Crystal structure and functional analysis of Drosophila Wind,a protein-disulfide isomerase-related protein

In the developing Drosophila melanogaster embryo, dorsal-ventral patterning displays an absolute requirement for the product of the essential windbeutel gene, Wind. In homozygous windbeutel mutant flies, dorsal-ventral patterning fails to initiate because of the failure of the Golgi-resident proteoglycan-modifying protein, Pipe, to exit the endoplasmic reticulum, and this leads to the death of the embryo. Here, we describe the three-dimensional structure of Wind at 1.9-A resolution and identify a candidate surface for interaction with Pipe. This represents the first crystal structure of a eukaryotic protein-disulfide isomerase-related protein of the endoplasmic reticulum to be described. The dimeric protein is composed of an N-terminal thioredoxin domain and a C-terminal alpha-helical domain unique to protein-disulfide isomerase D proteins. Although Wind carries a CXXC motif that is partially surface accessible, this motif is redox inactive, and the cysteines are not required for the targeting of Pipe to the Golgi. However, both domains are required for targeting Pipe to the Golgi, and, although the mouse homologue ERp28 cannot replace the function of Wind, exchange of the Wind D-domain with that of ERp28 allows for efficient Golgi transport of Pipe.     

Mapping of a substrate binding site in the protein disulfide isomerase-related chaperone wind based on protein function and crystal structure

The protein disulfide isomerase (PDI)-related protein Wind is essential in Drosophila melanogaster, and is required for correct targeting of Pipe, an essential Golgi transmembrane 2-O-sulfotransferase. Apart from a thioredoxin fold domain present in all PDI proteins, Wind also has a unique C-terminal D-domain found only in PDI-D proteins. Here, we show that Pipe processing requires dimeric Wind, which interacts directly with the soluble domain of Pipe in vitro, and we map an essential substrate binding site in Wind to the vicinity of an exposed cluster of tyrosines within the thioredoxin fold domain. In vitro, binding occurs to multiple sites within the Pipe polypeptide and shows specificity for two consecutive aromatic residues. A second site in Wind, formed by a cluster of residues within the D-domain, is likewise required for substrate processing. This domain, expressed separately, impairs Pipe processing by the full-length Wind protein, indicating competitive binding to substrate. Our data represent the most accurate map of a peptide binding site in a PDI-related protein available to date and directly show peptide specificity for a naturally occurring substrate.     

Crystal Structure and Functional Analysis of the Protein Disulfide Isomerase-Related Protein ERp29

The protein disulfide isomerase-related protein ERp29 is a putative chaperone involved in processing and secretion of secretory proteins. Until now, however, both the structure and the exact nature of interacting substrates remained unclear. We provide for the first time a crystal structure of human ERp29, refined to 2.9 Å, and show that the protein has considerable structural homology to its Drosophila homolog Wind. We show that ERp29 binds directly not only to thyroglobulin and thyroglobulin-derived peptides in vitro but also to the Wind client protein Pipe and Pipe-derived peptides, although it fails to process Pipe in vivo. A monomeric mutant of ERp29 and a D domain mutant in which the second peptide binding site is inactivated also bind protein substrates, indicating that the monomeric thioredoxin domain is sufficient for client protein binding. Indeed, the b domains of ERp29 or Wind, expressed alone, are sufficient for binding proteins and peptides. Interacting peptides have in common two or more aromatic residues, with stronger binding for sequences with overall basic character. Thus, the data allow a view of the two putative peptide binding sites of ERp29 and indicate that the apparent, different processing activity of the human and Drosophila proteins in vivo does not stem from differences in peptide binding properties.     

ERp27, a new non-catalytic endoplasmic reticulum-located human protein disulfide isomerase family member, interacts with ERp57

Protein folding and quality control in the endoplasmic reticulum are critical processes for which our current understanding is far from complete. Here we describe the functional characterization of a new human 27.7-kDa protein (ERp27). We show that ERp27 is a two-domain protein located in the endoplasmic reticulum that is homologous to the non-catalytic b and b' domains of protein disulfide isomerase. ERp27 was shown to bind Delta-somatostatin, the standard test peptide for protein disulfide isomerase-substrate binding, and this ability was localized to the second domain of ERp27. An alignment of human ERp27 and human protein disulfide isomerase allowed for the putative identification of the peptide binding site of ERp27 indicating conservation of the location of the primary substrate binding site within the protein disulfide isomerase family. NMR studies revealed a significant conformational change in the b'-like domain of ERp27 upon substrate binding, which was not just localized to the substrate binding site. In addition, we report that ERp27 is bound by ERp57 both in vitro and in vivo by a similar mechanism by which ERp57 binds calreticulin.     

Structure of the Non-Catalytic Domain of the Protein Disulfide Isomerase-Related Protein (PDIR) Reveals Function in Protein Binding

Protein disulfide isomerases comprise a large family of enzymes responsible for catalyzing the proper oxidation and folding of newly synthesized proteins in the endoplasmic reticulum (ER). Protein disulfide isomerase-related (PDIR) protein (also known as PDIA5) is a specialized member that participates in the folding of α₁-antitrypsin and N-linked glycoproteins. Here, the crystal structure of the non-catalytic domain of PDIR was determined to 1.5 Å resolution. The structure adopts a thioredoxin-like fold stabilized by a structural disulfide bridge with a positively charged binding surface for interactions with the ER chaperones, calreticulin and ERp72. Crystal contacts between molecules potentially mimic the interactions of PDIR with misfolded substrate proteins. The results suggest that the non-catalytic domain of PDIR plays a key role in the recognition of protein partners and substrates.     

Dimerization of ERp29, a PDI-like protein, is essential for its diverse functions

Protein disulfide isomerase (PDI)-like proteins act as oxido-reductases and chaperones in the endoplasmic reticulum (ER). How oligomerization of the PDI-like proteins control these activities is unknown. Here we show that dimerization of ERp29, a PDI-like protein, regulates its protein unfolding and escort activities. We have demonstrated previously that ERp29 induces the local unfolding of polyomavirus in the ER, a step required for viral infection. We now find that, in contrast to wild-type ERp29, a mutant ERp29 (D42A) that dimerizes inefficiently is unable to unfold polyomavirus or stimulate infection. A compensatory mutation that partially restores dimerization to the mutant ERp29 (G37D/D42A) rescues ERp29 activity. These results indicate that dimerization of ERp29 is crucial for its protein unfolding function. ERp29 was also suggested to act as an escort factor by binding to the secretory protein thyroglobulin (Tg) in the ER, thereby facilitating its secretion. We show that this escort function likewise depends on ERp29 dimerization. Thus our data demonstrate that dimerization of a PDI-like protein acts to regulate its diverse ER activities.     

Crystal structure of the bb' domains of the protein disulfide isomerase ERp57

The synthesis of proteins in the endoplasmic reticulum (ER) is limited by the rate of correct disulfide bond formation. This process is carried out by protein disulfide isomerases, a family of ER proteins which includes general enzymes such as PDI that recognize unfolded proteins and others that are selective for specific proteins or classes. Using small-angle X-ray scattering and X-ray crystallography, we report the structure of a selective isomerase, ERp57, and its interactions with the lectin chaperone calnexin. Using isothermal titration calorimetry and NMR spectroscopy, we show that the b' domain of ERp57 binds calnexin with micromolar affinity through a conserved patch of basic residues. Disruption of this binding site by mutagenesis abrogates folding of RNase B in an in vitro assay. The relative positions of the ERp57 catalytic sites and calnexin binding site suggest that activation by calnexin is due to substrate recruitment rather than a direct stimulation of ERp57 oxidoreductase activity.     

Endoplasmic reticulum protein 29 (ERp29): An emerging role in cancer

The endoplasmic reticulum protein 29 (ERp29) is a molecule that facilitates processing and transport of proteins in the early secretory pathway. Structural and functional analyses have suggested a biological role as a putative chaperone in the endoplasmic reticulum. The N-terminal domain of ERp29 resembles the thioredoxin domain of protein disulfide isomerase, but lacks its redox-active function due to the absence of an active motif consisting of double cysteines. In the context of carcinogenesis, the role of ERp29 in cancer progression has not been fully elucidated. However, recent studies indicate that high expression of ERp29 inversely correlates to tumor progression. In addition, over-expression of ERp29 significantly inhibits proliferation and suppresses tumorigenesis by modulating ER stress signaling and the mesenchymal-epithelial transition in breast cancer cells. In this review, we summarize the biological properties of ERp29 and its novel function as a tumor suppressor.     

The primary substrate binding site in the b' domain of ERp57 is adapted for endoplasmic reticulum lectin association

ERp57 is a member of the protein disulfide isomerase (PDI) family that is located in the endoplasmic reticulum (ER) and characterized by its specificity for glycoproteins. Substrate selection by ERp57 is dependent upon its formation of discrete complexes with two ER resident lectins, soluble calreticulin and membrane-bound calnexin. It is these two lectins that directly associate with glycoproteins bearing correctly trimmed oligosaccharide side chains. Thus, ERp57 is presented with a preselected set of substrates upon which it can act, and the specific binding of calreticulin and calnexin to ERp57 is pivotal to the functions of the resulting complexes. To gain further insights into the formation of these ERp57-ER lectin complexes, we have investigated the regions of ERp57 that are specifically required for its binding to calreticulin. Using a quantitative pull-down assay to investigate the binding of ERp57/PDI chimeras to calreticulin, we define the b and b' domains of ERp57 as the minimal elements that are sufficient for complex formation. This analysis further identifies a novel role for the distinctive C-terminal extension of ERp57 in reconstituting complex formation to wild type levels. Using our understanding of substrate binding to the b' domain of PDI as a paradigm, we show that alterations to specific residues in the b' domain of ERp57 dramatically reduce or completely abolish its binding to calreticulin. On the basis of these data, we propose a model where the region of ERp57 equivalent to the primary substrate binding site of archetypal PDI is occupied by calreticulin and suggest that the ER lectins act as adaptor molecules that define the substrate specificity of ERp57.     

ERp29 is a ubiquitous resident of the endoplasmic reticulum with a distinct role in secretory protein production.

ERp29 was recently characterized biochemically as a novel protein that resides in mammalian endoplasmic reticulum (ER). Here we applied immunochemical procedures at the cellular level to investigate the hypothesized role of ERp29 in secretory protein production. ERp29 was localized exclusively to the ER/nuclear envelope of MDCK cells using confocal immunocytochemistry and comparative markers of the ER lumen, ER/Golgi membrane, nuclei, and mitochondria. A predominant association with rough ER was revealed by sucrose-gradient analysis of rat liver microsomes. Immunohistochemistry showed ERp29 expression in 35 functionally distinct cell types of rat, establishing ERp29 as a general ER marker. The ERp29 expression profile largely paralleled that of protein disulfide isomerase (PDI), the closest relative of ERp29, consistent with a role in secretory protein production. However strikingly different ERp29/PDI ratios were observed in various cell types, suggesting independent regulation and functional roles. Together, these findings associate ERp29 primarily with the early stages of secretory protein production and implicate ERp29 in a distinct functional role that is utilized in most cells. Our identification of several ERp29-enriched cell types suggests a potential selectivity of ERp29 for non-collagenous substrates and provides a physiological foundation for future investigations.     

ERp57 is a multifunctional thiol-disulfide oxidoreductase

The thiol-disulfide oxidoreductase ERp57 is a soluble protein of the endoplasmic reticulum and the closest known homologue of protein disulfide isomerase. The protein interacts with the two lectin chaperones calnexin and calreticulin and thereby promotes the oxidative folding of newly synthesized glycoproteins. Here we have characterized several fundamental structural and functional properties of ERp57 in vitro, such as the domain organization, shape, redox potential, and the ability to catalyze different thiol-disulfide exchange reactions. Like protein disulfide isomerase, we find ERp57 to be comprised of four structural domains. The protein has an elongated shape of 3.4 +/- 0.1 nm in diameter and 16.8 +/- 0.5 nm in length. The two redox-active a and a' domains were determined to have redox potentials of -0.167 and -0.156 V, respectively. Furthermore, ERp57 was shown to efficiently catalyze disulfide reduction, disulfide isomerization, and dithiol oxidation in substrate proteins. The implications of these findings for the function of the protein in vivo are discussed.     

Identification of ERp29, an endoplasmic reticulum lumenal protein, as a new member of the thyroglobulin folding complex

Folding and post-translational modification of the thyroid hormone precursor, thyroglobulin (Tg), in the endoplasmic reticulum (ER) of the thyroid epithelial cells is facilitated by several molecular chaperones and folding enzymes, such as BiP, GRP94, calnexin, protein disulfide isomerase, ERp72, and others. They have been shown to associate simultaneously and/or sequentially with Tg in the course of its maturation, thus forming large heterocomplexes in the ER of thyrocytes. Here we present evidence that such complexes include a novel member, an ER-resident lumenal protein, ERp29, which is present in all mammalian tissues with exceptionally high levels of expression in the secretory cells. ERp29 was induced upon treatment of FRTL-5 rat thyrocytes with the thyroid-stimulating hormone, which is essential for the maintenance of thyroid cells and Tg biosynthesis. Chemical cross-linking followed by the cell lysis and immunoprecipitation of ERp29 or Tg revealed association of these proteins and additionally, immunocomplexes that also included major ER chaperones, BiP and GRP94. Sucrose density gradient analysis indicated co-localization of ERp29 with Tg and BiP in the fractions containing large macromolecular complexes. This was supported by immunofluorescent microscopy showing co-localization of ERp29 with Tg in the putative transport vesicular structures. Affinity chromatography using Tg as an affinity ligand demonstrated that ERp29 might be selectively isolated from the FRTL-5 cell lysate or purified lumenal fraction of rat liver microsomes along with the other ER chaperones. Preferential association with the urea-denatured Tg-Sepharose was indicative of either direct or circuitous ERp29/Tg interactions in a chaperone-like manner. Despite the presence of the C-terminal ER-retrieval signal, significant amounts of ERp29 were also recovered from the culture medium of stimulated thyrocytes, indicating ERp29 secretion. Based on these data, we suggest that the function of ERp29 in thyroid cells is connected with folding and/or secretion of Tg.     

PDI family protein ERp29 forms 1:1 complex with lectin chaperone calreticulin

Lectin chaperone calreticulin is well known to interact with ERp57 which is one of PDI family proteins. The interaction of ERp57 with calreticulin is believed to assist disulfide bond formation of nascent glycoprotein in the ER. Various kinds of PDI family proteins are present in the ER, however, their precise roles have been unclear. In this study, interaction assay between PDI family proteins and calreticulin by SPR analysis was performed. Our analysis revealed for the first time formation of a 1:1 complex between ERp29 and calreticulin. The dissociation constant of interaction between ERp29 and calreticulin was shown to be almost identical to ERp57–calreticulin interaction. We speculate that the recognition site of ERp29 within calreticulin is different from that of ERp57.     

The role of ERp44 in maturation of serotonin transporter protein

In heterologous and endogenous expression systems, we studied the role of ERp44 and its complex partner endoplasmic reticulum (ER) oxidase 1-α (Ero1-Lα) in mechanisms regulating disulfide bond formation for serotonin transporter (SERT), an oligomeric glycoprotein. ERp44 is an ER lumenal chaperone protein that favors the maturation of disulfide-linked oligomeric proteins. ERp44 plays a critical role in the release of proteins from the ER via binding to Ero1-Lα. Mutation in the thioredoxin-like domain hampers the association of ERp44C29S with SERT, which has three Cys residues (Cys-200, Cys-209, and Cys-109) on the second external loop. We further explored the role of the protein chaperones through shRNA knockdown experiments for ERp44 and Ero1-Lα. Those efforts resulted in increased SERT localization to the plasma membrane but decreased serotonin (5-HT) uptake rates, indicating the importance of the ERp44 retention mechanism in the proper maturation of SERT proteins. These data were strongly supported with the data received from the N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) labeling of SERT on ERp44 shRNA cells. MTSEA-biotin only interacts with the free Cys residues from the external phase of the plasma membrane. Interestingly, it appears that Cys-200 and Cys-209 of SERT in ERp44-silenced cells are accessible to labeling by MTSEA-biotin. However, in the control cells, these Cys residues are occupied and produced less labeling with MTSEA-biotin. Furthermore, ERp44 preferentially associated with SERT mutants (C200S, C209S, and C109A) when compared with wild type. These interactions with the chaperone may reflect the inability of Cys-200 and Cys-209 SERT mutants to form a disulfide bond and self-association as evidenced by immunoprecipitation assays. Based on these collective findings, we hypothesize that ERp44 together with Ero1-Lα plays an important role in disulfide formation of SERT, which may be a prerequisite step for the assembly of SERT molecules in oligomeric form.     

Replacement of domain b of human protein disulfide isomerase-related protein with domain b' of human protein disulfide isomerase dramatically increases its chaperone activity

We have reported that human protein disulfide isomerase-related protein (hPDIR) has isomerase and chaperone activities that are lower than those of the human protein disulfide isomerase (hPDI), and that the b domain of hPDIR is critical for its chaperone activity [J. Biol. Chem. 279 (2004) 4604]. To investigate the basis of the differences between hPDI and hPDIR, and to determine the functions of each hPDIR domain in detail, we constructed several hPDIR domain mutants. Interestingly, when the b domain of hPDIR was replaced with the b' domain of hPDI, a dramatic increase in chaperone activity that was close to that of hPDI itself was observed. However, this mutant showed decreased oxidative refolding of alpha1-antitrypsin. The replacement of the b domain of hPDIR with the c domain of hPDI also increased its chaperone activity. These observations suggest that putative peptide-binding sites of hPDI determine both its chaperone activity and its substrate specificity.     

Substrate Specificity of the Oxidoreductase ERp57 Is Determined Primarily by Its Interaction with Calnexin and Calreticulin

The formation of disulfides within proteins entering the secretory pathway is catalyzed by the protein disulfide isomerase family of endoplasmic reticulum localized oxidoreductases. One such enzyme, ERp57, is thought to catalyze the isomerization of non-native disulfide bonds formed in glycoproteins with unstructured disulfide-rich domains. Here we investigated the mechanism underlying ERp57 specificity toward glycoprotein substrates and the interdependence of ERp57 and the calnexin cycle for their correct folding. Our results clearly show that ERp57 must be physically associated with the calnexin cycle to catalyze isomerization reactions with most of its substrates. In addition, some glycoproteins only require ERp57 for correct disulfide formation if they enter the calnexin cycle. Hence, the specificity of ER oxidoreductases is not only determined by the physical association of enzyme and substrate but also by accessory factors, such as calnexin and calreticulin in the case of ERp57. These conclusions suggest that the calnexin cycle has evolved with a specialized oxidoreductase to facilitate native disulfide formation in complex glycoproteins.The ability to form disulfide bonds within proteins entering the secretory pathway is essential for cell survival and occurs within the endoplasmic reticulum (ER).³ For proteins with few disulfides, the process can be catalyzed by oxidation of cysteine residues to form the correct, native disulfide; however, for proteins with several disulfides, an isomerization reaction is also required to correct non-native disulfides formed following oxidation (1). Both these reactions are catalyzed by a group of ER-resident proteins that belong to the protein disulfide isomerase (PDI) family, which comprises over 17 members (2). It is well established that PDI and several other family members are able to catalyze the formation and isomerization of disulfides in vitro, although the exact function of each of the family members in vivo is unknown. It is still an open question as to whether they all catalyze similar reactions and have distinct substrate specificities or whether they have distinct enzymatic functions related to the breaking and formation of disulfides.For one member of the PDI family, the function and substrate specificity is a little clearer. ERp57 has been shown previously to interact specifically with glycoproteins during their folding (3). The enzyme is physically associated with either calnexin or calreticulin (4) and is therefore ideally placed to catalyze correct disulfide formation within proteins entering the calnexin/calreticulin cycle (referred to subsequently just as the calnexin cycle). In addition, the ability of ERp57 to catalyze the refolding of substrates in vitro is greatly enhanced if the substrate is bound to calnexin (5). Recently, substrates for the reduction or isomerization reaction catalyzed by ERp57 have been identified by trapping mixed disulfides between enzyme and substrate (6). Strikingly, there was an overrepresentation of substrate proteins with cysteine-rich domains containing little secondary structure, suggesting that the main function of ERp57 is in the isomerization of non-native disulfides. ERp57 has also been shown to function independently from the calnexin cycle. It is a component of the MHC class I loading complex where it forms a disulfide-linked complex with tapasin and is thought to either stabilize the complex or facilitate correct assembly of class I molecules (7, 8). Recently, ERp57 has been demonstrated to isomerize interchain disulfides in the major capsid protein, VP1, of simian virus 40 (9). The ability to dissociate VP1 pentamers by ERp57 does not require the substrate to interact with the calnexin cycle. Hence, it is still unclear how ERp57 recognizes its substrates, and in particular, whether this recognition is solely determined by an interaction with the calnexin cycle.The recognition of substrates by PDI is somewhat clearer in that one particular domain within the protein (the b′ domain) has been shown to be primarily responsible for substrate recognition and peptide binding (10). The corresponding domain within ERp57 has been shown to be responsible for interaction with the calnexin cycle (11), suggesting that for ERp57, substrate recognition must occur outside this domain or is determined solely by substrate interaction with calnexin via its oligosaccharide side chain. Hence, the aim of our study was to evaluate the necessity of the calnexin cycle both for ERp57 to recognize its substrates and for correct folding of glycoproteins. ERp57 was found to be required for the efficient folding of one substrate, influenza virus hemagglutinin (HA), but only when it entered the calnexin cycle. HA did not require ERp57 to fold if it was blocked from entering the calnexin cycle. In contrast, β1-integrin does not fold efficiently either if ERp57 was depleted or if ERp57 is blocked from entering the calnexin cycle (6). Although ERp57 may be dispensable for the folding of some glycoproteins, the interaction with calnexin commits them to an ERp57-dependent fate. We also found that the majority of ERp57 substrates need to enter the calnexin cycle to be acted upon by the enzyme, demonstrating that substrate specificity is primarily dependent upon substrate entry into the calnexin cycle.     

Independent and cooperative roles of N-glycans and molecular chaperones in the folding and disulfide bond formation of the low-density lipoprotein (LDL) receptor-related protein

The low-density lipoprotein receptor-related protein (LRP) is a large receptor that contains extensive glycosylation sites and disulfide bonds. Here we analyzed how N-linked glycosylation and molecular chaperones function during LRP folding. Treatment of cells with a glycosylation inhibitor tunicamycin significantly impaired LRP folding, although binding to receptor-associated protein (RAP), a specialized chaperone for LRP, was not affected. The effects of tunicamycin on LRP folding were not due to an inhibition of RAP glycosylation since a mutant RAP that harbors a mutation at its sole glycosylation site was still capable of promoting LRP folding. The roles of N-linked glycosylation and the lectin chaperone, calnexin, in LRP folding were further dissected using LRP minireceptors that carry mutations at individual glycosylation sites. Interestingly, we found that RAP interacts with oxidoreductase ERp57 and mediates its interaction with LRP. Since previous studies have shown that N-glycan-bound calnexin/calreticulin are also capable of recruiting ERp57, our results suggest that N-linked glycosylation and RAP can independently and cooperatively recruit oxidoreductases to facilitate protein folding and proper disulfide bond formation.     

Specific interaction of ERp57 and calnexin determined by NMR spectroscopy and an ER two-hybrid system

Calnexin and ERp57 act cooperatively to ensure a proper folding of proteins in the endoplasmic reticulum (ER). Calnexin contains two domains: a lectin domain and an extended arm termed the P-domain. ERp57 is a protein disulfide isomerase composed of four thioredoxin-like repeats and a short basic C-terminal tail. Here we show direct interactions between the tip of the calnexin P-domain and the ERp57 basic C-terminus by using NMR and a novel membrane yeast two-hybrid system (MYTHS) for mapping protein interactions of ER proteins. Our results prove that a small peptide derived from the P-domain is active in binding ERp57, and we determine the structure of the bound conformation of the P-domain peptide. The experimental strategy of using the MYTHS two-hybrid system to map interaction sites between ER proteins, together with NMR, provides a powerful new strategy for establishing the function of ER complexes.     

ERp57 Functions as a Subunit of Specific Complexes Formed with the ER Lectins Calreticulin and Calnexin

ERp57 is a lumenal protein of the endoplasmic reticulum (ER) and a member of the protein disulfide isomerase (PDI) family. In contrast to archetypal PDI, ERp57 interacts specifically with newly synthesized glycoproteins. In this study we demonstrate that ERp57 forms discrete complexes with the ER lectins, calnexin and calreticulin. Specific ERp57/calreticulin complexes exist in canine pancreatic microsomes, as demonstrated by SDS-PAGE after cross-linking, and by native electrophoresis in the absence of cross-linking. After in vitro translation and import into microsomes, radiolabeled ERp57 can be cross-linked to endogenous calreticulin and calnexin while radiolabeled PDI cannot. Likewise, radiolabeled calreticulin is cross-linked to endogenous ERp57 but not PDI. Similar results were obtained in Lec23 cells, which lack the glucosidase I necessary to produce glycoprotein substrates capable of binding to calnexin and calreticulin. This observation indicates that ERp57 interacts with both of the ER lectins in the absence of their glycoprotein substrate. This result was confirmed by a specific interaction between in vitro synthesized calreticulin and ERp57 prepared in solution in the absence of other ER components. We conclude that ERp57 forms complexes with both calnexin and calreticulin and propose that it is these complexes that can specifically modulate glycoprotein folding within the ER lumen.