Localization of the lectin,ERp57 binding,and polypeptide binding sites of calnexin and calreticulin

Calnexin and calreticulin are membrane-bound and soluble chaperones, respectively, of the endoplasmic reticulum (ER) which interact transiently with a broad spectrum of newly synthesized glycoproteins. In addition to sharing substantial sequence identity, both calnexin and calreticulin bind to monoglucosylated oligosaccharides of the form Glc(1)Man(5-9)GlcNAc(2), interact with the thiol oxidoreductase, ERp57, and are capable of acting as chaperones in vitro to suppress the aggregation of non-native proteins. To understand how these diverse functions are coordinated, we have localized the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin. Recent structural studies suggest that both proteins consist of a globular domain and an extended arm domain comprised of two sequence motifs repeated in tandem. Our results indicate that the primary lectin site of calnexin and calreticulin resides within the globular domain, but the results also point to a much weaker secondary site within the arm domain which lacks specificity for monoglucosylated oligosaccharides. For both proteins, a site of interaction with ERp57 is centered on the arm domain, which retains approximately 50% of binding compared with full-length controls. This site is in addition to a Zn(2+)-dependent site located within the globular domain of both proteins. Finally, calnexin and calreticulin suppress the aggregation of unfolded proteins via a polypeptide binding site located within their globular domains but require the arm domain for full chaperone function. These findings are integrated into a model that describes the interaction of glycoprotein folding intermediates with calnexin and calreticulin.     

Conformational requirements for glycoprotein reglucosylation in the endoplasmic reticulum

Newly synthesized glycoproteins interact during folding and quality control in the ER with calnexin and calreticulin, two lectins specific for monoglucosylated oligosaccharides. Binding and release are regulated by two enzymes, glucosidase II and UDP-Glc:glycoprotein:glycosyltransferase (GT), which cyclically remove and reattach the essential glucose residues on the N-linked oligosaccharides. GT acts as a folding sensor in the cycle, selectively reglucosylating incompletely folded glycoproteins and promoting binding of its substrates to the lectins. To investigate how nonnative protein conformations are recognized and directed to this unique chaperone system, we analyzed the interaction of GT with a series of model substrates with well defined conformations derived from RNaseB. We found that conformations with slight perturbations were not reglucosylated by GT. In contrast, a partially structured nonnative form was efficiently recognized by the enzyme. When this form was converted back to a nativelike state, concomitant loss of recognition by GT occurred, reproducing the reglucosylation conditions observed in vivo with isolated components. Moreover, fully unfolded conformers were poorly recognized. The results indicated that GT is able to distinguish between different nonnative conformations with a distinct preference for partially structured conformers. The findings suggest that discrete populations of nonnative conformations are selectively reglucosylated to participate in the calnexin/calreticulin chaperone pathway.     

The Number and Location of Glycans on Influenza Hemagglutinin Determine Folding and Association with Calnexin and Calreticulin

Calnexin and calreticulin are homologous molecular chaperones that promote proper folding, oligomeric assembly, and quality control of newly synthesized glycoproteins in the endoplasmic reticulum (ER). Both are lectins that bind to substrate glycoproteins that have monoglucosylated N-linked oligosaccharides. Their binding to newly translated influenza virus hemagglutinin (HA), and various mutants thereof, was analyzed in microsomes after in vitro translation and expression in live CHO cells. A large fraction of the HA molecules was found to occur in ternary HA– calnexin–calreticulin complexes. In contrast to calnexin, calreticulin was found to bind primarily to early folding intermediates. Analysis of HA mutants with different numbers and locations of N-linked glycans showed that although the two chaperones share the same carbohydrate specificity, they display distinct binding properties; calreticulin binding depends on the oligosaccharides in the more rapidly folding top/hinge domain of HA whereas calnexin is less discriminating. Calnexin's binding was reduced if the HA was expressed as a soluble anchor-free protein rather than membrane bound. When the co- and posttranslational folding and trimerization of glycosylation mutants was analyzed, it was observed that removal of stem domain glycans caused accelerated folding whereas removal of the top domain glycans (especially the oligosaccharide attached to Asn81) inhibited folding. In summary, the data established that individual N-linked glycans in HA have distinct roles in calnexin/calreticulin binding and in co- and posttranslational folding.     

N-linked oligosaccharides are necessary and sufficient for association of glycosylated forms of bovine RNase with calnexin and calreticulin.

Calnexin and calreticulin are lectin-like molecular chaperones that promote folding and assembly of newly synthesized glycoproteins in the endoplasmic reticulum. While it is well established that they interact with substrate monoglucosylated N-linked oligosaccharides, it has been proposed that they also interact with polypeptide moieties. To test this notion, glycosylated forms of bovine pancreatic ribonuclease (RNase) were translated in the presence of microsomes and their folding and association with calnexin and calreticulin were monitored. When expressed with two N-linked glycans in the presence of micromolar concentrations of deoxynojirimycin, this small soluble protein was found to bind firmly to both calnexin and calreticulin. The oligosaccharides were necessary for association, but it made no difference whether the RNase was folded or not. This indicated that unlike other chaperones, calnexin and calreticulin do not select their substrates on the basis of folding status. Moreover, enzymatic removal of the oligosaccharide chains using peptide N-glycosidase F or removal of the glucoses by ER glucosidase II resulted in dissociation of the complexes. This indicated that the lectin-like interaction, and not a protein-protein interaction, played the central role in stabilizing RNase-calnexin/calreticulin complexes.     

Calreticulin functions in vitro as a molecular chaperone for both glycosylated and non-glycosylated proteins

Calreticulin (CRT) is thought to be a molecular chaperone that interacts with glycoproteins exclusively through a lectin site specific for monoglucosylated oligosaccharides. However, this chaperone function has never been directly demonstrated nor is it clear how lectin-oligosaccharide interactions facilitate glycoprotein folding. Using purified components, we show that CRT suppresses the aggregation not only of a glycoprotein bearing monoglucosylated oligosaccharides but also that of non-glycosylated proteins. Furthermore, CRT forms stable complexes with unfolded, non-glycosylated substrates but does not associate with native proteins. ATP and Zn(2+) enhance CRT's ability to suppress aggregation of non- glycoproteins, whereas engagement of its lectin site with purified oligosaccharide attenuates this function. CRT also confers protection against thermal inactivation and maintains substrates in a folding-competent state. We conclude that in addition to being a lectin CRT possesses a polypeptide binding capacity capable of discriminating between protein conformational states and that it functions in vitro as a classical molecular chaperone.     

A polypeptide binding conformation of calreticulin is induced by heat shock, calcium depletion, or by deletion of the C-terminal acidic region

It is widely believed that the chaperone activity of calreticulin is mediated by its ability to bind glycoproteins containing monoglucosylated oligosaccharides. However, calreticulin is also a polypeptide binding protein. Here we show that heat shock, calcium depletion, or deletion of the C-terminal acidic domain enhance binding of purified calreticulin to polypeptide substrates and enhance calreticulin's chaperone activity. These conditions also enhance calreticulin oligomerization, but oligomerization per se is not required for enhanced polypeptide binding. In cells, calreticulin oligomerization intermediates accumulate in response to conditions that induce protein misfolding (heat shock and tunicamycin treatments), and upon calcium depletion. Additionally, in cells, calreticulin binds to deglycosylated major histocompatibility complex class I heavy chains when significant levels of calreticulin oligomerization intermediates are induced. Thus, cell stress conditions that generate nonnative substrates of calreticulin also affect the conformational properties of calreticulin itself, and enhance its binding to substrates, independent of substrate glucosylation.     

Calnexin discriminates between protein conformational states and functions as a molecular chaperone in vitro.

Although calnexin is thought to function as a molecular chaperone for glycoproteins, a prevalent view is that it cannot distinguish between protein conformational states, binding solely through its lectin site to monoglucosylated oligosaccharides. Using purified components in vitro, calnexin effectively prevented the aggregation not only of glycoproteins bearing monoglucosylated oligosaccharides but also proteins lacking N-glycans, an effect enhanced by ATP. It also suppressed the thermal denaturation of nonglycosylated proteins and enhanced their refolding in conjunction with other cellular components. Calnexin formed stable complexes with unfolded conformers of these proteins but not with the native molecules. Therefore, in addition to being a lectin, calnexin functions as a bona fide molecular chaperone capable of interacting with polypeptide segments of folding glycoproteins.     

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.     

Involvement of Endoplasmic Reticulum Chaperones in the Folding of Hepatitis C Virus Glycoproteins

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2) which interact noncovalently to form a heterodimer (E1-E2). During the folding and assembly of HCV glycoproteins, a large portion of these proteins are trapped in aggregates, reducing the efficiency of native E1-E2 complex assembly. To better understand this phenomenon and to try to increase the efficiency of HCV glycoprotein folding, endoplasmic reticulum chaperones potentially interacting with these proteins were studied. Calnexin, calreticulin, and BiP were shown to interact with E1 and E2, whereas no interaction was detected between GRP94 and HCV glycoproteins. The association of HCV glycoproteins with calnexin and calreticulin was faster than with BiP, and the kinetics of interaction with calnexin and calreticulin were very similar. However, calreticulin and BiP interacted preferentially with aggregates whereas calnexin preferentially associated with monomeric forms of HCV glycoproteins or noncovalent complexes. Tunicamycin treatment inhibited the binding of HCV glycoproteins to calnexin and calreticulin, indicating the importance of N-linked oligosaccharides for these interactions. The effect of the co-overexpression of each chaperone on the folding of HCV glycoproteins was also analyzed. However, the levels of native E1-E2 complexes were not increased. Together, our data suggest that calnexin plays a role in the productive folding of HCV glycoproteins whereas calreticulin and BiP are probably involved in a nonproductive pathway of folding.     

Glycan-dependent and -independent Interactions Contribute to Cellular Substrate Recruitment by Calreticulin

Calreticulin is an endoplasmic reticulum chaperone with specificity for monoglucosylated glycoproteins. Calreticulin also inhibits precipitation of nonglycosylated proteins and thus contains generic protein-binding sites, but their location and contributions to substrate folding are unknown. We show that calreticulin binds glycosylated and nonglycosylated proteins with similar affinities but distinct interaction kinetics. Although both interactions involve the glycan-binding site or its vicinity, the arm-like proline-rich (P-) domain of calreticulin contributes to binding non/deglycosylated proteins. Correspondingly, ensemble FRET spectroscopy measurements indicate that glycosylated and nonglycosylated proteins induce “open” and “closed” P-domain conformations, respectively. The co-chaperone ERp57 influences substrate-binding kinetics and induces a closed P-domain conformation. Together with analysis of the interactions of calreticulin with cellular proteins, these findings indicate that the recruitment of monoglucosylated proteins to calreticulin is kinetically driven, whereas the P-domain and co-chaperone contribute to stable substrate binding. Substrate sequestration in the cleft between the glycan-binding site and P-domain is a likely mechanism for calreticulin-assisted protein folding.     

The use of calnexin and calreticulin by cellular and viral glycoproteins

Calnexin and calreticulin are homologous lectin chaperones that assist maturation of cellular and viral glycoproteins in the mammalian endoplasmic reticulum. Calnexin and calreticulin share the same specificity for monoglucosylated protein-bound N-glycans but associate with a distinct set of newly synthesized polypeptides. We report here that most calnexin substrates do not associate with calreticulin even upon selective calnexin inactivation, while BiP associates more abundantly with nascent polypeptides under these conditions. Calreticulin associated more abundantly with orphan calnexin substrates only in infected cells and preferentially with polypeptides of viral origin, showing stronger dependence of model viral glycoproteins on endoplasmic reticulum lectins. This may explain why inactivation of the calnexin cycle affects viral replication and infectivity but not viability of mammalian cells.     

Reglucosylation by UDP-glucose:glycoprotein glucosyltransferase 1 delays glycoprotein secretion but not degradation

UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) is a central quality control gatekeeper in the mammalian endoplasmic reticulum (ER). The reglucosylation of glycoproteins supports their rebinding to the carbohydrate-binding ER molecular chaperones calnexin and calreticulin. A cell-based reglucosylation assay was used to investigate the role of UGT1 in ER protein surveillance or the quality control process. UGT1 was found to modify wild-type proteins or proteins that are expected to eventually traffic out of the ER through the secretory pathway. Trapping of reglucosylated wild-type substrates in their monoglucosylated state delayed their secretion. Whereas terminally misfolded substrates or off-pathway proteins were most efficiently reglucosylated by UGT1, the trapping of these mutant substrates in their reglucosylated or monoglucosylated state did not delay their degradation by the ER-associated degradation pathway. This indicated that monoglucosylated mutant proteins were actively extracted from the calnexin/calreticulin binding-reglucosylation cycle for degradation. Therefore trapping proteins in their monoglucosylated state was sufficient to delay their exit to the Golgi but had no effect on their rate of degradation, suggesting that the degradation selection process progressed in a dominant manner that was independent of reglucosylation and the glucose-containing A-branch on the substrate glycans.     

Transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins.

The soluble, calcium-binding protein calreticulin shares high sequence homology with calnexin, a transmembrane chaperone of glycoprotein folding. Our experiments demonstrated that calreticulin, like calnexin, associated transiently with numerous newly synthesized proteins in the endoplasmic reticulum. The population of proteins that bound to calreticulin was partially overlapping with those that bound to calnexin. Hemagglutinin (HA) of influenza virus was shown to associate with both calreticulin and calnexin. Using HA as a model substrate, it was found that both calreticulin- and calnexin-bound HA corresponded primarily to incompletely disulfide-bonded folding intermediates and conformationally trapped forms. Binding of all substrates was oligosaccharide-dependent and required the trimming of glucose residues from asparagine-linked core glycans by glucosidases I and II. In vitro, alpha-mannosidase digestion of calreticulin-bound HA indicated that calreticulin was specific for monoglucosylated glycans. Thus, calreticulin appeared to be a lectin with similar oligosaccharide specificity as its membrane-bound homologue, calnexin. Both are therefore likely to play an important role in glycoprotein maturation and quality control in the endoplasmic reticulum.     

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.     

Polypeptide substrate recognition by calnexin requires specific conformations of the calnexin protein

Calnexin is an endoplasmic reticulum chaperone that binds to substrates containing monoglucosylated oligosaccharides. Whether calnexin can also directly recognize polypeptide components of substrates is controversial. We found that calnexin displayed significant conformational lability for a chaperone and that heat treatment and calcium depletion induced the formation of calnexin dimers and higher order oligomers. These conditions enhanced the chaperone activity of calnexin toward glycosylated and non-glycosylated major histocompatibility complex (MHC) class I heavy chains, and enhanced calnexin binding to MHC class I heavy chains. In contrast to these observations, calnexin binding to oligosaccharide substrates has been reported to be impaired under calcium-depleting conditions. Calnexin dimers were induced in HeLa cells upon heat shock and under calcium-depleting conditions, and heat shock enhanced calnexin binding to MHC class I heavy chains in HeLa cells. Virus-induced endoplasmic reticulum stress also resulted in the appearance of calnexin dimers. Tunicamycin treatment of HeLa cells induced a slow accumulation of calnexin dimers, the appearance of which correlated with enhanced calnexin binding to deglycosylated MHC class I heavy chains. In vitro, the presence of calnexin-specific oligosaccharides inhibited the formation of calnexin dimers and higher order structures. Together, these data indicate that polypeptide binding is favored by conditions that induce partial unfolding of calnexin monomers, whereas oligosaccharide binding is favored by conditions that enhance the structural stability (folding) of calnexin monomers. Conditions that induce the calnexin "polypeptide-binding" conformation also induce self-association of calnexin if the concentration is sufficiently high; however, calnexin dimerization/oligomerization per se is not essential for polypeptide substrate binding.     

Tapasin and ERp57 form a stable disulfide-linked dimer within the MHC class I peptide-loading complex

We previously showed that the major histocompatibility complex (MHC) class I chaperone tapasin can be detected as a mixed disulfide with the thiol-oxidoreductase ERp57. Here we show that tapasin is a unique and preferred substrate, a substantial majority of which is disulfide-linked to ERp57 within the cell. Tapasin upregulation by interferon-gamma induces sequestration of the vast majority of ERp57 into the MHC class I peptide-loading complex. The rate of tapasin-ERp57 conjugate formation is unaffected by the absence of beta2-microglubulin (beta2m), and is independent of calnexin or calreticulin interactions with monoglucosylated N-linked glycans. The heterodimer forms spontaneously in vitro upon mixing recombinant ERp57 and tapasin. Noncovalent interactions between the native proteins inhibit the reductase activity of the thioredoxin CXXC motif within the N-terminal a domain of ERp57 to maintain its interaction with tapasin. Disruption of these interactions by denaturation allows reduction to proceed. Thus, tapasin association specifically inhibits the escape pathway required for disulfide-bond isomerization within conventional protein substrates, suggesting a specific structural role for ERp57 within the MHC class I peptide-loading complex.     

Endoplasmic reticulum glucosidase II is inhibited by its end products

The calnexin/calreticulin cycle is a quality control system responsible for promoting the folding of newly synthesized glycoproteins entering the endoplasmic reticulum (ER). The association of calnexin and calreticulin with the glycoproteins is regulated by ER glucosidase II, which hydrolyzes Glc 2Man X GlcNAc 2 glycans to Glc 1Man X GlcNAc 2 and further to Glc 0Man X GlcNAc 2 ( X represents any number between 5 and 9). To gain new insights into the reaction mechanism of glucosidase II, we developed a kinetic model that describes the interactions between glucosidase II, calnexin/calreticulin, and the glycans. Our model accurately reconstructed the hydrolysis of glycans with nine mannose residues and glycans with seven mannose residues, as measured by Totani et al. [Totani, K., Ihara, Y., Matsuo, I., and Ito, Y. (2006) J. Biol. Chem. 281, 31502-31508]. Intriguingly, our model predicted that glucosidase II was inhibited by its nonglucosylated end products, where the inhibitory effect of Glc 0Man 7GlcNAc 2 was much stronger than that of Glc 0Man 9GlcNAc 2. These predictions were confirmed experimentally. Moreover, our model suggested that glycans with a different number of mannose residues can be equivalent substrates of glucosidase II, in contrast to what had been previously thought. We discuss the possibility that nonglucosylated glycans, existing in the ER, might regulate the entry of newly synthesized glycoproteins into the calnexin/calreticulin cycle. Our model also shows that glucosidase II does not interact with monoglucosylated glycans while they are bound to calnexin or calreticulin.     

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.     

Trypanosoma cruzi calreticulin is a lectin that binds monoglucosylated oligosaccharides but not protein moieties of glycoproteins

Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse-chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin-cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-beta-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin-cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.     

Promotion of transferrin folding by cyclic interactions with calnexin and calreticulin.

Calnexin, an abundant membrane protein, and its lumenal homolog calreticulin interact with nascent proteins in the endoplasmic reticulum. Because they have an affinity for monoglucosylated N-linked oligosaccharides which can be regenerated from the aglucosylated sugar, it has been speculated that this repeated oligosaccharide binding may play a role in nascent chain folding. To investigate the process, we have developed a novel assay system using microsomes freshly prepared from pulse labeled HepG2 cells. Unlike the previously described oxidative folding systems which required rabbit reticulocyte lysates, the oxidative folding of transferrin in isolated microsomes could be carried out in a defined solution. In this system, addition of a glucose donor, UDP-glucose, to the microsomes triggered glucosylation of transferrin and resulted in its cyclic interaction with calnexin and calreticulin. When the folding of transferrin in microsomes was analyzed, UDP-glucose enhanced the amount of folded transferrin and reduced the disulfide-linked aggregates. Analysis of transferrin folding in briefly heat-treated microsomes revealed that UDP-glucose was also effective in elimination of heat-induced misfolding. Incubation of the microsomes with an alpha-glucosidase inhibitor, castanospermine, prolonged the association of transferrin with the chaperones and prevented completion of folding and, importantly, aggregate formation, particularly in the calnexin complex. Accordingly, we demonstrate that repeated binding of the chaperones to the glucose of the transferrin sugar moiety prevents and corrects misfolding of the protein.