associated degradation of glycoproteins. FEBS Lett 2004;577(3):422–6.
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Review
The EDEM and Yos9p families of lectin-like ERAD factors Kazue Kanehara, Shinichi Kawaguchi, Davis T.W. Ng ∗ Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore 117604, Singapore Available online 8 September 2007
Abstract Protein quality control pathways monitor the folding of newly synthesized proteins throughout the cell. Irreversibly misfolded proteins are sorted and degraded to neutralize their potential toxicity. In the secretory pathway, multiple strategies have evolved to test the wide diversity of molecules that traffic through the endoplasmic reticulum. The organelle has adapted the use of N-linked glycans to signal protein folding states. The signals are read by the EDEM and Yos9 protein families that take substrates out of folding cycles for degradation. © 2007 Elsevier Ltd. All rights reserved. Keywords: ER-associate degradation; ER quality control; ERAD; EDEM; Htm1p; Yos9p; Misfolded proteins; Lectin; N-linked glycosylation
Contents 1. 2. 3. 4. 5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-linked carbohydrates: active participants in ERQC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The ␣-mannosidase-like lectins of ERAD: EDEMs and Htm1p/Mnl1p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yos9p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Quality control systems that monitor folding operate everywhere that proteins are synthesized (for review, see [4,38,54]). Although the basic principle is simple—misfolded proteins are recognized and degraded—how cells accomplish the task remains mysterious. Protein synthesis in the endoplasmic reticulum (ER) is a particular challenge. The organelle’s dynamic nature and the sheer volume of cargo traversing it require that the flux be tightly regulated. ER localization of chaperones and modifying enzymes require unfolded proteins to be retained and the transport of folded proteins unfettered. The monitoring mechanisms must also contend with structurally diverse substrates. These include soluble luminal proteins, proteins linked to lipids, and integral membrane proteins. Membrane proteins can
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Corresponding author. Tel.: +65 6872 7822; fax: +65 6872 7000. E-mail address: [email protected] (D.T.W. Ng).
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be intrinsically more difficult because they can have structures in the lumen, the lipid bilayer, and the cytosol. This review will focus on the terminal phase of ER quality control (ERQC) called ER-associated degradation (ERAD). Substrates of all ERAD pathways are degraded by the cytosolic 26S proteasome. Thus, misfolded proteins are recognized in the ER and transported to the cytosol for degradation. Although ERAD appears to be the major mechanism of disposal, recent studies have reported that aberrant glycoproteins can sometimes take alternative routes [33]. The current understanding of ERAD comes mostly from studies of the budding yeast and mammalian model systems [16,17,30]. Given the evolutionary expanse between fungi and mammals, it was not surprising to observe differences in their specific strategies and compositions. To monitor the diversity of substrates, each organism relies on multiple ERAD pathways. Usually, the distinct steps of recognition and degradation are linked by dedicated protein complexes that receive substrates, ubiquitinate them, and facilitate their transfer to the proteasome.
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Studies from budding yeast indicate that their repertoire of ERAD pathways represent a basic conserved standard set [4,37,40]. Higher eukaryotes differ by an expansion of components and pathways, presumably to satisfy the needs of more complex organisms. In budding yeast, three ERQC pathways have been described that can account for much of the organelle’s needs. These are the ERAD-C (ER-associated degradation-Cytosol), ERAD-M (-Membrane), and ERAD-L (Lumen) pathways that monitor folding of cytosolic, membrane, and luminal domains of client proteins, respectively (Fig. 1). Two E3 ubiquitin ligases, Hrd1p and Doa10p, organize factors involved in substrate recognition, extraction, and ubiquitination for these pathways [7,8,15]. Doa10p organizes a complex that includes the E2 ubiquitin conjugating enzyme Ubc7p (tethered to the membrane via Cue1p) and the AAA-ATPase Cdc48p with its cofactors Npl4p and Ufd1p (tethered to the membrane via Ubx2p) [7]. These factors are required to initiate the degradation of membrane proteins bearing cytosolic domain lesions (Fig. 1, ERAD-C). The Cdc48p complex provides the mechanical energy to extraction substrates from the ER membrane [2,26,53,61]. It should be noted that the
factors of the Doa10p complex are localized almost entirely or on the cytosolic face of the ER membrane (Fig. 1, Doa10p). Although this organization might reflect its role in monitoring cytosolic domains, the large transmembrane domain of Doa10p has been proposed to serve as a conduit for substrate extraction [32]. Hrd1p, like Doa10p, also assembles with the Ubc7p/Cue1p dimer and the Cdc48p complex (Fig. 1, ERAD-M and ERAD-L). However, additional partners function to detect and process substrates with misfolded luminal domains. These include a direct 1:1 interaction with Hrd3p, a transmembrane protein with a large luminal domain [13]. Bound to Hrd3p is the lectin-like factor Yos9p. Also part of this complex is the integral membrane protein Der1p. Usa1p, whose function is unknown, links Der1p to Hrd1p [7]. A notable factor that works through the Hrd1p complex but is not a stable member is the lectin-like factor Htm1p (also known as Mnl1p). Nearly all known ERAD factors in yeast have homologues in mammals. Among E3 ubiquitin ligases, mammalian HRD1 (also known as synoviolin) is implicated in the turnover of several substrates including the Pael receptor, misfolded insulin,
Fig. 1. ERAD pathways in yeast and mammals. In yeast (unshaded) substrates bearing cytosolic lesions follow the ERAD-C pathway (blue arrows; glycanindependent), membrane lesions follow the ERAD-M pathway (green arrows; glycan-independent), and luminal lesions follow the ERAD-L pathway (pink arrows; glycan-dependent) pathways in yeast. In mammals (shaded green), EDEM family proteins take the substrate out of the calnexin folding cycle (lower right) to the Hrd1 complex (upper right). Other established mammalian ERAD pathways are not shown for the sake of simplicity. After binding to E3 complexes, the substrates are ubiquitinated and extracted from the membranes via the Cdc48p/p97 complex. Substrates are then deglycosylated by protein N-glycanase (Png1) and escorted to the proteasome for degradation.
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and the unassembled T-cell receptor subunits TCR-␣ and CD3␦ [1,27,52]. The human TEB4 protein, on the other hand, is a homolog of yeast Doa10p but its involvement in ERAD is yet to be established [32]. Other mammalian ERAD E3 enzymes have no counterparts in yeast. These include gp78 (which bears some resemblance to Hrd1p), CHIP, and RMA1 [9,39,64]. The list, yet to be completed, illustrates the expanded repertoire of pathways that make up mammalian ERAD. The Cdc48p complex and Ubc7p, common to both yeast E3 complexes, have mammalian counterparts with established roles in ERAD [27,53,62]. Of exclusive factors of the Hrd1p complex, mammalian homologs of Hrd3p (SEL1), Der1p (Derlin-1, -2, -3), and Yos9p (OS-9 and XTP3-B) are already known [44,49,57]. The role of Der1p is not yet clear but one of its mammalian homologues Derlin-1 was proposed to function as the protein channel that mediates retrotranslocation of substrates [35,63]. The mammalian homolog for Htm1p is EDEM (ER degradation enhancing ␣-mannosidaselike protein) [23]. At its essence, ERQC encompasses a series of pathways that integrates folding, traffic control, sorting and disposal of misfolded proteins. Understanding the system requires the characterization of individual pathways, the identification of components and their organization, the precise dissection of the sequential events, and how individual factors fit into each step. Several articles in this volume bring together the current understanding of one major aspect of ERQC—how misfolded glycoproteins are recognized and degraded. This article will review the functions of the lectin-like factors Htm1p/EDEM and Yos9p that act at an early step of glycoprotein ERAD. 2. N-linked carbohydrates: active participants in ERQC The attachment of N-linked carbohydrates is essential for the maturation of many proteins [19]. Some depend on the calnexin chaperone cycle while others use glycans to prevent protein aggregation or to provide the correct chemical composition at those sites. Glycans can play a critical role in the recognition and disposal of misfolded proteins. In Saccharomyces cerevisiae, a mutant carboxypeptidase Y enzyme called CPY* misfolds, is retained in the ER, and rapidly degraded by ERAD [10]. A non-glycosylated version of CPY* is subject to ER retention by ERQC but fails to degrade [29]. This suggested a role for N-linked glycans for entry into the ERAD pathway. In both mammalian and yeast cells, the carbohydrate structure seems to be important for ERAD (See Caramilo and Parodi, this issue). N-linked carbohydrates in all eukaryotes share the same core structure when first attached to the protein (Fig. 2). A series of carbohydrate trimming events begins with the removal of the terminal glucose residue by glucosidase I. Glucosidase II removes the next two progressively. This process is rapid in budding yeast but can be staggered in other organisms for the calnexin folding cycle [19]. In yeast, the ER ␣1,2-mannosidase Mns1p cleaves the terminal mannose residue on the B branch of the glycan leaving the structure Man8 GlcNAc2 [6]. In mammalian cells, the ER ␣-mannosidase I carries out the same reaction and can sometimes remove an additional mannose residue [18].
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Fig. 2. Schematic representation of the conserved core N-linked glycan. NAcetylglucosamine (GlcNAc) is represented as blue squares, mannose as pink circles, and glucose as lavender triangles. The glycosidic bond linkages between each sugar moiety are indicated.
It was observed that the ␣-mannosidase inhibitors kifunensine and 1-deoxymannojirimycin or cells deficient in ␣-mannosidase activity disrupt the degradation of misfolded glycoproteins [56]. Furthermore, overexpression of mammalian ER mannosidase I accelerates glycan trimming and degradation of misfolded substrates [22]. Since ER ␣-mannosidases are slow acting compared to the glucosidases, it was proposed that the enzyme might act as a molecular timer for folding [18,25]. It was an appealing hypothesis since it would enable the quality control system to recognize a trimmed glycan as a marker for degradation. However, it is unlikely that it could serve as a signal alone because glycans from folded proteins also proceed to Man8 GlcNAc2 [6]. A modification of the hypothesis emerged recently that might overcome the limitation. Processing to forms smaller than Man8 GlcNAc2 has been reported for some ERAD substrates [11]. The Man7 GlcNAc2 , Man6 GlcNAc2 , and Man5 GlcNAc2 glycans have been proposed as favored for ERAD since they are poor substrates for reentry into the calnexin cycle. Alternatively, studies of the model substrates CPY* and PrA*, misfolded versions of yeast vacuolar proteases, revealed that each harbored a single, specific carbohydrate that is necessary and sufficient for ERAD [31,55]. Since all four glycans of CPY* have the Man8 GlcNAc2 structure only (with no further trimming [58]), the findings provided evidence that the signal for ERAD-L is likely bipartite and composed of the glycan and a protein determinant. This provides the ERAD machinery with a means of differentiating folding (Man9 , unfolded determinant), folded (Man8 , folded determinant), and misfolded (Man8 , unfolded determinant) proteins. The nature of a putative protein determinant remains uncharacterized.
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Recently, the mannosidase timer hypothesis was challenged. Parodi and coworkers reported that although the fission yeast Schizosaccharomyces pombe expresses a functional ER ␣1,2mannosidase (a Mns1p homolog called Spmns1p), misfolded glycoproteins primarily possess Man9 GlcNAc2 glycans due its poor activity [43]. Despite the deficiency, CPY* degrades as efficiently as it does in S. cerevisiae. The data support the idea that a Man8 GlcNAc2 glycan is not required for ERAD in fission yeast. Surprisingly, deletion of the gene encoding the Spmns1 or addition of kifunensine led to strong stabilization of CPY*. To explain the seemingly contradictory results, the authors proposed that Spmns1p’s ERAD function is distinct from its enzymatic activity. One possibility is that it acts like a lectin factor similar to that proposed for Htm1/EDEM. This might help explain how kifunensine, a molecule that inserts into the carbohydrate-binding pocket of Spmns1p, would also inhibit ERAD. In budding yeast, strains lacking glucosidase I or glucosidase II degrade CPY* poorly [21,25]. The Mns1p ␣-mannosidase is present in both cases and active in a gls2 mutant. Similarly, alg3, alg9, and alg12 mutants that result in the attachment of Man5 GlcNAc2 , Man6 GlcNAc2 , and Man7 GlcNAc2 glycans (which were proposed to stimulate ERAD in mammals [11]), respectively, also resulted in the stabilization of CPY* [25]. Thus, whether the conversion from Man9 GlcNAc2 to Man8 GlcNAc2 is more incidental than causal for substrate entry into ERAD, the attachment of N-linked carbohydrates and their proper structures remain an essential element even as the optimal structure might vary among organisms. 3. The ␣-mannosidase-like lectins of ERAD: EDEMs and Htm1p/Mnl1p The importance of glycans led to a proposal that a lectin receptor could be involved in ERAD [25]. The subsequent discovery of EDEM and Htm1p/Mnl1p in mammals and yeast, respectively, confirmed the notion [23,24,41,46]. EDEM1 encodes a 652 amino acid protein with sequence similarity to class I ␣1,2-mannosidases. Initially, EDEM1 appeared to be a type II integral protein localized to the ER [23] but recent data from the Molinari group indicate that EDEM1 is a soluble luminal protein in some cell types [51]. Functionally, its regulation by the unfolded protein response (UPR) suggested a role in ER stress tolerance [23]. By contrast, ER mannosidase I transcription was unchanged under ER stress. Consistent with its possible role as a lectin, ␣1,2-mannosidase activity was undetectable for this ER protein. Although EDEM1 loss-offunctions experiments were not available at the time, EDEM1 overexpression accelerated the turnover of the NHK variant of ␣1-antitrypsin, an established ERAD substrate [23,41]. Furthermore, EDEM1 was found associated with the NHK substrate in co-immunopreciptation experiments. These data supported the view that EDEM1 is an ERAD factor. In parallel, two reports describing the yeast EDEM homolog provided complementary evidence for a role in ERAD [24,46]. The lone EDEM homolog, called HTM1/MNL1, encodes a protein of 796 amino acids with an N-terminal cleaved signal
sequence. HTM1/MNL1 deleted cells are viable and exhibit no defects in glycan trimming. They do, however, exhibit strong defects in the turnover of misfolded glycoproteins. The non-glycosylated ERAD substrates Gp␣F (misfolded pro␣factor) and Sec61-2p (unstable translocon subunit), however, are degraded normally in its absence. Interestingly, unlike mammalian EDEM1, HTM1/MNL1 expression is unresponsive to ER stress [59]. The significance of this difference is unknown but enhancement of ERAD by the UPR is not dependent on its upregulation in yeast [59]. Taken together, these reports established that the EDEM/Htm1p/Mnl1p lectin-like orthologues perform a conserved ERAD function, specifically in the degradation of misfolded glycoproteins. How does EDEM/Htm1p/Mnl1p fit into established ERQC pathways? In yeast, Htm1p/Mnl1p functions in the ERAD-L pathway through the Hrd1p complex [60]. It is not a stable member of the complex, and as a soluble protein, likely acts at a step upstream [7,8,14]. The function of mammalian EDEM1 is better defined. In a series of elegant studies from the Nagata and Molinari groups, EDEM1 was shown to be a factor that connects the calnexin cycle to ERAD [41,48]. Calnexin (CNX) is an ER lectin that assists the folding of some glycoproteins. The calnexin cycle is reviewed separately in this volume and therefore only briefly described here (See Caramilo and Parodi, this issue). During or immediately following protein translocation and glycosylation, N-linked glycans are rapidly trimmed of their two terminal glucose residues by glucosidases I and II (Fig. 2). Monoglucosylated glycoproteins are then substrates of calnexin or calreticulin. After a round of folding attempts, the substrate is released, deglucosylated by glucosidase II, and scanned for unfolded structures by UDP-glucose:glycoprotein glucotransferase (UGT). If the molecule not fully folded, UGT will reglucosylate the substrate so it can reenter the calnexin cycle for another round (Fig. 1). Not all proteins that enter the calnexin cycle will fold. If a protein becomes irreversibly misfolded, EDEM1 interrupts otherwise futile folding cycles and diverts the substrate to ERAD. EDEM and calnexin interact directly to facilitate this step [48]. Elevation of EDEM1 to its equivalent level under ER stress reduces substrate intervals in the calnexin cycle and accelerates their degradation [41,48]. Under these conditions, it was observed that substrates shift more to the unglucosylated state suggesting a role of EDEM1 is to shield or divert molecules from UGT. Kifunensine treatment blocks these effects altogether suggesting that mannose trimming is required for EDEM1 function [41]. The very act of taking misfolded proteins out of a chaperone environment would risk aggregation, a physical state that is incompatible with ERAD. In yeast, the ER chaperone BiP maintains substrates in a soluble, ERAD-competent state [47]. Similarly, EDEM1 overexpression inhibits the formation of disulfide-linked higher order structures for clients of the calnexin cycle, which can be less dependent on BiP for their folding [42]. Therefore, EDEM1 appears to play a dual role. First, as an escort of substrates out of the calnexin cycle, second, as a lectin chaperone maintaining substrate solubility for ERAD.
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In budding yeast, the function of Htm1p/Mnl1p was placed into the Hrd1 pathway genetically [60]. However, a direct interaction with factors of the complex has not been reported. In mammals, EDEM1 can be co-immunoprecipitated with Derlin2 and Derlin-3, homologs of the yeast Der1p ERAD protein [49]. The Derlin family of integral membrane proteins was proposed to form all or part of the membrane channel for substrate retrotranslocation [35,63]. They form complexes with the Cdc48p homolog p97 and with the mammalian Hrd1/SEL1 complex [35,44,63]. These data indicate that EDEM1 provides a link between the calnexin folding pathway and the Hrd1 ERAD substrate-processing site in the ER membrane (Fig. 1). Although less well characterized, the other members of the EDEM family, EDEM2 and EDEM3, also participate in ERQC. The homologues share significant sequence identity within the ER mannosidase I homology domain and vary sharply in other segments (Fig. 3 and [36]). The human EDEM2 gene encodes a soluble protein of 578 amino acids while mouse EDEM3 encodes the largest member at 931 amino acids. EDEM3 carries a long C-terminal extension beyond the mannosidase homology domain that includes a protease-like domain and the KDEL retention signal [20]. EDEM1 and EDEM2, on the other hand, lack any obvious retention signals, suggesting that their localization may require association with other ER resident proteins. The mouse and human orthologues share 90% or greater sequence identity [20,36]. EDEM2 is ubiquitously expressed in all tissues examined and it localizes to the ER. Like the earlier experiments performed with EDEM1, EDEM2 lacks a detectable ␣1,2-mannosidase
activity when examined in vitro and in vivo [51]. Overexpression of EDEM2 accelerated the degradation of two variants of ␣1-antitrypsin, NHK and PI Z, but not a non-glycosylated variant. Thus, EDEM2 shares several functional similarities with EDEM1. Their substrate overlap also suggests that the homologues might perform semi-redundant functions. This notion is supported by the modest effect on BACE457 degradation when only EDEM1 expression is knocked-down [41]. At first glance, EDEM3 appears to be most enigmatic member with its prominent C-terminal extension. However, its characterization revealed that EDEM family members share some characteristics [20]. All mammalian members encode soluble ER proteins that are transcriptionally regulated by the UPR. Like the others, EDEM3 overexpression accelerates the degradation of the glycosylated substrates NHK and the unassembled ␣ subunit of the T-cell receptor. One difference is the extent of demannosylation of N-linked glycans. In addition to the Man8 GlcNAc2 glycan, EDEM3 overexpression stimulated the formation of Man7 GlcNAc2 , Man6 GlcNAc2 glycans and to a lesser extent, Man5 GlcNAc2 structures. The finding was interesting because of other reports showing accumulated misfolded proteins can be demannosylated to structures smaller than Man8 GlcNAc2 in mammalian cells [11]. Based on structural insights gleaned from ␣-mannosidases, the Molinari group reexamined the issue of whether EDEM1 can itself act as an ␣1,2-mannosidase. They found that EDEM1 overexpression enhanced demannosylation of ERAD substrates [50]. Although the study could not rule out indirect effects of EDEM overexpression on mannosidase activity (e.g., activating other ␣-mannosidases), it was argued that all the critical residues required for catalysis are intact in the EDEMs and that the missing cysteine residues conserved in most ␣-mannosidases may not be essential for enzymatic activity [50]. Indeed, inserting a mutation in one of EDEM1’s conserved catalytic residues abolished enhanced ␣-mannosidase activity while largely retaining the factor’s ability to accelerate degradation. From these data, the authors propose an interesting hypothesis. Here, the ER mannosidase I (not a UPR target) slowly converts Man9 GlcNAc2 to Man8 GlcNAc2 for typical ER quality control. Under ER stress, the UPR upregulates EDEM1 and EDEM3. Their activation causes the rapid and extensive demannosylation of N-glycans (the A branch in particular). This reduces reglucosylation of substrates by UGT and accelerates the clearance of aberrant proteins that would otherwise overload the ER.
Fig. 3. Schematic representation of EDEM and Yos9 protein families. (A) MRH domain containing proteins. The mannose 6-phosphate receptor homology (MRH) domain is shown in lavender, the predicted signal sequence in black and the predicted transmembrane region in green. The C-terminal HDEL ER retention signal is indicated. CD-MPR; cation-dependent mannose 6-phosphate receptor (bovine, P11456), OS-9 (mouse, NP 808282), Yos9p (Saccharomyces cerevisiae, YDR057w). (B) ER mannosidase I and the EDEM family. The mannosidase homology domain (MHD) is shown in blue, the predicted signal sequence in black and the predicted transmembrane domain in green. The C-terminal KDEL ER retention signal is indicated. ER Man I; ER mannosidase I (human, NP 057303), EDEM1 (human, CAC69370), EDEM2 (human, NP 060687), EDEM3 (human, NP 079467) and Htm1/Mnl1 (Saccharomyces cerevisiae, YHR204w).
4. Yos9p First characterized in budding yeast, Yos9p represents a second class of lectin-like ERAD factors. This class is evolutionarily distinct from the EDEM family because it shares its lectin-like domain with the mannose 6-phosphate receptor (MPR) family (Fig. 3). The YOS9 (yeast osteosarcoma 9) gene was given its curious moniker because of its similarity to OS-9, a gene found in amplified chromosomal DNA from human tumors [57]. YOS9 predicts a 542 amino acid protein with four N-linked glycosylation sites, an amino terminal cleaved signal sequence,
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and a C-terminal HDEL ER retention signal. Initial biochemical analysis suggested that Yos9p is peripherally associated with the ER membrane [12]. Sean Munro first suggested that Yos9p might function by binding N-glycans [45]. His work on Mrl1p, a yeast relative of the mammalian MPRs, helped reveal the conservation of the MPR glycan-binding module across multiple protein classes and organisms. Appearance of the modules, called mannose 6phosphate homology domains (MRH), in otherwise dissimilar proteins, suggested an evolutionary assimilation of the domain for diverse functions. It would be a few years later before Yos9p’s role in ERAD was reported [3,5,28,58]. Although conserved evolutionarily, functional analysis of the homologues OS-9 and XTP3-B has not yet been reported. Yos9p was discovered variously through genetic screens for mutants that disrupted ERAD. The large number of substrates examined among the studies revealed a clear pattern. Glycosylated substrates bearing lesions in the ER lumen (both soluble and membrane bound) requires Yos9p function while those bearing lesions in their transmembrane or cytosolic domains do not. Engineered YOS9 mutants that alter conserved residues in the carbohydrate-binding domain abolish ERAD function. This provided indirect evidence that Yos9 functions through the binding of glycans [3,58]. Despite the obvious functional similarity with the EDEM family, genetic analyses indicated that Yos9p and Htm1p perform distinct functions within the same pathway. Both are equally indispensable for ERAD-L and strains deleted of both genes are no worse off than individual knock-outs [3,5,28,58]. Yos9p exhibits the expected characteristics of a recognition factor for misfolded proteins. In co-immunoprecipitation and crosslinking experiments, Yos9p binds to misfolded proteins and not to folded ones [3,28,58]. Unfortunately, the role of the glycans was not clarified through these analyses. In one study, Yos9p prefers CPY* molecules bearing Man8 GlcNAc2 and Man5 GlcNAc2 glycans [58]. This result mirrors the carbohydrate structures that are optimal for efficient degradation [25]. By contrast, the second study reported that Yos9p could bind to CPY* lacking any glycans [3]. Although the results appear contradictory, it is important to note that the experiments were performed differently. In the first study, the pull-down experiment utilized a Yos9p-protein A fusion expressed at endogenous levels. In the second, crosslinking was applied to the HA-tagged Yos9p expressed from a multi-copy plasmid. The difference could reflect a greater stringency inherent to the Yos9p-protein A approach that revealed differences in binding affinity. Conversely, the combination of chemical crosslinking and Yos9p overexpression may have increased sensitivity for weaker interactions. With Yos9p firmly established as a critical ERAD factor, a second wave of studies provided the best view to date into the inner workings of ERAD. The Sommer, Weissman, and Rapoport groups used Yos9p and the ERAD E3s as points of reference for detailed biochemical analyses. Tandem affinity tagged (TAP) versions of the E3 ubiquitin ligases Hrd1p and Doa10p revealed that they organize two distinct ERAD-specific protein complexes [7]. On the luminal side, Yos9p is part of complex that includes the Hrd1/Hrd3p dimer and the chaperone Kar2p
[7,8,14]. Hrd1p directly interacts with its E2 Cue1p/Ubc7p, Der1p, Usa1p, and the Cdc48p complex (Fig. 1). Cells deleted of HRD1 do not disrupt the Kar2p/Yos9p/Hrd3p luminal complex but these proteins no longer interact with the Cdc48p complex [8,15]. Thus, Hrd1p forms the critical link between recognition events in the lumen and the ubiquitination and protein extraction activities in the cytosol. Surprisingly, Yos9p and Hrd3p can bind substrates whether they are glycosylated or not [8,15]. If substrates can be recruited efficiently to Hrd3p (and therefore, the Hrd1 complex), what purpose does Yos9p serve? Despite the binding data, it should be emphasized that ERAD-L glycoprotein substrates like CPY* degrade very poorly in the absence of Yos9p. Non-glycosylated variants are degraded poorly whether or not Yos9p is present. Thus, the Hrd1 complex lectin and substrate glycans remain critical elements of ERAD-L even as binding experiments suggest that neither are required for substrate recruitment. To explain these data, it was proposed that Yos9p performs a “proofreading” or “gating function” [8,15]. In this scenario, Hrd3p can recruit misfolded proteins but Yos9p scans the substrate for Man8 GlcNAc2 glycans. Only if this requirement is satisfied will the substrate be passed through a pore complex (which remains unidentified) for ubiquitination and degradation. This extra level of screening would ensure that folding intermediates would not be degraded. Lending support for this idea, the Weissman group showed that overexpression of Hrd1p itself can partially overcome the loss of Hrd3p and Yos9p in degrading CPY*. More intriguingly, the non-glycosylated variant is now degraded equally to CPY*. These data suggest that while the Hrd1p complex can be made functional in the absence of these factors, the fidelity of substrate selection is impaired [8]. 5. Concluding remarks Early observations that the structures of N-linked glycans can be determinants in ERQC brought forth the hypothesis that glycan trimming could serve as a molecular timer for folding. The search for mediators uncovered the EDEM and Yos9 families of ER lectin-like ERAD factors. The studies presented in this review highlight the importance of glycans in ERQC. However, it should be emphasized that this system represents but a small part of the cellular strategies deployed for protein quality control. By definition, ERAD-M, ERAD-C, and quality control anywhere outside the ER lumen would employ entirely different mechanisms due to the absence of N-linked glycans. Indeed, even within the ER lumen exists a pathway defined by the nonglycosylated variant of the yeast pro␣-factor that depends on the proteasomal degradation but not ubiquitination [34]. Although the understanding of how lectin-like factors function in ER quality control has advanced dramatically in recent years, many questions remained unanswered. Perhaps most conspicuous is whether the EDEM and Yos9 proteins are, in fact, lectins. Although the accumulated data strongly suggest that they recognize glycans, direct biochemical evidence is needed to understand precisely what structures they recognize. Secondly, how the EDEM family functions in ERAD remains incomplete. Although it is clear that EDEM1 interacts with the calnexin
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cycle to take misfolded proteins out of futile folding cycles in mammalian cells, whether EDEMs work as functional ␣mannosidases is unclear. Its function in yeast remains a mystery since the canonical calnexin cycle does not appear to be used in budding yeast. The elegant models of luminal folding surveillance formulated by the Sommer and Weissman groups do not include a clear role for Htm1p. Yet, no doubt remains that it is an essential member of the pathway. Thus, uncovering Htm1p function should be considered a priority to understand the pathway. Since only a subset glycoproteins use the calnexin cycle for folding in mammals, principles learned from its role in yeast will likely be generally applicable. Beyond glycan recognition, there is ample evidence that sorting determinants are bipartite, composed of glycan and protein. What is the nature of this determinant? The view that they are simply glycans linked to a misfolded protein is insufficient, as there are ample examples of misfolded glycoproteins retained by ERQC but not subject to ERAD-L [31,55]. The use of a specifically positioned glycan in CPY*, even as the other glycans share the same Man8 GlcNAc2 structure, suggests that the protein determinant is distinctive. Finally, perhaps a reevaluation of the mannose timer hypothesis that has guided the field for years is in order. ER mannose trimming does not appear to be needed for efficient and accurate ERAD in fission yeast [43]. In budding yeast, deletion of the sole ER ␣-mannosidase in yeast leaves N-glycans entirely in Man9 GlcNAc2 form yet ERAD is only modestly affected [24,46]. The Parodi hypothesis that the ER ␣-mannosidase functions in ERAD, not as a glycosidase, but as an alternative lectin to Htm1p, could explain the curious phenotype. The field of glycoprotein quality control has experienced its share of twists and turns. However, with the tremendous strides witnessed over the past few years, it is well on its way to becoming one of the better-understood biological mechanisms. With its discoveries bearing implications for human conformational protein diseases, there is no doubt that the investigators of this field will bring this promise to fruition. Acknowledgements Research support was provided by the Temasek Life Sciences Laboratory and in part by the Japan Society for the Promotion of Science Postdoctoral Fellowships for Research Abroad (to KK). References [1] Allen JR, Nguyen LX, Sargent KE, Lipson KL, Hackett A, Urano F. High ER stress in beta-cells stimulates intracellular degradation of misfolded insulin. Biochem Biophys Res Commun 2004;324(1):166–70. [2] Bays NW, Wilhovsky SK, Goradia A, Hodgkiss-Harlow K, Hampton RY. HRD4/NPL4 is required for the proteasomal processing of ubiquitinated ER proteins. Mol Biol Cell 2001;12(12):4114–28. [3] Bhamidipati A, Denic V, Quan EM, Weissman JS. Exploration of the topological requirements of ERAD identifies Yos9p as a lectin sensor of misfolded glycoproteins in the ER lumen. Mol Cell 2005;19(6):741–51. [4] Bukau B, Weissman J, Horwich A. Molecular chaperones and protein quality control. Cell 2006;125(3):443–51. [5] Buschhorn BA, Kostova Z, Medicherla B, Wolf DH. A genome-wide screen identifies Yos9p as essential for ER-associated degradation of glycoproteins. FEBS Lett 2004;577(3):422–6.
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