Substrate-specific requirements for UGT1-dependent release from calnexin

Newly synthesized glycoproteins displaying monoglucosylated N-glycans bind to the endoplasmic reticulum (ER) chaperone calnexin, and their maturation is catalyzed by the calnexin-associated oxidoreductase ERp57. Folding substrates are eventually released from calnexin, and terminal glucoses are removed from N-glycans. The UDP-glucose:glycoprotein glucosyltransferase (UGT1, UGGT, GT) monitors the folding state of polypeptides released from calnexin and adds back a glucose residue on N-glycans of nonnative polypeptides, thereby prolonging retention in the calnexin chaperone system for additional folding attempts. Here we show that for certain newly synthesized glycoproteins UGT1 deletion has no effect on binding to calnexin. These proteins must normally complete their folding program in one binding event. Other proteins normally undergo multiple binding events, and UGT1 deletion results in their premature release from calnexin. For other proteins, UGT1 deletion substantially delays release from calnexin, unexpectedly showing that UGT1 activity might be required for a structural maturation needed for substrate dissociation from calnexin and export from the ER.     

Sequential assistance of molecular chaperones and transient formation of covalent complexes during protein degradation from the ER

BACE457 is a recently identified pancreatic isoform of human beta-secretase. We report that this membrane glycoprotein and its soluble variant are characterized by inefficient folding in the ER, leading to proteasome-mediated ER-associated degradation (ERAD). Dissection of the degradation process revealed that upon release from calnexin, extensively oxidized BACE457 transiently entered in disulfide-bonded complexes associated with the lumenal chaperones BiP and protein disulfide isomerase (PDI) before unfolding and dislocation into the cytosol for degradation. BACE457 and its lumenal variant accumulated in disulfide-bonded complexes, in the ER lumen, also when protein degradation was inhibited. The complexes were disassembled and the misfolded polypeptides were cleared from the ER upon reactivation of the degradation machinery. Our data offer new insights into the mechanism of ERAD by showing a sequential involvement of the calnexin and BiP/PDI chaperone systems. We report the unexpected transient formation of covalent complexes in the ER lumen during the ERAD process, and we show that PDI participates as an oxidoreductase and a redox-driven chaperone in the preparation of proteins for degradation from the mammalian ER.     

Palmitoylated calnexin is a key component of the ribosome-translocon complex

A third of the human genome encodes N-glycosylated proteins. These are co-translationally translocated into the lumen/membrane of the endoplasmic reticulum (ER) where they fold and assemble before they are transported to their final destination. Here, we show that calnexin, a major ER chaperone involved in glycoprotein folding is palmitoylated and that this modification is mediated by the ER palmitoyltransferase DHHC6. This modification leads to the preferential localization of calnexin to the perinuclear rough ER, at the expense of ER tubules. Moreover, palmitoylation mediates the association of calnexin with the ribosome-translocon complex (RTC) leading to the formation of a supercomplex that recruits the actin cytoskeleton, leading to further stabilization of the assembly. When formation of the calnexin-RTC supercomplex was affected by DHHC6 silencing, mutation of calnexin palmitoylation sites or actin depolymerization, folding of glycoproteins was impaired. Our findings thus show that calnexin is a stable component of the RTC in a manner that is exquisitely dependent on its palmitoylation status. This association is essential for the chaperone to capture its client proteins as they emerge from the translocon, acquire their N-linked glycans and initiate folding.     

Role of calnexin in the glycan-independent quality control of proteolipid protein

The endoplasmic (ER) quality control apparatus ensures that misfolded or unassembled proteins are not deployed within the cell, but are retained in the ER and degraded. A glycoprotein-specific system involving the ER lectins calnexin and calreticulin is well documented, but very little is known about mechanisms that may operate for non-glycosylated proteins. We have used a folding mutant of a non- glycosylated membrane protein, proteolipid protein (PLP), to examine the quality control of this class of polypeptide. We find that calnexin associates with newly synthesized PLP molecules, binding stably to misfolded PLP. Calnexin also binds stably to an isolated transmembrane domain of PLP, suggesting that this chaperone is able to monitor the folding and assembly of domains within the ER membrane. Notably, this glycan-independent interaction with calnexin significantly retards the degradation of misfolded PLP. We propose that calnexin contributes to the quality control of non-glycosylated polytopic membrane proteins by binding to misfolded or unassembled transmembrane domains, and discuss our findings in relation to the role of calnexin in the degradation of misfolded proteins.     

Lectin-deficient calnexin is capable of binding class I histocompatibility molecules in vivo and preventing their degradation

Calnexin is a membrane-bound lectin of the endoplasmic reticulum (ER) that binds transiently to newly synthesized glycoproteins. By interacting with oligosaccharides of the form Glc(1)Man(9)GlcNAc(2), calnexin enhances the folding of glycoprotein substrates, retains misfolded variants in the ER, and in some cases participates in their degradation. Calnexin has also been shown to bind polypeptides in vivo that do not possess a glycan of this form and to function in vitro as a molecular chaperone for nonglycosylated proteins. To test the relative importance of the lectin site compared with the polypeptide-binding site, we have generated six calnexin mutants defective in oligosaccharide binding using site-directed mutagenesis. Expressed as glutathione S-transferase fusions, these mutants were still capable of binding ERp57, a thiol oxidoreductase, and preventing the aggregation of a nonglycosylated substrate, citrate synthase. They were, however, unable to bind Glc(1) Man(9)GlcNAc(2) oligosaccharide and were compromised in preventing the aggregation of the monoglucosylated substrate jack bean alpha-mannosidase. Two of these mutants were then engineered into full-length calnexin for heterologous expression in Drosophila cells along with the murine class I histocompatibility molecules K(b) and D(b) as model glycoproteins. In this system, lectin site-defective calnexin was able to replace wild type calnexin in forming a complex with K(b) and D(b) heavy chains and preventing their degradation. Thus, at least for class I molecules, the lectin site of calnexin is dispensable for some of its chaperone functions.     

ER quality control: towards an understanding at the molecular level.

The process of 'quality control' in the endoplasmic reticulum (ER) involves a variety of mechanisms that collectively ensure that only correctly folded, assembled and modified proteins are transported along the secretory pathway. In contrast, non-native proteins are retained and eventually targeted for degradation. Recent work provides the first structural insights into the process of glycoprotein folding in the ER involving the lectin chaperones calnexin and calreticulin. Underlying principles governing the choice of chaperone system engaged by different proteins have also been discovered.     

Tyrosinase folding and copper loading in vivo: a crucial role for calnexin and alpha-glucosidase II.

Tyrosinase is the key enzyme of melanin biosynthesis. It is a multiply glycosylated metalloenzyme, which has a long maturation time making it an ideal in vivo model system to probe protein folding and metal loading events. The use of NB-DNJ, an alpha-glucosidase I and II inhibitor has allowed us to dissect these processes. Here we show that tyrosinase folds through several inactive intermediates, at least two of which are recognised by the ER chaperone, calnexin. If the association with calnexin is prevented, more rapid folding occurs, the resulting protein fails to bind copper and is inactive. If dissociation from calnexin is inhibited, folding is prevented; the protein does not go through the normal secretory pathway and is targeted for degradation. Thus, tyrosinase folds off calnexin, giving alpha-glucosidase II a critical role, but the association with calnexin is essential to promote the correct folding which enables it to acquire copper.     

EDEM1 regulates ER-associated degradation by accelerating de-mannosylation of folding-defective polypeptides and by inhibiting their covalent aggregation

Proteins expressed in the endoplasmic reticulum (ER) are covalently modified by co-translational addition of pre-assembled core glycans (glucose(3)-mannose(9)-N-acetylglucosamine(2)) to asparagines in Asn-X-Ser/Thr motifs. N-Glycan processing is essential for protein quality control in the ER. Cleavages and re-additions of the innermost glucose residue prolong folding attempts in the calnexin cycle. Progressive loss of mannoses is a symptom of long retention in the ER and elicits preparation of terminally misfolded polypeptides for dislocation into the cytosol and proteasome-mediated degradation. The ER stress-induced protein EDEM1 regulates disposal of folding-defective glycoproteins and has been described as a mannose-binding lectin. Here we show that elevation of the intralumenal concentration of EDEM1 accelerates ER-associated degradation (ERAD) by accelerating de-mannosylation of terminally misfolded glycoproteins and by inhibiting formation of covalent aggregates upon release of terminally misfolded ERAD candidates from calnexin. Acceleration of Man(9) or Man(5)N-glycans dismantling upon overexpression was fully blocked by substitution in EDEM1 of one catalytic residue conserved amongst alpha1,2-mannosidases, thus suggesting that EDEM1 is an active mannosidase. This mutation did not affect the chaperone function of EDEM1.     

Protein folding and quality control in the endoplasmic reticulum

The endoplasmic reticulum (ER) is a highly versatile protein factory that is equipped with chaperones and folding enzymes essential for protein folding. ER quality control guided by these chaperones is essential for life. Whereas correctly folded proteins are exported from the ER, misfolded proteins are retained and selectively degraded. At least two main chaperone classes, BiP and calnexin/calreticulin, are active in ER quality control. Folding factors usually are found in complexes. Recent work emphasises more than ever that chaperones act in concert with co-factors and with each other.     

Early postnatal death and motor disorders in mice congenitally deficient in calnexin expression

Calnexin is a ubiquitously expressed type I membrane protein which is exclusively localized in the endoplasmic reticulum (ER). In mammalian cells, calnexin functions as a chaperone molecule and plays a key role in glycoprotein folding and quality control within the ER by interacting with folding intermediates via their monoglucosylated glycans. In order to gain more insight into the physiological roles of calnexin, we have generated calnexin gene-deficient mice. Despite its profound involvement in protein folding, calnexin is not essential for mammalian-cell viability in vivo: calnexin gene knockout mice were carried to full term, although 50% died within 48 h and the majority of the remaining mice had to be sacrificed within 4 weeks, with only a very few mice surviving to 3 months. Calnexin gene-deficient mice were smaller than their littermates and showed very obvious motor disorders, associated with a dramatic loss of large myelinated nerve fibers. Thus, the critical contribution of calnexin to mammalian physiology is tissue specific.     

Functional relationship between calreticulin, calnexin, and the endoplasmic reticulum luminal domain of calnexin

Calnexin is a membrane protein of the endoplasmic reticulum (ER) that functions as a molecular chaperone and as a component of the ER quality control machinery. Calreticulin, a soluble analog of calnexin, is thought to possess similar functions, but these have not been directly demonstrated in vivo. Both proteins contain a lectin site that directs their association with newly synthesized glycoproteins. Although many glycoproteins bind to both calnexin and calreticulin, there are differences in the spectrum of glycoproteins that each binds. Using a Drosophila expression system and the mouse class I histocompatibility molecule as a model glycoprotein, we found that calreticulin does possess apparent chaperone and quality control functions, enhancing class I folding and subunit assembly, stabilizing subunits, and impeding export of assembly intermediates from the ER. Indeed, the functions of calnexin and calreticulin were largely interchangeable. We also determined that a soluble form of calnexin (residues 1-387) can functionally replace its membrane-bound counterpart. However, when calnexin was expressed as a soluble protein in L cells, the pattern of associated glycoproteins changed to resemble that of calreticulin. Conversely, membrane-anchored calreticulin bound to a similar set of glycoproteins as calnexin. Therefore, the different topological environments of calnexin and calreticulin are important in determining their distinct substrate specificities.     

Cleavage of calnexin caused by apoptotic stimuli: implication for the regulation of apoptosis

Calnexin is an endoplasmic reticulum (ER)-resident molecular chaperone that plays an essential role in the correct folding of membrane proteins. We found that calnexin is subjected to partial cleavage in apoptotic mouse cells. Both ER stress-inducing and ER stress-non-inducing apoptotic stimuli caused the cleavage of calnexin, indicating that this event does not always occur downstream of ER stress. The inhibition of caspases that target the amino acid sequence DXXD abrogated calnexin cleavage in apoptotic stimulus-treated cells. In addition, disruption of one of two DXXD sequences located in the cytoplasmic domain caused calnexin to escape cleavage during apoptosis. Furthermore, calnexin was cleaved in vitro by recombinant caspase-3 or caspase-7. Finally, the overexpression of a presumed cleavage product of calnexin partly inhibited apoptosis. These results collectively suggest that caspase-3 or caspase-7 cleaves calnexin, whose cleaved product leads to the attenuation of apoptosis.     

Diverse pathways for maturation of the Na,K-ATPase β1 and β2 subunits in the endoplasmic reticulum of Madin-Darby canine kidney cells

Proper folding of the Na,K-ATPase β subunits followed by assembly with the α subunits is necessary for their export from the endoplasmic reticulum (ER). Here we examine roles of the ER lectin chaperone, calnexin, and non-lectin chaperone, BiP, in folding and quality control of the β(1) and β(2) subunits in Madin-Darby canine kidney cells. Short term prevention of glycan-calnexin interactions by castanospermine slightly increases ER retention of β(1), suggesting minor involvement of calnexin in subunit folding. However, both prolonged incubation with castanospermine and removal of N-glycosylation sites do not affect the α(1)-assembly or trafficking of β(1) but increase the amount of the β(1)-bound BiP, showing that BiP can compensate for calnexin in assisting β(1) folding. In contrast to β(1), prevention of either N-glycosylation or glycan-calnexin interactions abolishes the α(1)-assembly and export of β(2) from the ER despite increased β(2)-BiP binding. Mutations in the α(1)-interacting regions of β(1) and β(2) subunits impair α(1) assembly but do not affect folding of the β subunits tested by their sensitivity to trypsin. At the same time, these mutations increase the amount of β-bound BiP but not of β-bound calnexin and increase ER retention of both β-isoforms. BiP, therefore, prevents the ER export of folded but α(1)-unassembled β subunits. These α(1)-unassembled β subunits are degraded faster than α(1)-bound β subunits, preventing ER overload. In conclusion, folding of the β(1) and β(2) subunits is assisted predominantly by BiP and calnexin, respectively. Folded β(1) and β(2) either assemble with α(1) or bind BiP. The α(1)-bound β subunits traffic to the Golgi, whereas BiP-bound β subunits are retained and degraded in the ER.     

Potent lectin-independent chaperone function of calnexin under conditions prevalent within the lumen of the endoplasmic reticulum

Calnexin is a membrane-bound chaperone of the endoplasmic reticulum (ER) that participates in the folding and quality control of newly synthesized glycoproteins. Binding to glycoproteins occurs through a lectin site with specificity for Glc1Man9GlcNAc2 oligosaccharides as well as through a polypeptide binding site that recognizes non-native protein conformations. The latter interaction is somewhat controversial because it is based on observations that calnexin can suppress the aggregation of non-glycosylated substrates at elevated temperature or at low calcium concentrations, conditions that may affect the structural integrity of calnexin. Here, we examine the ability of calnexin to interact with a non-glycosylated substrate under physiological conditions of the ER lumen. We show that the soluble ER luminal domain of calnexin can indeed suppress the aggregation of non-glycosylated firefly luciferase at 37 degrees C and at the normal resting ER calcium concentration of 0.4 mM. However, gradual reduction of calcium below the resting level was accompanied by a progressive loss of native calnexin structure as assessed by thermal stability, protease sensitivity, intrinsic fluorescence, and bis-ANS binding. These assays permitted the characterization of a single calcium binding site on calnexin with a Kd = 0.15 +/- 0.05 mM. We also show that the suppression of firefly luciferase aggregation by calnexin is strongly enhanced in the presence of millimolar concentrations of ATP and that the Kd for ATP binding to calnexin in the presence of 0.4 mM calcium is 0.7 mM. ATP did not alter the overall stability of calnexin but instead triggered the localized exposure of a hydrophobic site on the chaperone. These findings demonstrate that calnexin is a potent molecular chaperone that is capable of suppressing the aggregation of substrates through polypeptide-based interactions under conditions that exist within the ER lumen.     

Trimming and readdition of glucose to N-linked oligosaccharides determines calnexin association of a substrate glycoprotein in living cells

To analyze the role of glucose trimming and reglucosylation in the binding of substrate proteins to calnexin in the endoplasmic reticulum (ER) of living cells, we made use of the thermosensitive vesicular stomatitis virus tsO45 glycoprotein (G protein). At nonpermissive temperature the G protein failed to fold completely and remained bound to calnexin. When the cells were shifted to permissive temperature, complete folding occurred accompanied by glucosidase-mediated elimination of calnexin-G protein complexes. If release from calnexin was blocked during the temperature shift by inhibiting the glucosidases, folding occurred, albeit at a reduced rate. In contrast, when unfolded by a shift from permissive to nonpermissive temperature, the G protein was reglucosylated rapidly and became capable of rebinding to calnexin. The rate at which calnexin binding occurred showed a 20-min delay that was explained by accumulation of the G protein in calnexin-free exit sites of the ER. These contained the glucosyltransferase responsible for reglucosylation of misfolded glycoproteins but had little or no calnexin. After unfolding and reglucosylation, the G proteins moved slowly from these structures back to the ER where they reassociated with the chaperone. Taken together, these results in live cells fully supported the lectin-only model of calnexin function. The ER exit sites emerged as a potentially important location for components of the quality control system.     

Calnexin-dependent regulation of tunicamycin-induced apoptosis in breast carcinoma MCF-7 cells

The endoplasmic reticulum (ER) has evolved specific mechanisms to ensure protein folding as well as the maintenance of its own homeostasis. When these functions are not achieved, specific ER stress signals are triggered to activate either adaptive or apoptotic responses. Here, we demonstrate that MCF-7 cells are resistant to tunicamycin-induced apoptosis. We show that the expression level of the ER chaperone calnexin can directly influence tunicamycin sensitivity in this cell line. Interestingly, the expression of a calnexin lacking the chaperone domain (DeltaE) partially restores their sensitivity to tunicamycin-induced apoptosis. Indeed, we show that DeltaE acts as a scaffold molecule to allow the cleavage of Bap31 and thus generate the proapoptotic p20 fragment. Utilizing the ability of MCF-7 cells to resist tunicamycin-induced apoptosis, we have characterized a molecular mechanism by which calnexin regulates ER-stress-mediated apoptosis in a manner independent of its chaperone functions but dependent of its binding to Bap31.     

Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control

Calreticulin and calnexin are homologous lectins that serve as molecular chaperones for glycoproteins in the endoplasmic reticulum of eukaryotic cells. Here we show that calreticulin depletion specifically accelerates the maturation of cellular and viral glycoproteins with a modest decrease in folding efficiency. Calnexin depletion prevents proper maturation of some proteins such as influenza hemagglutinin but does not interfere appreciably with the maturation of several others. A dramatic loss of stringency in the ER quality control with transport at the cell surface of misfolded glycoprotein conformers is only observed when substrate access to both calreticulin and calnexin is prevented. Although not fully interchangeable during assistance of glycoprotein folding, calreticulin and calnexin may work, independently, as efficient and crucial factors for retention in the ER of nonnative polypeptides.     

Glycoprotein folding and the role of EDEM1, EDEM2 and EDEM3 in degradation of folding-defective glycoproteins

Proteins synthesized in the endoplasmic reticulum (ER) lumen are exposed to several dedicated chaperones and folding factors that ensure efficient maturation. Nevertheless, protein folding remains error-prone and mutations in the polypeptide sequence may significantly reduce folding-efficiency. Folding-incompetent proteins carrying N-glycans are extracted from futile folding cycles in the calnexin chaperone system upon intervention of EDEM1, EDEM2 and EDEM3, three ER-stress-induced members of the glycosyl hydrolase 47 family. This review describes current knowledge about mechanisms regulating folding and disposal of glycoproteins.     

Mechanisms of pharmacological rescue of trafficking-defective hERG mutant channels in human long QT syndrome

Long QT syndrome type 2 is caused by mutations in the human ether-a-go-go-related gene (hERG). We previously reported that the N470D mutation is retained in the endoplasmic reticulum (ER) but can be rescued to the plasma membrane by hERG channel blocker E-4031. The mechanisms of ER retention and how E-4031 rescues the N470D mutant are poorly understood. In this study, we investigated the interaction of hERG channels with the ER chaperone protein calnexin. Using coimmunoprecipitation, we showed that the immature forms of both wild type hERG and N470D associated with calnexin. The association required N-linked glycosylation of hERG channels. Pulse-chase analysis revealed that N470D had a prolonged association with calnexin compared with wild type hERG and E-4031 shortened the time course of calnexin association with N470D. To test whether the prolonged association of N470D with calnexin is due to defective folding of mutant channels, we studied hERG channel folding using the trypsin digestion method. We found that N470D and the immature form of wild type hERG were more sensitive to trypsin digestion than the mature form of wild type hERG. In the presence of E-4031, N470D became more resistant to trypsin even when its ER-to-Golgi transport was blocked by brefeldin A. These results suggest that defective folding of N470D contributes to its prolonged association with calnexin and ER retention and that E-4031 may restore proper folding of the N470D channel leading to its cell surface expression.