OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD

Terminally misfolded or unassembled proteins in the early secretory pathway are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). How substrates of this pathway are recognized within the ER and delivered to the cytoplasmic ubiquitin-conjugating machinery is unknown. We report here that OS-9 and XTP3-B/Erlectin are ER-resident glycoproteins that bind to ERAD substrates and, through the SEL1L adaptor, to the ER-membrane-embedded ubiquitin ligase Hrd1. Both proteins contain conserved mannose 6-phosphate receptor homology (MRH) domains, which are required for interaction with SEL1L, but not with substrate. OS-9 associates with the ER chaperone GRP94 which, together with Hrd1 and SEL1L, is required for the degradation of an ERAD substrate, mutant alpha(1)-antitrypsin. These data suggest that XTP3-B and OS-9 are components of distinct, partially redundant, quality control surveillance pathways that coordinate protein folding with membrane dislocation and ubiquitin conjugation in mammalian cells.     

Characterization of the Grp94/OS-9 Chaperone–Lectin Complex

Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum (ER) of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER-associated degradation quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins. To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full-length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal mannose recognition homology lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains “mammalian-specific insets” that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate and it additionally hints at a plausible biological role for the Grp94/OS-9 complex.     

GRP94 in ER quality control and stress responses

A system of endoplasmic reticulum (ER) chaperones has evolved to optimize the output of properly folded secretory and membrane proteins. An important player in this network is Glucose Regulated Protein 94 (GRP94). Over the last decade, new structural and functional data have begun to delineate the unique characteristics of GRP94 and have solidified its importance in ER quality control pathways. This review describes our current understanding of GRP94 and the four ways in which it contributes to the ER quality control: (1) chaperoning the folding of proteins; (2) interacting with other components of the ER protein folding machinery; (3) storing calcium; and (4) assisting in the targeting of malfolded proteins to ER-associated degradation (ERAD).     

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

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

Mammalian OS-9 Is Upregulated in Response to Endoplasmic Reticulum Stress and Facilitates Ubiquitination of Misfolded Glycoproteins

Proteins that fail to fold or assemble with partner subunits are selectively removed from the endoplasmic reticulum (ER) via the ER-associated degradation (ERAD) pathway. Proteins selected for ERAD are polyubiquitinated and retrotranslocated into the cytosol for degradation by the proteasome. Although it is unclear how proteins are initially identified by the ERAD system in mammalian cells, OS-9 was recently proposed to play a key role in this process. Here we show that OS-9 is upregulated in response to ER stress and is associated both with components of the ERAD machinery and with ERAD substrates. Using RNA interference, we show that OS-9 is required for efficient ubquitination of glycosylated ERAD substrates, suggesting that it helps transfer misfolded proteins to the ubiquitination machinery. We also find that OS-9 binds to a misfolded nonglycosylated protein destined for ERAD, but not to the properly folded wild-type protein. Surprisingly, however, OS-9 is not required for ubiquitination or degradation of this nonglycosylated ERAD substrate. We propose a model in which OS-9 recognises terminally misfolded proteins via polypeptide-based rather than glycan-based signals, but is only required for transferring those bearing N-glycans to the ubiquitination machinery.     

Role of the SEL1L:LC3-I complex as an ERAD tuning receptor in the mammalian ER

Several regulators of endoplasmic reticulum (ER)-associated degradation (ERAD) have a shorter half-life compared to conventional ER chaperones. At steady state, they are selectively removed from the ER by poorly defined events collectively referred to as ERAD tuning. Here we identify the complex comprising the type-I transmembrane protein SEL1L and the cytosolic protein LC3-I as an ERAD tuning receptor regulating the COPII-independent, vesicle-mediated removal of the lumenal ERAD regulators EDEM1 and OS-9 from the ER. Expression of?folding-defective polypeptides enhances the lumenal content of EDEM1 and OS-9 by inhibiting their SEL1L:LC3-I-mediated segregation. This raises ERAD activity in the absence of UPR-induction. The mouse hepatitis virus (MHV) subverts ERAD tuning for replication. Consistently, SEL1L or LC3 silencing impair the MHV life cycle. Collectively, our data provide new molecular information about the ERAD tuning mechanisms that regulate ERAD in mammalian cells at the post translational level and how these mechanisms are hijacked by a pathogen.     

The activities and function of molecular chaperones in the endoplasmic reticulum

Most proteins in the secretory pathway are translated, folded, and subjected to quality control at the endoplasmic reticulum (ER). These processes must be flexible enough to process diverse protein conformations, yet specific enough to recognize when a protein should be degraded. Molecular chaperones are responsible for this decision making process. ER associated chaperones assist in polypeptide translocation, protein folding, and ER associated degradation (ERAD). Nevertheless, we are only beginning to understand how chaperones function, how they are recruited to specific substrates and assist in folding/degradation, and how unique chaperone classes make quality control "decisions".     

Probing for membrane domains in the endoplasmic reticulum: retention and degradation of unassembled MHC class I molecules

Quality control of protein biosynthesis requires ER-retention and ER-associated degradation (ERAD) of unassembled/misfolded molecules. Although some evidence exists for the organization of the ER into functionally distinct membrane domains, it is unknown if such domains are involved in the retention and ERAD of unassembled proteins. Here, it is shown that unassembled MHC class I molecules are retained in the ER without accumulating at ER-exit sites or in the ERGIC of beta2m-/- cells. Furthermore, these molecules did not cluster in the ER membrane and appeared to be highly mobile even when ERAD or their association with calnexin were inhibited. However, upon ATP depletion, they were reversibly segregated into an ER membrane domain, distinct from ER exit sites, which included calnexin and COPII, but not the ERGIC marker protein p58. This quality control domain was also observed upon prolonged inhibition of proteasomes. Microtubules were required for its appearance. Segregation of unfolded proteins, ER-resident chaperones, and COPII may be a temporal adaptation to cell stress.     

Sweet bays of ERAD

Proteins that improperly mature in the endoplasmic reticulum (ER) are dislocated to the cytoplasm for proteasome-mediated destruction. A recent study provides insight into the incompletely understood processes for selection and targeting of aberrant proteins for ER-associated protein degradation. The identification of the ER chaperones GRP94 and BiP as binding partners for the mannose-binding proteins OS-9 and XTP3-B, indicates that these protein complexes bind to aberrant proteins and direct them to the Hrd1 dislocation and ubiquitylation complex in the ER membrane.     

Herp coordinates compartmentalization and recruitment of HRD1 and misfolded proteins for ERAD

A functional unfolded protein response (UPR) is essential for endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded secretory proteins, reflecting the fact that some level of UPR activation must exist under normal physiological conditions. A coordinator of the UPR and ERAD processes has long been sought. We previously showed that the PKR-like, ER-localized eukaryotic translation initiation factor 2α kinase branch of the UPR is required for the recruitment of misfolded proteins and the ubiquitin ligase HRD1 to the ER-derived quality control compartment (ERQC), a staging ground for ERAD. Here we show that homocysteine-induced ER protein (Herp), a protein highly upregulated by this UPR branch, is responsible for this compartmentalization. Herp localizes to the ERQC, and our results suggest that it recruits HRD1, which targets to ERAD the substrate presented by the OS-9 lectin at the ERQC. Predicted overall structural similarity of Herp to the ubiquitin-proteasome shuttle hHR23, but including a transmembrane hairpin, suggests that Herp may function as a hub for membrane association of ERAD machinery components, a key organizer of the ERAD complex.     

Multi-chaperone complexes regulate the folding of interferon-gamma in the endoplasmic reticulum

The quality control mechanisms directing the folding of cytokines in the endoplasmic reticulum (ER) are poorly understood. We have investigated ER chaperone usage by the cytokine interferon-gamma (IFN-gamma). ATP-depletion or inhibition of N-glycosylation was found to cause IFN-gamma to accumulate into detergent-insoluble aggregates in the ER. Six chaperones, GRP94, GRP78, ERp72, PDI, CaBP1/P5 and CRT were found to associate with IFN-gamma during its steady state folding. Interaction of the five first chaperones with IFN-gamma was regulated co-ordinately by ATP. These chaperones were recently reported to be part of a multi-chaperone complex involved in the folding of complex, multi-subunit proteins. Our data suggest that also proteins with a relatively simple quaternary structure such as cytokines may fold in association with this complex. In addition, we identified calreticulin as the major chaperone interacting with IFN-gamma, and the related class II cytokine interleukin-10, during heat-shock in vivo. IFN-gamma was maintained in a folding-competent form by calreticulin during heat-shock and released during subsequent recovery at 37 degrees C. This interaction was observed in both recombinant (CHO-F11) and natural producer cells (Jurkat, NK-92MI) of IFN-gamma. Since cytokines such as IFN-gamma and IL-10 are frequently produced in the course of inflammatory conditions associated with fever, the thermo-protective effect of calreticulin may constitute a previously unrecognized component of the cellular cytokine production machinery, of likely relevance in sustaining cytokine folding and secretion in pathophysiological conditions.     

Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation

The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.     

GRP94 hyperglycosylation and phosphorylation in Sf21 cells

GRP94 is an inducible resident endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) glycoprotein that functions as a protein chaperone and Ca(2+) regulator. GRP94 has been reported to be a substrate for protein kinase CK2 in vitro, although its phosphorylation in intact cells remains unreported. In Sf21 insect cells, overexpression of canine GRP94 led to the appearance of a multiplet of three or more molecular-mass isoforms which was reduced to a single mobility form following treatment of cells with tunicamycin, suggesting stable accumulations of consecutively modified protein. Metabolic labeling of Sf21 cells with (32)P(i) led to a constitutive phosphorylation of GRP94 which, based upon phosphopeptide mapping, occurred specifically on CK2-sensitive sites. Among the GRP94 multiplet, however, only the lowest mobility form of GRP94 was phosphorylated, even though in vitro phosphorylation of GRP94 by CK2 led to phosphorylation of all glycosylated forms. The (32)P(i) incorporation into GRP94 indicated a slow turnover of phosphate incorporation that was unaffected by inhibition of biosynthesis, resulting in a steady-state level of phospho-GRP94 on CK2 sites. These data support a role for protein kinase CK2 in the cell biology for GRP94 and other resident ER/SR proteins that may occur in ER compartments.