The endoplasmic reticulum (ER) quality control system ensures that newly synthesized proteins in the early secretory pathway are in the correct conformation. Polypeptides that have failed to fold into native conformers are subsequently retrotranslocated and degraded by the cytosolic ubiquitin–proteasome system, a process known as endoplasmic reticulum-associated degradation (ERAD). Most of the polypeptides that enter the ER are modified by the addition of N-linked oligosaccharides, and quality control of these glycoproteins is assisted by lectins that recognize specific sugar moieties and molecular chaperones that recognize unfolded proteins, resulting in proper protein folding and ERAD substrate selection. In Saccharomycescerevisiae, Yos9p, a lectin that contains a mannose 6-phosphate receptor homology (MRH) domain, was identified as an important component of ERAD. Yos9p was shown to associate with the membrane-embedded ubiquitin ligase complex, Hrd1p–Hrd3p, and provide a proofreading mechanism for ERAD. Meanwhile, the function of the mammalian homologues of Yos9p, OS-9 and XTP3-B remained elusive until recently. Recent studies have determined that both OS-9 and XTP3-B are ER resident proteins that associate with the HRD1–SEL1L ubiquitin ligase complex and are important for the regulation of ERAD. Moreover, recent studies have identified the N-glycan species with which both yeast Yos9p and mammalian OS-9 associate as M7A, a Man7GlcNAc2 isomer that lacks the α1,2-linked terminal mannose from both the B and C branches. M7A has since been demonstrated to be a degradation signal in both yeast and mammals.
The endoplasmic reticulum (ER) is a eukaryotic intracellular organelle where most secretory and membrane proteins are synthesized. Upon entry into the ER through the Sec61 channel, nascent polypeptides can be modified by the addition of N-linked oligosaccharides on asparagines (Asn) in the consensus sequence Asn-Xaa-Ser/Thr (Xaa: any amino acid except Pro), and inter- and intramolecular disulphide bonds are introduced by oxidoreductases. Correct folding and oligomerization of the polypeptides are assisted and monitored by the ER quality control mechanism, in which only proteins that have acquired the proper conformation are sorted further through the secretory pathway (Ellgaard and Helenius 2003; van Anken and Braakman 2005; Anelli and Sitia 2008). Although cells and organisms have developed a refined network in the ER to support efficient and proper folding of diverse proteins, some portions of newly synthesized proteins still fail to obtain the correct structure. Moreover, under circumstances that evoke ER stress, misfolded proteins accumulate in the ER (Malhotra and Kaufman 2007; Ron and Walter 2007). In addition, several genetic mutations result in the generation of terminally misfolded polypeptides. Proteins that fail to acquire the correct conformation are subsequently degraded by a mechanism known as endoplasmic reticulum-associated degradation (ERAD) (Bonifacino and Weissman 1998; Vembar and Brodsky 2008). In this pathway, polypeptides destined for disposal are removed from the ER through a currently unidentified dislocon or retrotranslocation channel and ubiquitinated, followed by proteasomal degradation in the cytosol (Raasi and Wolf 2007; Hirsch et al. 2009).
During the process of protein synthesis and retention, glycoproteins in a nonnative conformation are recognized by molecular chaperones and lectins in the ER. This N-glycan-dependent quality control system was first identified as an essential step during the folding of glycoproteins in mammals (Hammond et al. 1994) and for ERAD in yeast (Knop et al. 1996). N-glycan synthesis consists of multiple steps that occur both at the cytoplasmic surface and inside the ER. Immediately after the transfer of a high-mannose oligosaccharide consisting of 14 sugar residues of Glc3Man9GlcNAc2 (G3M9) to the nascent polypeptides, glucose trimming is initiated (Trombetta and Parodi 2003; Helenius and Aebi 2004). Subsequently, glucose and mannose residues on the N-linked oligosaccharides are processed. To explain this enigmatic phenomenon, an attractive model was proposed in which N-glycans serve as tags for the quality control of various glycoproteins (Jakob et al. 1998; Helenius and Aebi 2001). The lectin chaperones calnexin and calreticulin recognize monoglucosylated N-glycans to facilitate the productive folding of glycoproteins (Schrag et al. 2003; Helenius and Aebi 2004; Caramelo and Parodi 2008), and the lectins ERGIC-53, VIP-L and VIP36 bind to high-mannose glycans and act as cargo receptors for ER–Golgi transport (Ito et al. 2005; Appenzeller-Herzog and Hauri 2006; Kato and Kamiya 2007). Mannose trimming of N-glycans plays an important role in glycoprotein ERAD (Cabral et al. 2001; Helenius and Aebi 2001). The Man8GlcNAc2 isomer B (M8B) on misfolded polypeptides has been proposed to act as the glycan signal for degradation in both budding yeast and mammals. Htm1p/Mnl1p in budding yeast and ER degradation enhancing α-mannosidase-like proteins (EDEMs) in mammals were originally cloned as potential lectins involved in ERAD, although their sugar-binding ability in vitro has not been confirmed (Herscovics et al. 2002; Kanehara et al. 2007; Olivari and Molinari 2007). In addition, the smaller M7, M6 and M5 N-glycans have also been characterized as degradation signals in the ER in mammalian cells (Lederkremer and Glickman 2005; Molinari 2007).
Yos9p was recently identified as a lectin involved in glycoprotein ERAD in Saccharomycescerevisiae (Buschhorn et al. 2004; Bhamidipati et al. 2005; Kim et al. 2005; Szathmary et al. 2005; reviewed by Kanehara et al. 2007), followed by the identification of OS-9 and XTP3-B as mammalian homologues. Recent analysis has revealed that the N-glycan recognized by mammalian OS-9 and yeast Yos9p is most probably M7A, an isomer of Man7GlcNAc2 that lacks the two terminal mannoses on both the B and C branches (Quan et al. 2008; Hosokawa et al. 2009). These findings shed light on the importance of the terminal mannose on the C branch of N-glycans. In this review, we focus on the recently identified function of mammalian OS-9 and XTP3-B in ERAD based on their N-glycan recognition specificities.
Mammalian mannose 6-phosphate receptor homology domain-containing lectins
In 2001, Munro identified six mannose 6-phosphate receptor homology (MRH) domain-containing lectins in the human genome (Munro 2001; Figure 1). Mannose 6-phosphate receptors (MPRs) are well characterized as receptors that sort lysosomal proteins through the recognition of N-glycans that have been modified with phosphomannosyl residues (Dahms et al. 1989; Ghosh et al. 2003). The crystal structure of the lectin domain of cation-dependent MPR (CD-MPR) was determined in complex with mannose 6-phosphate (Man-6-P) (Roberts et al. 1998); thus, the interaction of Man-6-P and its binding domain became clear. The MRH domain was defined by sequence homology to MPR, and proteins containing an MRH domain were proposed to act as lectins. In addition to four previously characterized proteins (CD-MPR, cation-independent MPR (CI-MPR), GlcNAc-phosphotransferase γ-subunit and ER glucosidase II β-subunit), two proteins of unknown function were also classified in this family: OS-9 and an expressed sequence tag (EST) clone, which was later named XTP3-B or Erlectin.
CI-MPR, also known as insulin-like growth factor-II (IGF-II), is a 300-kDa multifunctional protein that contains 15 MRH domain repeats (Dahms et al. 1989; Ghosh et al. 2003). In addition to transporting lysosomal proteins from the trans-Golgi network to the endosomes based on recognition of Man-6-P, CI-MPR also localizes to the plasma membrane and regulates Man-6-P-containing serum proteins and cytokines. Recently, the crystal structures of several domains of CI-MPR were determined, which revealed that they have tertiary structures similar to CD-MPR (Dahms et al. 2008; Kim et al. 2009).
GlcNAc-1-phosphotransferase catalyzes the transfer of GlcNAc-1-P to the mannose residues on N-glycans and is the first enzyme necessary for the creation of Man-6-P. It consists of six subunits: α2, β2 and γ2 (Bao et al. 1996). The α- and β-subunits have enzyme activity, whereas the γ-subunit (GNPTG), which contains the MRH domain, lacks catalytic activity and is believed to participate in substrate binding of the enzyme (Lee et al. 2007). However, the sugar-binding property of GNPTG is currently unknown.
Glucosidase II (GII) is an ER luminal protein that removes the α1,3-linked glucoses from N-glycans (Trombetta et al. 1996). Because this gene was previously cloned as a cDNA encoding a substrate for protein kinase C named protein kinase C substrate 80K-H (PRKCSH; Sakai et al. 1989), the MRH domain is also known as the PRKCSH domain. GII is an α-β heterodimer; the α chain (GIIα) is the catalytic subunit, while the β chain (GIIβ) contains the MRH domain as well as the C-terminal ER retrieval sequence (Pelham 1990). Therefore, GIIβ should retain the enzymatically active α-subunit in the ER (Trombetta et al. 1996). Recently, analysis of the sugar-binding specificity of the human GIIβ MRH domain revealed that the terminal α1,2-linked mannobiose on the B or C arm is required for binding (Hu et al. 2009). Consistent with this report, the GIIβ MRH domain in fission yeast has been reported to recognize the mannose residues on the B and C branches for glucose hydrolysis (Stigliano et al. 2009).
OS-9 was originally identified as one of the genes of unknown function amplified in osteosarcoma (Su et al. 1994, 1996; Kimura et al. 1997, 1998). OS-9 mRNA is expressed ubiquitously and is sometimes upregulated in human sarcomas. Localization studies of OS-9 indicated that the protein resides in the nucleus and cytosol or at the cytoplasmic surface of the ER (Nakayama et al. 1999; Litovchick et al. 2002; Baek et al. 2005; Wang et al. 2007). The intracellular localization of OS-9 suggests that its primary function is not likely to be in the ER. In addition, although OS-9 and XTP3-B both contain a stretch of hydrophobic residues at the N terminus, they lack the ER retrieval sequence at the C terminus (Su et al. 1996; Munro 2001).
The sequence of XTP3-B was submitted under the name XTP3-transactivated gene B (Wang et al. 2003) and was later designated Erlectin in a study of ER genes involved in Xenopus development (Cruciat et al. 2006). In this study, XTP3-B/Erlectin was shown to be an ER luminal protein that binds to a specific domain of the plasma membrane protein, Krm2, in an N-glycan-dependent manner in the ER and was suggested to regulate the glycoprotein traffic. Morpholino oligo injection revealed the significance of XTP3-B/Erlectin in Xenopus embryo development.
Yos9p is a yeast lectin that recognizes misfolded glycoproteins in ERAD
The first evidence of the contribution of MRH domain-containing lectins to ERAD came from the genome-wide screening of S. cerevisiae (Buschhorn et al. 2004). Buschhorn and coworkers demonstrated that Yos9p is a luminal, membrane-associated ER protein required for ERAD of glycoproteins but not of non-glycosylated proteins. Yos9p had been identified previously as a yeast homologue of human OS-9, sharing 12% overall identity and 20% identity in the N-terminal MRH domain (Friedmann et al. 2002). Unlike its mammalian homologue, Yos9p contains a carboxy-terminal tetrapeptide His-Asp-Glu-Leu (HDEL) ER retrieval signal (Munro 2001). Subsequent analyses confirmed that the lectin activity of Yos9p is necessary for ERAD of luminal glycoproteins (Bhamidipati et al. 2005; Kim et al. 2005; Szathmary et al. 2005). Yos9p does not bind to correctly folded carboxypeptidase Y (CPY) but does associate with misfolded glycoprotein CPY*, although it was unclear whether Yos9p interacts only with aberrant glycoproteins that bear specific N-glycans or if it also recognizes ERAD substrates that lack N-glycans.
Next, it was demonstrated that Yos9p forms a complex with the membrane-embedded ubiquitin ligase complex in the ER (Carvalho et al. 2006; Denic et al. 2006; Gauss et al. 2006). Hrd1p/Der3p is a multispanning transmembrane ubiquitin ligase (E3) with a cytosolic RING-H2 domain (Bays et al. 2001; Deak and Wolf 2001). The protein is stabilized through the formation of a complex with the type-I transmembrane protein, Hrd3p (Plemper et al. 1999; Gardner et al. 2000). Hrd3p has a relatively large luminal domain and has been confirmed to physically interact with Yos9p. The proposed mechanism whereby yeast Yos9p participates in ERAD is schematically illustrated in Figure 2A. ER quality control should be able to discriminate between nascent polypeptides and terminally misfolded conformers, both of which have exposed stretches of hydrophobic residues on their surfaces. To prevent the premature degradation of nascent polypeptides that have become mistakenly associated with Hrd3p, the polypeptides are reconfirmed by the proofreading activity of Yos9p, thereby ensuring that only unfolded polypeptides bearing specific N-glycans recognized by Yos9p are degraded. This model also explains why misfolded non-glycosylated proteins are retained undegraded in the ER. Alternatively, Yos9p may deliver the ERAD substrates directly to the membrane ubiquitin ligase complex for retrotranslocation.
OS-9 and XTP3-B are involved in mammalian ERAD
As is summarized in Figure 2C, most of the components of the ERAD pathway have orthologues in S. cerevisiae and mammals. Although a relatively clear model had been proposed to explain the mechanism by which Yos9p ensures glycoprotein quality control in yeast, it remained unknown whether the mammalian homologues of Yos9p also participated in ERAD. Recent works have now clarified the involvement of OS-9 and XTP3-B in this process (Bernasconi et al. 2008; Christianson et al. 2008; Hosokawa et al. 2008, 2009; Mueller et al. 2008; Alcock and Swanton 2009). The possible mechanisms proposed in these studies are summarized schematically in Figure 2B, although many issues must still be resolved. Overall, OS-9 functions in a manner analogous to yeast Yos9p. OS-9 is characterized as an ER luminal protein that interacts with the membrane-embedded ubiquitin ligase HRD1–SEL1L complex, which is the mammalian orthologue of the yeast Hrd1p–Hrd3p complex (Figure 2C). The primary binding site of OS-9 is in the luminal domain of SEL1L. OS-9 associates with misfolded proteins, but not correctly folded proteins, and is required for the ERAD of glycoproteins. Recent studies have shown that the degradation kinetics of ERAD substrates are affected by the overexpression or RNA interference-mediated knockdown of OS-9, although the current results are not completely consistent. Some reports indicate that the degradation of misfolded glycoproteins is partially inhibited by OS-9 knockdown (Bernasconi et al. 2008; Christianson et al. 2008; Hosokawa et al. 2009), but this effect seems to depend on the ERAD substrates (Christianson et al. 2008). However, overexpression of OS-9 does not seem to accelerate the ERAD of misfolded glycoproteins; rather, it delays (Bernasconi et al. 2008; Mueller et al. 2008) or has no effect (Hosokawa et al. 2009) on the rate of intracellular degradation. This stabilization effect on ERAD substrates may be caused by the binding of the substrates to overexpressed OS-9 that is not functionally associated with the HRD1–SEL1L complex. Furthermore, the significance of the lectin activity of the MRH domain of OS-9 in vivo is also controversial and likely depends on the levels of overexpressed OS-9 and its sugar-binding defective mutants (Bernasconi et al. 2008; Mueller et al. 2008; Hosokawa et al. 2009).
Two models of the function of OS-9 are suggested at present. In the first, OS-9 stably associates with the SEL1L–HRD1 complex (Hosokawa et al. 2008, 2009; Mueller et al. 2008; Figure 2B, left). In the second, OS-9 dynamically transfers the misfolded proteins to membrane-localized SEL1L (Christianson et al. 2008; Figure 2B, right). GRP94 is an ER resident chaperone protein of the HSP90 family that has no homologue in budding yeast. Christianson and colleagues have demonstrated that OS-9 binds to SEL1L and GRP94 in a mutually exclusive manner, suggesting that OS-9 transports misfolded luminal glycoproteins to the membrane dislocation machinery. Furthermore, it has also been shown that the MRH domains of both OS-9 and XTP3-B are required for association with SEL1L through recognition of the N-glycans on SEL1L (Christianson et al. 2008), since SEL1L is an ER resident glycoprotein with five N-glycosylation sites (Mueller et al. 2006). This result supports the hypothesis that OS-9 transports terminally misfolded polypeptides to the retrotranslocation machinery but, at the same time, revises the function of the MRH domain-containing protein, Yos9p (and possibly OS-9) to designate misfolded glycoproteins for dislocation by decoding the specific oligosaccharides on the substrates. On the other hand, our results support the hypothesis that the OS-9 MRH domain is necessary for the recognition of N-glycans on ERAD substrates rather than for SEL1L binding activity (Hosokawa et al. 2009).
It has also been reported that XTP3-B is involved in ERAD (Christianson et al. 2008; Hosokawa et al. 2008), but less is known about its function. There are two splice variants of XTP3-B (Figure 1, inset), and the short isoform lacks the N-terminal cysteine that is conserved in the MRH domain. The two XTP3-B isoforms seem to have different functions, and only the long splice variant is incorporated into the HRD1–SEL1L complex. The interaction of the XTP3-B long form and SEL1L may be mediated by the region between the two MRH domains or by an overall conformational change induced by this region (Hosokawa et al. 2008).
N-glycan recognition by mammalian OS-9
Thus far, the requirement for lectin activity of the OS-9 MRH domain in glycoprotein ERAD is controversial. To fully understand the function of OS-9, it is important to identify the oligosaccharide structures recognized by the OS-9 lectin domain. We have recently identified the oligosaccharide structures that the human OS-9 (hOS-9) MRH domain binds to in vitro (Hosokawa et al. 2009). To detect the relatively weak interaction between the sugar-binding domain of mammalian lectins and specific oligosaccharides, we used frontal affinity chromatography (FAC) analysis (Kasai et al. 1986; Kamiya et al. 2005, 2008; Figure 3B). A bacterially expressed recombinant hOS-9 MRH domain was immobilized on Ni2-Sepharose and packed into a small column. Pyridylaminated oligosaccharides were applied to this column, and the elution profile was monitored. The hOS-9 MRH domain clearly showed affinity for high-mannose N-glycans lacking the C branch terminal mannose (Figure 3C, upper panel). No specific binding was observed in Glc1Man9GlcNAc2 (G1M9) and Man9GlcNAc2 (M9), which are in the calnexin/calreticulin folding cycle (Helenius and Aebi 2001; Kamiya et al. 2009). When the three M8 isoforms were compared, it was clear that the hOS-9 MRH domain has a specific affinity for M8C, which lacks the C branch terminal mannose, but not for M8A or M8B, which lacks either the A or B branch terminal mannose, respectively.
We next analyzed the significance of N-glycan structures on ERAD substrates that are degraded through the OS-9 pathway in vivo. To modify the N-linked oligosaccharides on the misfolded α1-antitrypsin null Hong Kong variant (NHK), we overexpressed ER α1,2-mannosidase I (ER ManI) or EDEM3 in cultured HEK293 cells. In combination with OS-9 knockdown, we confirmed that degradation of NHK bearing M7 and M6 N-glycans created by overexpressed EDEM3 is dependent on OS-9, whereas the degradation of NHK with M8B and G1M8B N-glycans generated by transfected ER ManI is independent of OS-9 activity. Taken together, these findings indicate that the presence of N-glycans lacking the C branch terminal mannose is a structural requirement for OS-9 sugar recognition. This probably occurs most often in the form of M7A, an isomer that lacks the α1,2-linked terminal mannose from both the B and C branches (see below).
N-glycan recognition by yeast Yos9p
Recently, Quan and colleagues characterized the oligosaccharide structures that are recognized by yeast Yos9p in vivo and in vitro by FAC analysis of the sugar-binding specificity of Yos9p (Quan et al. 2008; Figure 3C, lower panel). Importantly, the oligosaccharides that Yos9p associates with are essentially the same as those recognized by the human OS-9 MRH domain, with the exception of an extraordinarily high binding affinity of Yos9p with the M5 glycan. To create an M7A glycan that lacks the two terminal mannoses on both the B and C branches in yeast cells, a glycosyltransferase was overexpressed in genetic mutants with a defective N-glycan synthesis pathway. Analysis of the degradation kinetics of CPY*, a well-characterized ERAD-luminal substrate glycoprotein in yeast (Wolf and Fink 1975; Finger et al. 1993), showed that CPY* with the M7A glycan is recognized by Yos9p and subjected to ERAD.
Furthermore, deletion of Htm1p/Mnl1p in these yeast strains demonstrated that Htm1p acts upstream of Yos9p, possibly as a processing α-mannosidase, and that Yos9p is essential for proofreading of N-glycans containing α1,6-linked mannose residues.
Significance of N-glycan trimming in ERAD
Once the specificities of N-glycans recognized by mammalian OS-9 and yeast Yos9p were characterized, the next question to be answered was which enzyme is responsible for the trimming of the terminal mannose on the C branch. In S. cerevisiae, the overexpression of Htm1p/Mnl1p demonstrated that it was the processing α1,2-mannosidase responsible for removal of the terminal mannose on the C branch, and this process occurs downstream of ER mannosidase I (Clerc et al. 2009). In yeast, terminal α1,6-linked mannose residues on N-glycans are generated by manipulating the Asn-linked glycosylation biosynthesis pathway, which modulates the N-glycan conformers on lipid-linked oligosaccharides (Burda et al. 1999). By transferring sugars bearing terminal α1,6-linked mannose residues to the misfolded glycoproteins, the mutant yeast does not require Htm1p/Mnl1p for the efficient degradation of the ERAD substrate, although Yos9p is still required (Quan et al. 2008; Clerc et al. 2009). These results support the idea that Htm1p/Mnl1p acts as the processing α-mannosidase that exposes the α1,6-linked mannose residue on the C branch, which is then recognized by Yos9p and degraded (Figure 4A). This also explains previous observations indicating that Yos9p and Htm1p/Mnl1p are genetically in the same pathway. These findings have helped to clarify the N-glycan degradation signal, the decoding lectin and the mannosidases responsible for signal creation in S. cerevisiae (Figure 4A).
In mammals, we propose that the N-glycan degradation signal is essentially the same as in budding yeast (Figure 4B). In addition to M7A, M6 and M5 glycans are also recognized as degradation signals in mammals (Lederkremer and Glickman 2005). However, the processing α-mannosidases responsible for the exposure of α1,6-linked mannose on the C branch in mammals are still intriguing. ER ManI concentrated in the ER quality control compartment, a specialized subcompartment of the ER where ERAD machinery and substrates are enriched, has been shown to play an important role in this process (Avezov et al. 2008). We believe that EDEM3 also participates in the removal of mannoses, since the increase of M7, M6 and M5 N-glycans on misfolded NHK in ER ManI-overexpressing cells is relatively small compared to the increase seen in EDEM3-overexpressing cells (Hosokawa et al. 2003; Hirao et al. 2006). At present, the enzyme activity of EDEM3 has not been demonstrated in vitro, and the specificity of the mannose residues that are processed by EDEM3 is not clear. We have recently shown that EDEM1 is also capable of trimming mannose on the C branch of N-glycans (Hosokawa et al. 2010). Based on the sequence similarity of the glycosidase domains of EDEM1 and EDEM3, we believe that EDEM3 is responsible for trimming the α1,2-linked mannose on the C branch during ERAD. On the other hand, the ability of EDEM1 to process A branch mannose has also been reported (Olivari et al. 2006). Further analysis is required to identify the specific α-mannosidases responsible for this trimming.
Concluding remarks and remaining questions
The mammalian MRH domain-containing lectins OS-9 and XTP3-B have recently been implicated as regulators of the degradation of misfolded proteins. Both proteins are confirmed to reside in the ER, and some, but not all, physically interact with SEL1L through incorporation into a large complex centered on ubiquitin ligase HRD1–SEL1L. Thus, OS-9 and XTP3-B functionally cooperate with the degradation machinery to process misfolded proteins. Furthermore, the oligosaccharide specificities of mammalian OS-9 and yeast Yos9p have been characterized, suggesting that the primary glycan signal for glycoprotein ERAD in both yeast and mammals is M7A lacking the terminal mannoses on the B and C branches, as well as isomers of M6 and M5 (Figure 4).
Because the functional analyses of mammalian OS-9 and XTP3-B in ERAD are very recent, many questions remain unanswered. First, do OS-9 and XTP3-B bind to the N-glycans on ERAD substrates or to those on SEL1L? Second, for both OS-9 and XTP3-B, substrate specificity seems to exist. How are misfolded glycoproteins that are not recognized by OS-9/XTP3-B degraded? Furthermore, free oligosaccharides exist in the cytosol, and most of them are believed to originate from the N-glycans of ERAD substrates (Suzuki and Funakoshi 2006; Chantret and Moore 2008). In wild-type S. cerevisiae, the free oligosaccharide that is predominantly detected in the cytosol is Man8GlcNAc2 (Chantret et al. 2003). The major free oligosaccharides detected in the cytosol of human HepG2 cells treated with swainsonine, an inhibitor of cytosolic α-mannosidase, are Man9GlcNAc and Man8BGlcNAc. This suggests that ERAD substrates bearing M9 and M8 glycans are removed from the ER (Yanagida et al. 2006). If these are true, it must also be determined whether these N-glycans are removed via a Yos9p/OS-9 independent pathway. Third, in S. cerevisiae, ERAD substrates that lack N-glycans are usually stabilized, whereas in mammals, the NHK-QQQ mutant that is deficient of N-glycosylation sites is more rapidly degraded than N-glycosylated NHK (Hirao et al. 2006). However, misfolded glycoproteins and aberrant non-glycosylated proteins are surveyed by MRH domain-containing lectin(s) and the Hrd1p–Hrd3p/HRD1–SEL1L ubiquitin ligase complex in both yeast and mammals. How is this difference in the degradation of glycosylated and non-glycosylated proteins between yeast and mammals explained? Can it be attributed to unidentified components or proteins of undetermined function included in the complex? Furthermore, substrate binding and dislocation enhancement seem to be separate steps. How are these steps regulated at the molecular level?
The oligosaccharide structures that are recognized by XTP3-B have not been clarified yet. It remains to be determined whether the two MRH domains of XTP3-B have a repertoire similar to that of the lectin domain of OS-9. The functional difference between OS-9 and XTP3-B also remains unclear. Are they redundant, or do they have discrete functions in ERAD? Are there functional differences among the four OS-9 transcription variants? It has been reported that Yos9p binds to non-glycosylated proteins to recognize the bipartite signal on misfolded glycoproteins. Similarly, the binding of OS-9 and XTP3-B to non-glycosylated ERAD substrates has been reported in mammals (Bernasconi et al. 2008; Christianson et al. 2008; Hosokawa et al. 2008, 2009). Based on the recent report showing that recognition of unfolded polypeptides is mediated by Kar2 in yeast (Xie et al. 2009), it will be interesting to determine whether mammalian OS-9 and XTP3-B recognize unfolded polypeptide directly or in cooperation with BiP. Finally, which enzymes are responsible for the processing of the C branch mannose in ERAD in mammals? N-glycan profiles from total glycoproteins in cells overexpressing each of the three EDEM proteins (EDEM 1, 2 and 3) are different, and EDEM2 appears to lack enzyme activity (Mast et al. 2005). The discovery that Yos9p and OS-9/XTP3-B recognize specific N-glycan signals has advanced our understanding of the ER quality control process, but many questions remain to be answered.
This work was supported by Hayashi Memorial Foundation for Female Natural Scientists (N.H.) and by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (N.H. [19GS0314], Y.K. , and K.K. [21370050, 20107004]).
ER degradation enhancing α-mannosidase-like protein
- ER ManI
ER α1,2-mannosidase I
ER-associated (protein) degradation
frontal affinity chromatography
Man7GlcNAc2 isomer A
Man7GlcNAc2 isomer C
Man8GlcNAc2 isomer A
Man8GlcNAc2 isomer B
Man8GlcNAc2 isomer C
- MRH domain
mannose 6-phosphate receptor homology domain
α1-antitrypsin null (Hong Kong)
protein kinase C substrate 80K-H