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The endoplasmic reticulum (ER) represents the entry point into the secretory pathway where nascent proteins encounter a specialized environment for their folding and maturation. Inherent to these processes is a dedicated quality-control system that detects proteins that fail to mature properly and targets them for cytosolic degradation. An imbalance in protein folding and degradation can result in the accumulation of unfolded proteins in the ER, resulting in the activation of a signaling cascade that restores proper homeostasis in this organelle. The ER heat shock protein 70 (Hsp70) family member BiP is an ATP-dependent chaperone that plays a critical role in these processes. BiP interacts with specific ER-localized DnaJ family members (ERdjs), which stimulate BiP's ATP-dependent substrate interactions, with several ERdjs also binding directly to unfolded protein clients. Recent structural and biochemical studies have provided detailed insights into the allosteric regulation of client binding by BiP and have enhanced our understanding of how specific ERdjs enable BiP to perform its many functions in the ER. In this review, we discuss how BiP's functional cycle and interactions with ERdjs enable it to regulate protein homeostasis in the ER and ensure protein quality control., (© 2019 Pobre et al.)

Mutations in SIL1 , a cofactor for the endoplasmic reticulum (ER)-localized Hsp70 chaperone, BiP, cause Marinesco-Sjögren syndrome (MSS), an autosomal recessive disorder. Using a mouse model, we characterized molecular aspects of the progressive myopathy associated with MSS. Proteomic profiling of quadriceps at the onset of myopathy revealed that SIL1 deficiency affected multiple pathways critical to muscle physiology. We observed an increase in ER chaperones prior to the onset of muscle weakness, which was complemented by upregulation of multiple components of cellular protein degradation pathways. These responses were inadequate to maintain normal expression of secretory pathway proteins, including insulin and IGF-1 receptors. There was a paradoxical enhancement of downstream PI3K-AKT-mTOR signaling and glucose uptake in SIL1-disrupted skeletal muscles, all of which were insufficient to maintain skeletal muscle mass. Together, these data reveal a disruption in ER homeostasis upon SIL1 loss, which is countered by multiple compensatory responses that are ultimately unsuccessful, leading to trans -organellar proteostasis collapse and myopathy., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Protein maturation in the endoplasmic reticulum is controlled by multiple chaperones, but how they recognize and determine the fate of their clients remains unclear. We developed an in vivo peptide library covering substrates of the ER Hsp70 system: BiP, Grp170, and three of BiP's DnaJ-family co-factors (ERdj3, ERdj4, and ERdj5). In vivo binding studies revealed that sites for pro-folding chaperones BiP and ERdj3 were frequent and dispersed throughout the clients, whereas Grp170, ERdj4, and ERdj5 specifically recognized a distinct type of rarer sequence with a high predicted aggregation potential. Mutational analyses provided insights into sequence recognition characteristics for these pro-degradation chaperones, which could be readily introduced or disrupted, allowing the consequences for client fates to be determined. Our data reveal unanticipated diversity in recognition sequences for chaperones; establish a sequence-encoded interplay between protein folding, aggregation, and degradation; and highlight the ability of clients to co-evolve with chaperones, ensuring quality control., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In eukaryotic cells, secretory pathway proteins must pass stringent quality control checkpoints before exiting the endoplasmic reticulum (ER). Acquisition of native structure is generally considered to be the most important prerequisite for ER exit. However, structurally detailed protein folding studies in the ER are few. Furthermore, aberrant ER quality control decisions are associated with a large and increasing number of human diseases, highlighting the need for more detailed studies on the molecular determinants that result in proteins being either secreted or retained. Here we used the clonotypic αβ chains of the T cell receptor (TCR) as a model to analyze lumenal determinants of ER quality control with a particular emphasis on how proper assembly of oligomeric proteins can be monitored in the ER. A combination of in vitro and in vivo approaches allowed us to provide a detailed model for αβTCR assembly control in the cell. We found that folding of the TCR α chain constant domain Cα is dependent on αβ heterodimerization. Furthermore, our data show that some variable regions associated with either chain can remain incompletely folded until chain pairing occurs. Together, these data argue for template-assisted folding at more than one point in the TCR α/β assembly process, which allows specific recognition of unassembled clonotypic chains by the ER chaperone machinery and, therefore, reliable quality control of this important immune receptor. Additionally, it highlights an unreported possible limitation in the α and β chain combinations that comprise the T cell repertoire., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

DnaK/Hsp70 chaperones form oligomers of poorly understood structure and functional significance. Site-specific proteolysis and crosslinking were used to probe the architecture of oligomers formed by the endoplasmic reticulum (ER) Hsp70, BiP. These were found to consist of adjacent protomers engaging the interdomain linker of one molecule in the substrate binding site of another, attenuating the chaperone function of oligomeric BiP. Native gel electrophoresis revealed a rapidly-modulated reciprocal relationship between the burden of unfolded proteins and BiP oligomers and slower equilibration between oligomers and inactive, covalently-modified BiP. Lumenal ER calcium depletion caused rapid oligomerization of mammalian BiP and a coincidental diminution in substrate binding, pointing to the relative inertness of the oligomers. Thus, equilibration between inactive oligomers and active monomeric BiP is poised to buffer fluctuations in ER unfolded protein load on a rapid timescale attainable neither by inter-conversion of active and covalently-modified BiP nor by the conventional unfolded protein response.

BiP (immunoglobulin heavy-chain binding protein) is the endoplasmic reticulum (ER) orthologue of the Hsp70 family of molecular chaperones and is intricately involved in most functions of this organelle through its interactions with a variety of substrates and regulatory proteins. Like all Hsp70 family members, the ability of BiP to bind and release unfolded proteins is tightly regulated by a cycle of ATP binding, hydrolysis, and nucleotide exchange. As a characteristic of the Hsp70 family, multiple DnaJ-like co-factors can target substrates to BiP and stimulate its ATPase activity to stabilize the binding of BiP to substrates. However, only in the past decade have nucleotide exchange factors for BiP been identified, which has shed light not only on the mechanism of BiP-assisted folding in the ER but also on Hsp70 family members that reside throughout the cell. We will review the current understanding of the ATPase cycle of BiP in the unique environment of the ER and how it is regulated by the nucleotide exchange factors, Grp170 (glucose-regulated protein of 170kDa) and Sil1, both of which perform unanticipated roles in various biological functions and disease states., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Sil1 is a nucleotide exchange factor for the endoplasmic reticulum chaperone BiP, and mutations in this gene lead to Marinesco-Sjögren syndrome (MSS), a debilitating autosomal recessive disease characterized by multisystem defects. A mouse model for MSS was previously produced by disrupting Sil1 using gene-trap methodology. The resulting Sil1Gt mouse phenocopies several pathologies associated with MSS, although its ability to assemble and secrete antibodies, the best-characterized substrate of BiP, has not been investigated. In vivo antigen-specific immunizations and ex vivo LPS stimulation of splenic B cells revealed that the Sil1Gt mouse was indistinguishable from wild-type age-matched controls in terms of both the kinetics and magnitude of antigen-specific antibody responses. There was no significant accumulation of BiP-associated Ig assembly intermediates or evidence that another molecular chaperone system was used for antibody production in the LPS-stimulated splenic B cells from Sil1Gt mice. ER chaperones were expressed at the same level in Sil1WT and Sil1Gt mice, indicating that there was no evident compensation for the disruption of Sil1. Finally, these results were confirmed and extended in three human EBV-transformed lymphoblastoid cell lines from individuals with MSS, leading us to conclude that the BiP cofactor Sil1 is dispensable for antibody production., (© 2015 Ichhaporia et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Cell-surface multiprotein complexes are synthesized in the endoplasmic reticulum (ER), where they undergo cotranslational membrane integration and assembly. The quality control mechanisms that oversee these processes remain poorly understood. We show that less hydrophobic transmembrane (TM) regions derived from several single-pass TM proteins can enter the ER lumen completely. Once mislocalized, they are recognized by the Hsp70 chaperone BiP. In a detailed analysis for one of these proteins, the αβT cell receptor (αβTCR), we show that unassembled ER-lumenal subunits are rapidly degraded, whereas specific subunit interactions en route to the native receptor promote membrane integration of the less hydrophobic TM segments, thereby stabilizing the protein. For the TCR α chain, both complete ER import and subunit assembly depend on the same pivotal residue in its TM region. Thus, membrane integration linked to protein assembly allows cellular quality control of membrane proteins and connects the lumenal ER chaperone machinery to membrane protein biogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

DnaJ proteins often bind to unfolded substrates and recruit their Hsp70 partners. This induces a conformational change in the Hsp70 that stabilizes its binding to substrate. By some unknown mechanism, the DnaJ protein is released. We examined the requirements for the release of ERdj3, a mammalian ER DnaJ, from substrates and found that BiP promoted the release of ERdj3 only in the presence of ATP. Mutations in ERdj3 or BiP that disrupted their interaction interrupted the release of ERdj3. BiP mutants that were defective in any step of the ATPase cycle were also unable to release ERdj3. These results demonstrate that a functional interaction between ERdj3 and BiP, including both a direct interaction and the ability to stimulate BiP's ATPase activity are required to release ERdj3 from substrate and support a model where ERdj3 must recruit BiP and stimulate its high-affinity association with the substrate through activation of ATP hydrolysis to trigger its own release from substrates. On the basis of similarities among DnaJs and Hsp70s, this is likely to be applicable to other Hsp70-DnaJ pairs.

The heat shock protein (Hsp)70 family of molecular chaperones interacts with unfolded proteins through a C-terminal substrate-binding domain (SBD) that is controlled by nucleotide binding to the N-terminal domain. The ATPase cycle is regulated by cochaperones, including DnaJ proteins that accelerate ATP hydrolysis to stabilize the Hsp70-substrate complex. We found that R197 in hamster BiP, which resides at the surface of the nucleotide-binding domain, is critical for both association with endoplasmic reticulum DnaJ proteins and interaction with the SBD. Decreasing the positive charge at this residue enhanced basal ATPase activity, destabilized interaction with the SBD, and reduced substrate release both in vitro and in vivo. Mutation of three glutamic acids in the SBD mimicked many of these effects. Our data provide insights into communications between the two domains and suggest a mechanism by which DnaJ proteins increase ATP hydrolysis.

To investigate the disposal of nonglycosylated BiP substrates, we used a nonsecreted kappa LC, which exists in partially (ox1) and completely (ox2) oxidized states. The ox2 form is partially reduced in order to be degraded, and only the ox1 form is ubiquitinated and associates with both Herp and Derlin-1. Herp is in a complex with ubiquitinated proteins and with the 26S proteasome, suggesting that it plays a role in linking substrates with the proteasome. Overexpressed Herp also interacts with two other BiP substrates, but not with two calnexin substrates. Either expression of p97 or Hrd1 mutants, which are in a complex with Herp and Derlin-1, or reduction of Herp levels inhibited the degradation of the BiP substrates, whereas the latter had no effect on the degradation of the calnexin substrates. This result suggests that there is some distinction in the pathways used to dispose of these two types of ERAD substrates.

The Sec61 translocon of the endoplasmic reticulum membrane forms an aqueous pore that is gated by the lumenal Hsp70 chaperone BiP. We have explored the molecular mechanisms governing BiP-mediated gating activity, including the coupling between gating and the BiP ATPase cycle, and the involvement of the substrate-binding and J domain-binding regions of BiP. Translocon gating was assayed by measuring the collisional quenching of fluorescent probes incorporated into nascent chains of translocation intermediates engaged with microsomes containing various BiP mutants and BiP substrate. Our results indicate that BiP must assume the ADP-bound conformation to seal the translocon, and that the reopening of the pore requires an ATP binding-induced conformational change. Further, pore closure requires functional interactions between both the substrate-binding region and the J domain-binding region of BiP and membrane proteins. The mechanism by which BiP mediates translocon pore closure and opening is therefore similar to that in which Hsp70 chaperones associate with and dissociate from substrates.

We recently identified ERdj3 as a component of unassembled immunoglobulin (Ig) heavy chain:BiP complexes. ERdj3 also associates with a number of other protein substrates, including unfolded light chains, a nonsecreted Ig light chain mutant, and the VSV-G ts045 mutant at the nonpermissive temperature. We produced an ERdj3 mutant that was unable to stimulate BiP's ATPase activity in vitro or to bind BiP in vivo. This mutant retained the ability to interact with unfolded protein substrates, suggesting that ERdj3 binds directly to proteins instead of via interactions with BiP. BiP remained bound to unfolded light chains longer than ERdj3, which interacted with unfolded light chains initially, but quickly disassociated before protein folding was completed. This suggests that ERdj3 may bind first to substrates and serve to inhibit protein aggregation until BiP joins the complex, whereas BiP remains bound until folding is complete. Moreover, our findings support a model where interactions with BiP help trigger the release of ERdj3 from the substrate:BiP complex.

The endoplasmic reticulum (ER) is a command center of the cell that is second only to the nucleus in terms of the breadth of its influence on other organelles and activities. It is a major site of protein synthesis, contains the cellular calcium stores that are an essential component of many signaling pathways, and is the proximal site of a signal transduction cascade that responds to cellular stress conditions and serves to maintain homeostasis of the cell. All eucaryotic cells possess an ER, which can comprise nearly 50% of the membranes of a cell. Its functions can be divided into those that occur on the cytosolic side of the membrane (where protein translation and signal transduction cascades occur) and the luminal space (where most other ER functions take place). Our studies during the past several years have revealed that the ER molecular chaperone BiP is a master regulator of ER function. It is responsible for maintaining the permeability barrier of the ER during protein translocation, directing protein folding and assembly, targeting misfolded proteins for retrograde translocation so they can be degraded by the proteasome, contributing to ER calcium stores, and sensing conditions of stress in this organelle, to activate the mammalian unfolded protein response.

The mammalian unfolded protein response (UPR) includes two major branches: one(s) specific to ER stress (Ire1/XBP-1 and ATF6-dependent), and one(s) shared by other cellular stresses (PERK/eIF-2alpha phosphorylation-dependent). Here, we demonstrate that the ER-localized protein Herp represents a second target, in addition to CHOP, that is dually regulated by both the shared and the ER stress-specific branches during UPR activation. For the first time, we are able to assess the contribution of each branch of the UPR in the induction of these targets. We demonstrate that activation of the shared branch of the UPR alone was sufficient to induce Herp and CHOP. ATF4 was not required during ER stress when both branches were used but did contribute significantly to their induction. Conversely, stresses that activated only the shared branch of the UPR were completely dependent on ATF4 for CHOP and Herp induction. Thus, the shared and the ER stress-specific branches of the UPR diverge to regulate two groups of targets, one that is ATF6 and Ire1/XBP-1-dependent, which includes BiP and XBP-1, and another that is eIF-2alpha kinase-dependent, which includes ATF4 and GADD34. The two branches also converge to maximally up-regulate targets like Herp and CHOP. Finally, our studies reveal that a PERK-dependent target other than ATF4 is contributing to the cross-talk between the two branches of the UPR that has previously been demonstrated.

We identified a mammalian BiP-associated protein, BAP, using a yeast two-hybrid screen that shared low homology with yeast Sls1p/Sil1p and mammalian HspBP1, both of which regulate the ATPase activity of their Hsp70 partner. BAP encoded an approximately 54-kDa protein with an N-terminal endoplasmic reticulum (ER) targeting sequence, two sites of N-linked glycosylation, and a C-terminal ER retention sequence. Immunofluorescence staining demonstrated that BAP co-localized with GRP94 in the endoplasmic reticulum. BAP was ubiquitously expressed but showed the highest levels of expression in secretory organ tissues, a pattern similar to that observed with BiP. BAP binding was affected by the conformation of the ATPase domain of BiP based on in vivo binding studies with BiP mutants. BAP stimulated the ATPase activity of BiP when added alone or together with the ER DnaJ protein, ERdj4, by promoting the release of ADP from BiP. Together, these data demonstrate that BAP serves as a nucleotide exchange factor for BiP and provide insights into the mechanisms that control protein folding in the mammalian ER.

The activity of Hsp70 proteins is regulated by accessory proteins, which include members of the DnaJ-like protein family. Characterized by the presence of a highly conserved 70-amino acid J domain, DnaJ homologues activate the ATPase activity of Hsp70 proteins and stabilize their interaction with unfolded substrates. DnaJ homologues have been identified in most organelles where they are involved in nearly all aspects of protein synthesis and folding. Within the endoplasmic reticulum (ER), DnaJ homologues have also been shown to assist in the translocation, secretion, retro-translocation, and ER-associated degradation (ERAD) of secretory pathway proteins. By using bioinformatic methods, we identified a novel mammalian DnaJ homologue, ERdj4. It is the first ER-localized type II DnaJ homologue to be reported. The signal sequence of ERdj4 remains uncleaved and serves as a membrane anchor, orienting its J domain into the ER lumen. ERdj4 co-localized with GRP94 in the ER and associated with BiP in vivo when they were co-expressed in COS-1 cells. In vitro experiments demonstrated that the J domain of ERdj4 stimulated the ATPase activity of BiP in a concentration-dependent manner. However, mutation of the hallmark tripeptide HPD (His --> Gln) in the J domain totally abolished this activation. ERdj4 mRNA expression was detected in all human tissues examined but showed the highest level of the expression in the liver, kidney, and placenta. We found that ERdj4 was highly induced at both the mRNA and protein level in response to ER stress, indicating that this protein might be involved in either protein folding or ER-associated degradation.

Unassembled Ig heavy chains are retained in the ER via the binding of BiP to the C(H)1 domain, which remains unoxidized. Interestingly, this domain folds rapidly, albeit nonproductively, when heavy chains are released from BiP in vitro with ATP. The in vivo cycling of BiP from heavy chains was monitored using BiP ATPase mutants as kinetic traps. Our data suggest that BiP does not cycle from the C(H)1 domain of free heavy chains. However, heavy and light chain assembly occurs rapidly and requires the ATP-dependent release of BiP. We propose that BiP's ATPase cycle is stalled or nonproductive when it is bound to free heavy chains. The binding of light chains to the complex reactivates the cycle and releases BiP.

Collagen biosynthesis involves a complex series of post-translational modifications, controlled by a number of general and specific molecular chaperones. A recent study has shed new light on the role played in this process by the procollagen-specific chaperone Hsp47.

Lymphoma proprotein convertase (LPC) is a subtilisin-like serine protease of the mammalian proprotein convertase family. It is synthesized as an inactive precursor protein, and propeptide cleavage occurs via intramolecular cleavage in the endoplasmic reticulum. In contrast to other convertases like furin and proprotein convertase-1, propeptide cleavage occurs slowly. Also, both a glycosylated and an unglycosylated precursor are detected. Here we demonstrate that the unglycosylated precursor form of LPC is localized in the cytosol due to the absence of a signal peptide. Using a reducible cross-linker, we found that glycosylated pro-LPC is associated with the molecular chaperone BiP. In addition, we show that pro-LPC is prone to aggregation and forms large complexes linked via interchain disulfide bonds. BiP is associated mainly with non-aggregated pro-LPC and pro-LPC dimers and trimers, suggesting that BiP prevents aggregation. Overexpression of wild-type BiP or a dominant-negative BiP ATPase mutant resulted in reduced processing of pro-LPC. Taken together, these results suggest that binding of BiP to pro-LPC prevents aggregation, but results in slower maturation.

PERK and IRE1 are type-I transmembrane protein kinases that reside in the endoplasmic reticulum (ER) and transmit stress signals in response to perturbation of protein folding. Here we show that the lumenal domains of these two proteins are functionally interchangeable in mediating an ER stress response and that, in unstressed cells, both lumenal domains form a stable complex with the ER chaperone BiP. Perturbation of protein folding promotes reversible dissociation of BiP from the lumenal domains of PERK and IRE1. Loss of BiP correlates with the formation of high-molecular-mass complexes of activated PERK or IRE1, and overexpression of BiP attenuates their activation. These findings are consistent with a model in which BiP represses signalling through PERK and IRE1 and protein misfolding relieves this repression by effecting the release of BiP from the PERK and IRE1 lumenal domains.

The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP-heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 family of chaperones and one of the most abundant proteins in the ER lumen. It is known to interact transiently with many nascent proteins as they enter the ER and more stably with protein subunits produced in stoichiometric excess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To begin to understand what controls the likelihood that a nascent protein entering the ER will associate with BiP, we have examined the in vivo folding of a murine lambdaI immunoglobulin (Ig) light chain (LC). This LC is composed of two Ig domains that can fold independent of the other and that each possess multiple potential BiP-binding sequences. To detect BiP binding to the LC during folding, we used BiP ATPase mutants, which bind irreversibly to proteins, as "kinetic traps." Although both the wild-type and mutant BiP clearly associated with the unoxidized variable region domain, we were unable to detect binding of either BiP protein to the constant region domain. A combination of in vivo and in vitro folding studies revealed that the constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond. Thus, the simple presence of a BiP-binding site on a nascent chain does not ensure that BiP will bind and play a role in its folding. Instead, it appears that the rate and stability of protein folding determines whether or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.

Not much is known about the features that determine the biological stability of a molecule retained in the endoplasmic reticulum (ER). Ig light (L) chains that are not secreted in the absence of Ig heavy (H) chain expression bind to the ER chaperone BiP as partially folded molecules until they are degraded. Although all Ig L chains have the same three-dimensional structure when part of an antibody molecule, the degradation rate of unassembled Ig L chains is not identical. For instance, the two nonsecreted murine Ig L chains, kappaNS1 and lambdaFS62, are degraded with half-lives of approximately 1 and 4 hr, respectively, in the same NS1 myeloma cells. Furthermore, the BiP/lambdaFS62 Ig L chain complex appears to be more stable than the BiP/kappaNS1 complex. Here, we used the ability of single Ig domains to form an internal disulfide bond after folding as a measure of the folding state of kappaNS1 and lambdaFS62 Ig L chains. Both of these nonsecreted L chains lack the internal disulfide bond in the variable (V) domain, whereas the constant (C) domain was folded in that respect. In both cases the unfolded V domain provided the BiP binding site. The stability of BiP binding to these two nonsecreted proteins was quite different, and both the stability of the BiP:Ig L chain complex and the half-life of the Ig L chain could be transferred from one Ig L chain isotype to the other by swapping the V domains. Our data suggest that the physical stability of BiP association with an unfolded region of a given light chain determines the half-life of that light chain, indicating a direct link between chaperone interaction and delivery of partially folded substrates to the mammalian degradation machinery.

The stress-induced unfolded protein response (UPR) is the only signaling pathway known to regulate expression of genes encoding the resident endoplasmic reticulum (ER) molecular chaperones and folding enzymes, yet these genes are constitutively expressed in all cells. We have examined the expression of ER chaperones in several cell lines that are dependent on a variety of cytokines for growth and survival. When the various cell lines were deprived of essential growth factors, mRNA levels of the ER chaperones BiP and GRP94 decreased dramatically. Re-stimulation of ligand-deprived cells with the appropriate growth factor induced BiP and GRP94 as delayed-early response genes. Cytokine induction of BiP and GRP94 biosynthesis was not preceded by a burst of glycoprotein traffic through the ER nor accompanied by expression of the CHOP transcription factor. The glycosylation inhibitor tunicamycin potently induced expression of both ER chaperones and CHOP in ligand-deprived cells, demonstrating that the UPR pathway remains functionally intact in the absence of growth factor-mediated signaling. Therefore, basal expression of ER chaperones is dependent upon and regulated by a mitogenic pathway distinct from the stress-inducible UPR cascade and this probably controls expression of ER chaperones and folding enzymes needed to assist protein biogenesis in the ER of normal, non-stressed cells.

BiP/GRP78 is a lumenal stress protein of the endoplasmic reticulum (ER) that interacts with polypeptide folding intermediates transiting the secretory compartment. We have studied the secretion and the stress response in Chinese hamster ovary (CHO) cells that overexpress either wild-type immunoglobulin binding protein (BiP) or a BiP deletion molecule (residues 175-201) that can bind peptides and ATP but is defective in ATP hydrolysis and concomitant peptide release. Overexpressed wild-type BiP was localized to the ER and unique vesicles within the nucleus, whereas overexpressed ATPase-defective BiP was localized to the ER and cytoplasmic vesicles but was absent from the nucleus. Compared with wild-type CHO cells, overexpression of ATPase-defective BiP prevented secretion of factor VIII, a coagulation factor that extensively binds BiP in the lumen of the ER. Under these conditions factor VIII was stably associated with the ATPase-defective BiP. In contrast, the secretion of monocyte/macrophage colony stimulating factor, a protein that is not detected in association with BiP, was not affected by overexpression of ATPase-defective BiP. These results show that BiP function is not required for secretion of some proteins and suggest that some proteins do not interact with BiP upon transport through the ER. The presence of unfolded protein in the ER induces transcription of BiP and also elicits a general inhibition of protein synthesis. Overexpression of wild-type BiP prevented the stress-mediated transcriptional induction of BiP in response to either calcium ionophore A23187 treatment or tunicamycin treatment. In contrast, overexpression of ATPase-defective BiP did not prevent the stress induction of BiP, showing that the ATPase activity is required to inhibit transcriptional induction. Overexpression of wild-type BiP, but not ATPase-defective BiP, increased survival of cells treated with A23187. The increased survival mediated by overexpressed wild-type BiP correlated with reduced translation inhibition in response to the stress condition. These results indicate that overexpressed BiP alleviated the stress in the ER to prevent BiP transcriptional induction and permit continued translation of cellular mRNAs.

The gene encoding C/EBP-homologous protein (CHOP), also known as growth arrest and DNA-damage-inducible gene 153 (GADD153), is activated by agents that adversely affect the function of the endoplasmic reticulum (ER). Because of the pleiotropic effects of such agents on other cellular processes, the role of ER stress in inducing CHOP gene expression has remained unclear. We find that cells with conditional (temperature-sensitive) defects in protein glycosylation (CHO K12 and BHK tsBN7) induce CHOP when cultured at the nonpermissive temperature. In addition, cells that are defective in initiating the ER stress response, because of overexpression of an exogenous ER chaperone, BiP/GRP78, exhibit attenuated inducibility of CHOP. Surprisingly, attenuated induction of CHOP was also noted in BiP-overexpressing cells treated with methyl methanesulfonate, an agent thought to activate CHOP by causing DNA damage. The roles of DNA damage and growth arrest in the induction of CHOP were therefore reexamined. Induction of growth arrest by culture to confluence or treatment with the enzymatic inhibitor N-(phosphonacetyl)-L-aspartate did not induce CHOP. Furthermore, both a DNA-damage-causing nucleoside analog (5-hydroxymethyl-2'-deoxyuridine) and UV light alone did not induce CHOP. These results suggest that CHOP is more responsive to ER stress than to growth arrest or DNA damage and indicate a potential role for CHOP in linking stress in the ER to alterations in gene expression.

In the present study, we produced single point mutations in the ATP binding site of hamster BiP, isolated recombinant proteins, and characterized them in terms of their affinity for ATP and ADP, their ability to undergo a conformational change upon nucleotide binding, and their rate of ATP hydrolysis. These analyses allowed us to classify the mutants into three groups: ATP hydrolysis (T229G), ATP binding (G226D, G227D), and ATP-induced conformation (T37G) mutants, and to test the role of these activities in the in vitro ATP-mediated release of proteins from BiP. All three classes of mutants were still able to bind peptide demonstrating that nucleotide is not involved in this function. Addition of ATP to either wild-type BiP or the T229G mutant caused the in vitro release of bound peptide, confirming that ATP hydrolysis is not required for protein release. ATP did not dissociate G226D, G227D, or T37G mutant BiP-peptide complexes, suggesting that ATP binding to BiP is not sufficient for the release of bound peptides, but that an ATP-induced conformational change in BiP is necessary. The identification of BiP mutants that are defective in each of these steps of ATP hydrolysis will allow the in vivo dissection of the role of nucleotide in BiP's activity.

HSP70 family proteins bind ATP and hydrolyze it, but the precise role of these activities in their in vivo chaperoning function has not been determined. In this report, we characterized wild-type hamster BiP isolated from bacteria in terms of its ATP binding and ATPase activities. Recombinant BiP behaved essentially the same as endogenous BiP in terms of oligomeric status, protease digestion patterns, and ATPase properties. By engineering a Factor Xa cleavable site following the His tag which was used for affinity purification, we demonstrated that the six histidines had no effect on either the structural or ATPase properties of recombinant BiP. We also found that bacteria-synthesized BiP had a tightly bound ADP that was resistant to dialysis. Removal of the bound nucleotide allowed us to directly measure the binding affinity of ATP and ADP to BiP (Kd of 0.2 microM for ATP and 0.29 microM for ADP) by equilibrium dialysis. Careful characterization of wild-type BiP will allow us to use this system to characterize BiP ATP binding site mutants that can be used to probe the role of ATP binding and ATPase activity in BiP functions.

BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly in the endoplasmic reticulum (ER). We have produced a series of point mutations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic inactivation of BiP did not interfere with its ability to bind to Ig heavy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy revealed vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a "44K" fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to target proteins as an inactive chaperone is responsible for the ER disruption. This is the first report on the in vivo expression of mammalian BiP mutants and is demonstration that in vitro-identified ATPase mutants behave as dominant negative mutants when expressed in vivo.

BiP/GRP78 is a member of the HSP70 family involved in the folding and assembly of proteins in the endoplasmic reticulum. Using PCR amplification of DNA from human x rodent somatic hybrids that segregate human chromosomes in conjunction with fluorescence in situ hybridization, we have assigned GRP78 to chromosome 9q34. This is in agreement with the localization of murine and bovine homologues based on the high degree of synteny in this region. Several interesting genes and disorders map to this region and are discussed.

In vitro incubation of immunoprecipitated immunoglobulin-binding protein (BiP) complexes with calcium and [gamma-32P]ATP resulted in the phosphorylation of BiP on a threonine residue. This autophosphorylation activity did not occur in the presence of magnesium but had the same pH optimum as reported for its magnesium-dependent ATPase activity. This suggested the possibility that both activities could occur through ATP hydrolysis at the same site. In support of this, mutation of either Thr-37 or Thr-229 to a glycine eliminated both autophosphorylation and ATPase activities, and mutation of either residue to a serine significantly reduced both activities. Glutamic acid 175 in HSC71 has been hypothesized to flank the divalent cation complexed with ATP. Mutation of the analogous glutamic acid, Glu-201, in BiP abolished ATPase activity but still supported some autophosphorylation. The in vitro phosphorylation site was mapped to Thr-229 by mutational analysis. This threonine has been hypothesized to interact with the gamma-phosphate of ATP through a polarized water molecule and would be in a position to act as a phosphate acceptor in the ATP hydrolysis reaction. These data imply that both ATPase and autophosphorylation result from ATP hydrolysis at the same site and that the cation associated with BiP determines which activity is observed. Comparison of partial protease digestion or cyanogen bromide cleavage products of in vitro and in vivo phosphorylated BiP demonstrated that Thr-229 is not a detectable site of phosphorylation in cells. Therefore, whatever functional role phosphorylation may have in vivo, it cannot be attributed to autophosphorylation of Thr-229.

The immunoglobulin heavy chain binding protein BiP/GRP78 is post-translationally modified by phosphorylation and ADP ribosylation. In cells induced to synthesize higher levels of BiP, either due to the accumulation of nontransported proteins or to glucose starvation, both BiP phosphorylation and ADP ribosylation are reduced. BiP bound to other proteins is unmodified, suggesting that both phosphorylation and ADP ribosylation are restricted to the unbound BiP pool. In the present study, both modifications were further characterized in terms of their stability, the pool of BiP that harbored these modifications, and the relationship between the modified and unmodified forms of BiP. While levels of BiP synthesis vary according to the physiological state of a cell, we found that both induced and uninduced cells contain similar amounts of free BiP. However, free BiP in uninduced cells was found primarily in an aggregated state, whereas in cells that accumulate nontransported proteins, it was predominantly monomeric. Both phosphorylation and ADP ribosylation were restricted to the aggregated form of free BiP. These post-translational modifications occurred upon release of BiP from associated proteins, and could be reversed upon induction of BiP synthesis. Therefore, BiP exists either (1) complexed to other proteins, (2) as a free unmodified monomer, or (3) as free modified aggregates. Our data suggest that BiP can be interconverted from one state to another, and that the various forms are functionally distinct.

Immunoglobulin heavy chain binding protein (BiP, GRP78) associates stably with the free, nonsecreted Ig heavy chains synthesized by Abelson virus transformed pre-B cell lines. In cells synthesizing both Ig heavy and light chains, the Ig subunits assemble rapidly and are secreted. Only incompletely assembled Ig molecules can be found bound to BiP in these cells. In addition to Ig heavy chains, a number of mutant and incompletely glycosylated transport-defective proteins are stably complexed with BiP. When normal proteins are examined for combination with BiP, only a small fraction of the intracellular pool of nascent, unfolded, or unassembled proteins can be found associated. It has been difficult to determine whether these BiP-associated molecules represent assembly intermediates which will be displaced from BiP and transported from the cell, or whether these are aberrant proteins that are ultimately degraded. In order for BiP to monitor and aid in normal protein transport, its association with these proteins must be reversible and the released proteins should be transport competent. In the studies described here, transient heterokaryons were formed between a myeloma line producing BiP-associated heavy chains and a myeloma line synthesizing the complementary light chain. Introduction of light chain synthesis resulted in assembly of prelabeled heavy chains with light chains, displacement of BiP from heavy chains, and secretion of Ig into the culture supernatant. These data demonstrate that BiP association can be reversible, with concordant release of transportable proteins. Thus, BiP can be considered a component of the exocytic secretory pathway, regulating the transport of both normal and abnormal proteins.

Immunoglobulin light chains are usually secreted from cells when they are synthesized alone or in molar excess of heavy chains, but, there have been reports of nonsecreted light chains. We wished to determine whether immunoglobulin heavy chain binding protein (BiP), which blocks the transport of free heavy chains, might be responsible for the lack of secretion of some light chains. In two murine lymphoid cell lines that synthesize but do not secrete immunoglobulin light chains, the free light chain polymers were found bound to BiP. Examination of 20 other cell lines and hybridomas failed to disclose any cells synthesizing free or excess light chains that associated with BiP, in all cases the free light chains were secreted as dimers. Despite their association with BiP and their blocked secretion, the aberrant light chains could combine with heavy chains and could be secreted as intact Ig molecules. Thus, while light chains do not usually express signals which allow them to bind to BiP, it appears that such signals can be expressed on certain light chains, resulting in their combination with BiP and blocked secretion. When single chain mutant cell lines are isolated from parental lines producing both heavy and light chains, they are almost always light chain producers suggesting that free heavy chains are much more toxic than free light chains. In both PC700 and P3X63Ag cells, however, clones that have lost either heavy chains or transport-defective light chains are present at the same frequency. Our findings that the light chains in both of these lines are associated with BiP raise the possibility that BiP actually contributes to heavy chain toxicity instead of preventing it.

Human EBV transformed lymphoblastoid cell lines and lymphomas representing various stages of B cell development were examined for heavy chain binding protein (BiP) expression and its association with immunoglobin (Ig) heavy chains. Human BiP was shown to migrate with an apparent mol. wt of 79,000 and to have a pI of approximately 5.5 in all the human cell lines examined. Both the mum and the mus heavy chains synthesized in a pre-B cell line (mu+, LC-) remained associated with BiP and were all found to be endo H sensitive, suggesting that this association occurred in the endoplasmic reticulum (ER). Surface Ig+ B cell lines produce membrane type heavy chains which are expressed on the cell surface and secretory type heavy chains which remain intracellular. The membrane type mu heavy chains produced by a surface Ig+ B cell line were not associated with BiP after assembling with light chains and processing in the Golgi. However, the secretory type mu heavy chains synthesized by these same cells did not combine efficiently with LC and a significant quantity remained associated with BiP and were not secreted suggesting that BiP is involved in the divergent transport of membrane and secretory mu heavy chains in surface Ig+ B cell lines. In Ig secreting plasmacytoid lines the heavy chains were only associated with BiP prior to assembling with LC. When LC assembly was inhibited, the association of heavy chains with BiP was prolonged and Ig secretion was blocked. Therefore, BiP was found to participate in the post-translational processing of mu heavy chains synthesized by human lymphoid cell lines representing all stages of B cell development. Further, heavy chains that remained associated with BiP were not transported to the cell surface or secreted while heavy chains that were only transiently associated with BiP chains were expressed on the cell surface or secreted.

The 78,000-dalton glucose-regulated protein (GRP78) and the immunoglobulin heavy-chain-binding protein (BiP) were shown to be the same protein by NH2-terminal sequence comparison. Immunoprecipitation of GRP78-BiP induced by glucose starvation and a temperature-sensitive mutation in a hamster fibroblast cell line demonstrated the association of GRP78-BiP with other cellular proteins. In both fibroblasts and lymphoid cells, GRP78-BiP was found to label with 32Pi and [3H]adenosine. Phosphoamino acid analysis demonstrated that GRP78-BiP is phosphorylated on serine and threonine residues. Conditions which induce increased production of GRP78-BiP resulted in decreased incorporation of 32Pi and [3H]adenosine into GRP78-BiP. Furthermore, we report here that the phosphorylated form of BiP resides in the endoplasmic reticulum and that BiP which is associated with heavy chains is not phosphorylated or labeled with [3H]adenosine, whereas free BiP is. This suggests that posttranslational modifications may be important in regulating the synthesis and binding of BiP.

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.