KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene

The yeast KAR2 gene was isolated by complementation of a mutation that blocks nuclear fusion. The predicted KAR2 protein sequence is most homologous to mammalian BiP/GRP78 and has several structural features in common with it: a functional secretory signal sequence, a yeast endoplasmic reticulum retention signal (HDEL) at the carboxyl terminus, and the absence of potential N-linked glycosylation sites. Moreover KAR2 is regulated like BiP/GRP78: the level of mRNA is increased by drug treatments and mutations that cause accumulation of secretory precursors in the endoplasmic reticulum. However, unlike BiP/GRP78, KAR2 is also regulated by heat shock. Deletion of the KAR2 gene generated a recessive lethal mutation, showing that BiP/GRP78 function is required for cell viability.     

Unfolded protein response-induced BiP/Kar2p production protects cell growth against accumulation of misfolded protein aggregates in the yeast endoplasmic reticulum

Overproduction of delta(pro), a mutated secretory proteinase derived from a filamentous fungus Rhizopus niveus, results in formation of gross aggregates (delta(pro) aggregates) in the yeast endoplasmic reticulum (ER) lumen, activation of the unfolded protein response (UPR) and ER membrane proliferation. To investigate the roles of the UPR against the delta(pro) aggregates, we constructed an IRE1-deleted ((delta)ire1) strain. In contrast to wild-type cells, (delta)ire1 cells ceased to grow several hours after the overproduction of (delta)pro. Two lines of evidence argued against the possibility that the growth defect was due to the inability to make extra ER membrane which accommodates the (delta)pro aggregates. First, by electron microscopy, ER membrane proliferation was observed in (delta)ire1 cells overproducing (delta)pro. Second, disruption of the OPI1 gene in the (delta)ire1 mutant, which is considered to derepress the activities of phospholipid-synthesizing enzymes, did not restore the growth upon the overproduction of (delta)pro. Instead, the growth was restored when an extra copy of the KAR2 gene, which encodes yeast BiP, was introduced, indicating that an increase in the amount of BiP is essential for cell growth when the (delta)pro aggregates accumulate in the ER. Since BiP is included in the insoluble (delta)pro aggregates, it is likely that the amount of free BiP in the ER lumen is insufficient without the UPR to fully exert its functions. Consistently, overproduction of (delta)pro impaired protein translocation and folding in (delta)ire1 cells but not in wild-type cells. The tunicamycin sensitivity of (delta)ire1 cells was also suppressed by extra expression of KAR2, suggesting that BiP plays a principal role in protecting cell growth against misfolded proteins accumulated in the ER.     

Bip/GRP78-induced production of cytokines and uptake of amyloid-beta(1-42) peptide in microglia

In the brains of Alzheimer's disease (AD) patients, fibrillar amyloid-beta peptides (Abeta) are markedly accumulated and the microglia associate with the amyloid plaques. However, the regulation of Abeta clearance is still unclear. In the present study, we examined the effect of a chaperone protein BiP/GRP78 on the microglial function. Exogenous addition of recombinant BiP/GRP78 induced the production of cytokines such as interleukin-6 and tumor necrosis factor-alpha, but heat treatment of this protein abolished the activity. Although Abeta(1-42) did not induce cytokine production, it was taken up by the microglia. In addition, the amount of Abeta(1-42) uptake and the number of microglia that phagocytosed Abeta(1-42) were markedly increased by BiP/GRP78. Exogenous BiP/GRP78 also translocated to the endoplasmic reticulum (ER). These results suggest that BiP/GRP78 stimulates Abeta clearance in the microglia, and that dysfunction in the ER may cause the accumulation of extracellular Abeta(1-42).     

Host response to the subtilase cytotoxin produced by locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli

Shiga-toxigenic Escherichia coli (STEC) is a major bacterium responsible for disease resulting from foodborne infection, including bloody diarrhea and hemolytic uremic syndrome. STEC produces important virulence factors such as Shiga toxin (Stx) 1 and/or 2. In the STEC family, some locus of enterocyte effacement-negative STEC produce two different types of cytotoxins, namely, Stx2 and subtilase cytotoxin (SubAB). The Stx2 and SubAB cytotoxins are structurally similar and composed of one A subunit and pentamer of B subunits. The catalytically active A subunit of SubAB is a subtilase-like serine protease and specifically cleaves an endoplasmic reticulum (ER) chaperone 78-kDa glucose-regulated protein (GRP78/BiP), a monomeric ATPase that is crucial in protein folding and quality control. The B subunit binds to cell surface receptors. SubAB recognizes sialic carbohydrate-modified cell surface proteins as a receptor. After translocation into cells, SubAB is delivered to the ER, where it cleaves GRP78/BiP. SubAB-catalyzed BiP cleavage induces ER stress, which causes various cell events including inhibition of protein synthesis, suppression of nuclear factor-kappa B activation, apoptotic cell death, and stress granules formation. In this review, we describe SubAB, the SubAB receptor, and the mechanism of cell response to the toxin.     

ADP-ribosylation of the molecular chaperone GRP78/BiP

Starvation of mouse hepatoma cells for essential amino acids or glucose results in the ADP-ribosylation of the molecular chaperone BiP/GRP78. Addition of the missing nutrient to the medium reverses the reaction. The signal mediating the response to environmental nutrients involves the translational efficiency. An inhibitor of proteins synthesis, cycloheximide, or reduced temperature, both of which reduce translational efficiency, stimulate the ADP-ribosylation of BiP/GRP78. Inhibition of N-linked glycosylation of proteins results in the overproduction of BiP/GRP78. The over produced protein is not ADP-ribosylated suggesting that this is the functional form of BiP/GRP78. The over produced BiP/GRP78 can, however, be ADP-ribosylated if the cells are starved for an essential amino acid. BiP/GRP78 resides in the lumen of the endoplasmic reticulum where it participates in the assembly of secretory and integral membrane proteins. ADP-ribosylation of BiP/GRP78 during starvation is probably part of a nutritional stress response which conserves limited nutrients by slowing flow through the secretory pathway.     

Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78

Alterations in Ca(2+) homeostasis and accumulation of unfolded proteins in the endoplasmic reticulum (ER) lead to an ER stress response. Prolonged ER stress may lead to cell death. Glucose-regulated protein (GRP) 78 (Bip) is an ER lumen protein whose expression is induced during ER stress. GRP78 is involved in polypeptide translocation across the ER membrane, and also acts as an apoptotic regulator by protecting the host cell against ER stress-induced cell death, although the mechanism by which GRP78 exerts its cytoprotective effect is not understood. The present study was carried out to determine whether one of the mechanisms of cell death inhibition by GRP78 involves inhibition of caspase activation. Our studies indicate that treatment of cells with ER stress inducers causes GRP78 to redistribute from the ER lumen with subpopulations existing in the cytosol and as an ER transmembrane protein. GRP78 inhibits cytochrome c-mediated caspase activation in a cell-free system, and expression of GRP78 blocks both caspase activation and caspase-mediated cell death. GRP78 forms a complex with caspase-7 and -12 and prevents release of caspase-12 from the ER. Addition of (d)ATP dissociates this complex and may facilitate movement of caspase-12 into the cytoplasm to set in motion the cytosolic component of the ER stress-induced apoptotic cascade. These results define a novel protective role for GRP78 in preventing ER stress-induced cell death.     

Condensation-sorting events in the rough endoplasmic reticulum of exocrine pancreatic cells

In guinea pig exocrine pancreatic cells intracisternal granules (ICGs) occur at a low frequency within the lumen of the RER. By starving and refeeding guinea pigs or injecting them in CoCl2 solution, the number of these granules is greatly increased. We show here that ICGs contain the complete set of secreted pancreatic digestive enzymes and proenzymes. Two other soluble proteins in the lumen of the RER, GRP 78/BiP and protein disulphide isomerase (PDI), are specifically excluded from ICGs. The formation of ICGs, which occurs without acidification of the RER cisternae, is therefore a sorting event involving the cocondensation of a complete set of secretory enzymes and proenzymes, which for brevity we refer to collectively as the zymogens. With the exception of approximately 50% of the RNase, the zymogens in ICGs are covalently cross-linked by intermolecular disulphide bonds. The synthesis of all three resident ER cisternal proteins--PDI, GRP 78/BiP, and GRP 94--with the carboxy-terminal sequence KDEL, is induced in response to the accumulation of massive amounts of misfolded secretory protein in the ICGs in the lumen of the RER. After injection of rats with large doses of parachlorophenylalanine-methylester, crystals form in the lumen of the RER. We show that these crystals appear to be a lattice of amylase with the other zymogens incorporated between the layers. Both GRP 78/BiP and PDI are excluded from these crystals. The formation of these amylase crystals within the RER and the inclusion of other zymogens is, therefore, also a sorting event. These data establish that in exocrine pancreatic cells zymogens can cocondense in the RER into either amorphous aggregates or crystals that exclude other soluble RER proteins. This demonstrates that cocondensation is a mechanism capable of sorting zymogens within the secretory pathway.     

Genetic interactions between KAR2 and SEC63, encoding eukaryotic homologues of DnaK and DnaJ in the endoplasmic reticulum.

KAR2 encodes the yeast homologue of mammalian BiP, the endoplasmic reticulum (ER) resident member of the HSP70 family. Kar2p has been shown to be required for the translocation of proteins across the ER membrane as well as nuclear fusion. Sec63, an ER integral membrane protein that shares homology with the Escherichia coli DnaJ protein, is also required for translocation. In this paper we describe several specific genetic interactions between these two proteins, Kar2p and Sec63p. First, temperature-sensitive mutations in KAR2 and SEC63 form synthetic lethal combinations. Second, dominant mutations in KAR2 are allele-specific suppressors for the temperature-sensitive growth and translocation defect of sec63-1. Third, the sec63-1, unlike other translocation defective mutations, results in the induction of KAR2 mRNA levels. Taken together, these genetic interactions suggest that Kar2p and Sec63p interact in vivo in a manner similar to that of the E. coli HSP70, DnaK, and DnaJ. We propose that the interaction between these two proteins is critical to their function in protein translocation.     

Thapsigargin down-regulates protein levels of GRP78/BiP in INS-1E cells

Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) and express large amounts of chaperones and protein disulfide isomerases (PDI) to meet the high demand for synthesis of proteins. We have observed an unexpected decrease in chaperone protein level in the β-cell model INS-1E after exposure to the ER stress inducing agent thapsigargin. As these cells are a commonly used model for primary β-cells and has been shown to be vulnerable to ER stress, we hypothesize these cells are incapable of mounting a chaperone defense upon activation of ER stress. To investigate the chaperone expression during an ER stress response, induced by thapsigargin in INS-1E cells, we used quantitative mass spectrometry based proteomics. The results displayed a decrease of GRP78/BiP, PDIA3 and PDIA6. Decrease of GRP78/BiP was verified by Western blot and occurred in parallel with enhanced levels of p-eIF2α and CHOP. In contrast to INS-1E cells, GRP78/BiP was not decreased in MIN6 cell or rat and mouse islets after thapsigargin exposure. Investigation of the decreased protein levels of GRP78/BiP indicates that this is not a consequence of reduced mRNA expression. Rather the reduction results from the combined effect of reduced protein synthesis and enhanced proteosomal degradation and possibly also degradation via autophagy. Induction of ER stress with thapsigargin leads to lower protein levels of GRP78/BiP, PDIA3 and PDIA6 in INS-1E cells which may contribute to the susceptibility of ER stress in this β-cell model.     

Regulation and recovery of functions of Saccharomyces cerevisiae chaperone BiP/Kar2p after thermal insult

We described earlier a novel mode of regulation of Hsp104, a cytosolic chaperone directly involved in the refolding of heat-denatured proteins, and designated it delayed upregulation, or DUR. When Saccharomyces cerevisiae cells grown at the physiological temperature of 24 degrees C, preconditioned at 37 degrees C, and treated briefly at 50 degrees C were shifted back to 24 degrees C, Hsp104 expression was strongly induced after 2.5 h of recovery and returned back to normal after 5 h. Here we show that the endoplasmic reticulum (ER) chaperones BiP/Kar2p and Lhs1p and the mitochondrial chaperone Hsp78 were also upregulated at the physiological temperature during recovery from thermal insult. The heat shock element (HSE) in the KAR2 promoter was found to be sufficient to drive DUR. The unfolded protein element could also evoke DUR, albeit weakly, in the absence of a functional HSE. BiP/Kar2p functions in ER translocation and assists protein folding. Here we found that the synthesis of new BiP/Kar2p molecules was negligible for more than an hour after the shift of the cells from 50 degrees C to 24 degrees C. Concomitantly, ER translocation was blocked, suggesting that preexisting BiP/Kar2p molecules or other necessary proteins were not functioning. Translocation resumed concomitantly with enhanced synthesis of BiP/Kar2p after 3 h of recovery, after which ER exit and protein secretion also resumed. For a unicellular organism like S. cerevisiae, conformational repair of denatured proteins is the sole survival strategy. Chaperones that refold proteins in the cytosol, ER, and mitochondria of S. cerevisiae appear to be subject to DUR to ensure survival after thermal insults.     

Interaction of murine BiP/GRP78 with the DnaJ homologue MTJ1

The activity of Hsp70 proteins is regulated by accessory proteins, among which the most studied are the members of the DnaJ-like protein family. BiP/GRP78 chaperones the translocation and maturation of secreted and membrane proteins in the endoplasmic reticulum. No DnaJ-like partner has been described so far to regulate the function of mammalian BiP/GRP78. We show here that murine BiP/GRP78 interacts with the lumenal J domain of the murine transmembrane protein MTJ1 (J-MTJ1). J-MTJ1 stimulates the ATPase activity of BiP/GRP78 at stoichiometric concentrations. The C-terminal tail of BiP/GRP78 is not required for the interaction with J-MTJ1, leaving the function of this portion of the molecule still unclear. Physical interactions between J-MTJ1 and BiP/GRP78 are stable and can be abolished by a single histidine --> glutamine substitution in the highly conserved HPD motif shared by all DnaJ-like proteins. The J-MTJ1 fragment, but not the mutant J-MTJ1:H89Q fragment, stimulates the ATPase activity of Escherichia coli DnaK, although at a higher concentration than its genuine partner DnaJ. Full-length DnaJ does not stimulate BiP over the range of concentrations investigated. These results indicate that the J domain of MTJ1 is sufficient for its interaction with BiP/GRP78 and cannot be substituted by E. coli DnaJ.     

Regulation of endoplasmic reticulum stress proteins in COS cells transfected with immunoglobulin mu heavy chain cDNA

The mechanism by which endoplasmic reticulum (ER) stress proteins are induced by the accumulation of incompletely assembled or malfolded proteins in the ER is poorly understood. The 78-kDa glucose-regulated protein (BiP), one of the ER stress proteins, has often been detected in stable complexes with these accumulated proteins. We have transfected COS cells with an immunoglobulin (Ig) mu heavy chain expression plasmid. Expressed mu-chain accumulated in the cells and formed stable complexes with BiP. As a result, the synthesis of three ER stress proteins, BiP, the 94-kDa glucose-regulated protein (GRP94/ERp99), and ERp72, was increased as were their mRNA levels. In addition, the degradation rate of BiP was increased, possibly because of its interaction with mu-chain. Cotransfection of the mu-chain plasmid with an Ig lambda light chain expression plasmid resulted in the appearance of mu-chain in the media in a covalent complex with lambda-chain. An intracellular consequence of this was a reduction in the levels of BiP.mu-chain complex, and a diminished stimulation in the synthesis of the ER stress proteins. These results suggest that the BiP.mu-chain complex in the ER may be involved in the signaling pathway for the induction of ER stress proteins and may represent one regulatory mechanism operating in differentiating B-lymphocytes.     

Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

Disturbance of endoplasmic reticulum (ER) homeostasis causes ER stress and leads to activation of the unfolded protein response, which reduces the stress and promotes cell survival at the early stage of stress, or triggers cell death and apoptosis when homeostasis is not restored under prolonged ER stress. Here, we report that Cab45S, a member of the CREC family, inhibits ER stress-induced apoptosis. Depletion of Cab45S increases inositol-requiring kinase 1 (IRE1) activity, thus producing more spliced forms of X-box-binding protein 1 mRNA at the early stage of stress and leads to phosphorylation of c-Jun N-terminal kinase, which finally induces apoptosis. Furthermore, we find that Cab45S specifically interacts with 78-kDa glucose-regulated protein/immunoglobulin heavy chain binding protein (GRP78/BiP) on its nucleotide-binding domain. Cab45S enhances GRP78/BiP protein level and stabilizes the interaction of GRP78/BiP with IRE1 to inhibit ER stress-induced IRE1 activation and apoptosis. Together, Cab45S, a novel regulator of GRP78/BiP, suppresses ER stress-induced IRE1 activation and apoptosis by binding to and elevating GRP78/BiP, and has a role in the inhibition of ER stress-induced apoptosis.     

The role of endoplasmic reticulum‐related BiP/GRP78 in interferon gamma‐induced persistent Chlamydia pneumoniae infection

Direct interaction of Chlamydiae with the endoplasmic reticulum (ER) is essential in intracellular productive infection. However, little is known about the interplay between Chlamydiae and the ER under cellular stress conditions that are observed in interferon gamma (IFN‐γ) induced chlamydial persistent infection. ER stress responses are centrally regulated by the unfolded protein response (UPR) under the control of the ER chaperone BiP/GRP78 to maintain cellular homeostasis. In this study, we could show that the ER directly contacted with productive and IFN‐γ‐induced persistent inclusions of Chlamydia pneumoniae (Cpn). BiP/GRP78 induction was observed in the early phase but not in the late phase of IFN‐γ‐induced persistent infection. Enhanced BiP/GRP78 expression in the early phase of IFN‐γ‐induced persistent Cpn infection was accompanied by phosphorylation of the eukaryotic initiation factor‐2α (eIF2α) and down‐regulation of the vesicle‐associated membrane protein‐associated protein B. Loss of BiP/GRP78 function resulted in enhanced phosphorylation of eIF2α and increased host cell apoptosis. In contrast, enhanced BiP/GRP78 expression in IFN‐γ‐induced persistent Cpn infection attenuated phosphorylation of eIF2α upon an exogenous ER stress inducer. In conclusion, ER‐related BiP/GRP78 plays a key role to restore cells from stress conditions that are observed in the early phase of IFN‐γ‐induced persistent infection.     

FAD-linked presenilin-1 mutants impede translation regulation under ER stress

FAD mutations in presenilin-1 (PS1) cause attenuation of the induction of the endoplasmic reticulum (ER)-resident chaperone GRP78/BiP under ER stress, due to disturbed function of IRE1, the sensor for accumulation of unfolded protein in the ER lumen. PERK, an ER-resident transmembrane protein kinase, is also a sensor for the unfolded protein response (UPR), causing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit translation initiation. Here, we report that the FAD mutant PS1 disturbs the UPR by attenuating both the activation of PERK and the phosphorylation of eIF2alpha. Consistent with the results of a disturbed UPR, inhibition of protein synthesis under ER stress was impaired in cells expressing PS1 mutants. These results suggest that mutant PS1 impedes general translational attenuation regulated by PERK and eIF2alpha, resulting in an increased load of newly synthesized proteins into the ER and subsequently increasing vulnerability to ER stress.     

Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.