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Query: UNIPROT:P11021 (BiP)
 2,049 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The kappa-chain of the myeloma MOPC 21 is an unusual L chain, in that it is not secreted unless complexed with a H chain. This nonsecreted kappa-chain seems to be retained in the endoplasmic reticulum in association with the protein BiP/GRP78, both in myeloma cells and when expressed in COS-1 fibroblasts. By assaying the fate of the MOPC 21 kappa-chain and its mutated derivatives in COS-1 cells, we show that the cause of the nonsecreted phenotype is the presence of histidine in position 87 of the variable domain. When this amino acid is changed back to the tyrosine that usually occupies position 87, secretion of the unassembled kappa-chain is restored. As in B lymphoid cells, co-expression of gamma H chains in COS-1 cells complements the mutation in the L chain, and rescues secretion of the arrested kappa. Thus, the presence of histidine at position 87 creates a conditional L chain secretory mutant: it is not compatible with normal transport of free L chain, but can be rescued in the presence of H chain.
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PMID:A conditional secretory mutant in an Ig L chain is caused by replacement of tyrosine/phenylalanine 87 with histidine. 151 62

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.
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PMID:Characterization of the nucleotide binding properties and ATPase activity of recombinant hamster BiP purified from bacteria. 759 93

A panel of monoclonal antibodies (MAbs) was raised against Semliki Forest virus (SFV) nonstructural protein nsP2, which is a protease, an NTPase, a putative RNA helicase, and a regulator of the synthesis of the subgenomic 26S mRNA encoding the structural proteins. nsP2, used for immunization, was expressed as a histidine fusion protein in Escherichia coli and purified by metal affinity chromatography. Dot-blot assay, using a membrane fraction from SFV-infected cells as antigen, gave 33 positive clones. Of these, 30 MAbs recognized nsP2 in Western immunoblotting, and 25 showed positive indirect immunofluorescence (IFAT) in SFV-infected cells; 15 MAbs stained the cytoplasmic vacuoles (CPVI), which are the sites of viral RNA synthesis in alphavirus-infected cells. MAb 3B5 recognized only CPVIs, as shown by double immunofluorescence staining with polyclonal anti-nsP3 antiserum. Most of the MAbs (20/33) recognized the nuclear form of nsP2, which may be associated with SFV neurovirulence. Immunoprecipitation with MAbs revealed that the SFV nonstructural proteins are associated with each other. None of the MAbs recognized Sindbis virus nsP2 in immunoblotting, indicating that they were directed to non-conserved sequences specific for SFV. Interestingly, these epitopes were located mostly within the N-terminal half of nsP2. Unexpectedly, the anti-nsP2 MAb 1E9 cross-reacted strongly with a host protein of 78 kDa from uninfected human, murine, avian and insect cells. This protein was identified as the immunoglobulin binding protein, BiP, by 2-D gel mapping and reaction with anti-BiP antiserum.
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PMID:Monoclonal antibodies specific for Semliki Forest virus replicase protein nsP2. 901 56

DnaJ-like proteins are functional partners for Hsp70 molecular chaperones. Complete nucleotide sequencing of yeast chromosome X has revealed that an open reading frame YJL073w encodes a novel member of the DnaJ-like protein family. The open reading frame represents a protein of 692 amino acids with a J-domain and one putative membrane-spanning segment. An epitope-tagged version of the protein was anchored in the endoplasmic reticulum (ER) membrane and its J-domain faced the ER lumen. We therefore propose to designate this gene JEM1 (DnaJ-like protein of the ER membrane) and to designate its gene product JEM1p. The JEM1 gene is not essential for cell growth, but double disruption of the JEM1 gene and the SCJ1 gene, which encodes another DnaJ-like protein in the ER lumen, causes growth arrest at elevated temperature. The Deltajem1 mutant is defective in nuclear fusion, karyogamy, during mating. A mutant JEM1p carrying a mutation in the highly conserved His-Pro-Asp sequence in the J-domain could not complement either temperature-sensitive growth of the Deltajem1 Deltascj1 double mutant or defects in karyogamy of the Deltajem1 mutant. JEM1p likely assists the functions of BiP, Hsp70 in the ER, including karyogamy.
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PMID:The yeast JEM1p is a DnaJ-like protein of the endoplasmic reticulum membrane required for nuclear fusion. 914 90

DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and glyoxysomes possess a membrane-anchored form of DnaJ protein located on the cytosolic face. Hints as to how the membrane-bound co-chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family. Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His-tagged DnaJ proteins and glutathione S-transferase-Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His-tagged form of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the glyoxysome-bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the BiP-related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase-modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.
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PMID:The membrane-bound DnaJ protein located at the cytosolic site of glyoxysomes specifically binds the cytosolic isoform 1 of Hsp70 but not other Hsp70 species. 1065 11

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.
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PMID:Interaction of murine BiP/GRP78 with the DnaJ homologue MTJ1. 1077 98

The C-terminal tail of the calcium receptor (CaR) regulates the affinity of the receptor for ligand, desensitization, and membrane localization. To determine the role of specific amino acids in the bovine parathyroid CaR in mediating signal transduction and cell-surface expression, we transfected truncated and mutated CaR cDNAs into HEK-293 cells. The ability of high extracellular [Ca(2+)] ([Ca(2+)](o)) to increase total inositol phosphate (InsP) production, an index of phospholipase C (PLC) activation, was determined. Receptor expression was assessed by immunoblotting and immunocytochemistry. In cells transiently or stably expressing receptors with the C-terminal tail truncated after residue 895 (CaR-(1-895)) or 929 (CaR-(1-929)), raising [Ca(2+)](o) increased InsPs to levels comparable with those of cells expressing wild-type CaRs. There were no PLC responses to high [Ca(2+)](o) (up to 30 mm) in cells expressing CaRs with C-terminal tails of only 3 residues (CaR-(1-866)), even though these receptors were expressed in the membrane. We scanned the residues between Ser(866) and Val(895) using tandem-Ala and single-site mutagenesis. Two point mutants (His(880) --> Ala and Phe(882) --> Ala CaR) showed 50-70% reductions in high [Ca(2+)](o)-induced InsP production. The levels of expression and glycosylation of these mutants were comparable with wild-type CaRs, but both receptors were profoundly retained in intracellular organelles and co-localized with the endoplasmic reticulum marker BiP. This suggested that the signaling defects of these receptors were likely because of defective trafficking of receptors to the cell surface. Modeling of the C-terminal domain of the CaR indicated that His(880) and Phe(882) are situated in a putative alpha-helical structure of 15 amino acids between residues 877 and 891 in the C-terminal tail. Our studies support the idea that specific amino acids, and possibly a unique secondary structure in the C-terminal tail, are required for the efficient targeting of the CaR to the cell surface required for PLC activation.
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PMID:Amino acids in the cytoplasmic C terminus of the parathyroid Ca2+-sensing receptor mediate efficient cell-surface expression and phospholipase C activation. 1153 93

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.
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PMID:Identification and characterization of a novel endoplasmic reticulum (ER) DnaJ homologue, which stimulates ATPase activity of BiP in vitro and is induced by ER stress. 1183 48

Mammalian glycerol uptake/transporter 1 (Gup1), a homolog of Saccharomyces cerevisiae Gup1, is predicted to be a member of the membrane-bound O-acyltransferase family and is highly homologous to mammalian hedgehog acyltransferase, known as Skn, the homolog of the Drosophila skinny hedgehog gene product. Although mammalian Gup1 has a sequence conserved among the membrane-bound O-acyltransferase family, the histidine residue in the motif that is indispensable to the acyltransferase activity of the family has been replaced with leucine. In this study, we cloned Gup1 cDNA from adult mouse lung and examined whether Gup1 is involved in the regulation of N-terminal palmitoylation of Sonic hedgehog (Shh). Subcellular localization of mouse Gup1 was indistinguishable from that of mouse Skn detected using the fluorescence of enhanced green fluorescent protein that was fused to each C terminus of these proteins. Gup1 and Skn were co-localized with an endoplasmic reticulum marker, 78 kDa glucose-regulated protein, suggesting that these two molecules interact with overlapped targets, including Shh. In fact, full-length Shh coprecipitated with FLAG-tagged Gup1 by immunoprecipitation using anti-FLAG IgG. Ectopic expression of Gup1 with full-length Shh in cells lacking endogenous Skn showed no hedgehog acyltransferase activity as determined using the monoclonal antibody 5E1, which was found to recognize the palmitoylated N-terminal signaling domain of Shh under denaturing conditions. On the other hand, Gup1 interfered with the palmitoylation of Shh catalyzed by endogenous Skn in COS7 and NSC34. These results suggest that Gup1 is a negative regulator of N-terminal palmitoylation of Shh and may contribute to the variety of biological actions of Shh.
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PMID:Mammalian Gup1, a homolog of Saccharomyces cerevisiae glycerol uptake/transporter 1, acts as a negative regulator for N-terminal palmitoylation of Sonic hedgehog. 1808 66

To improve the existing human papillomavirus type 16 (HPV16) virus-like particle (VLP) preparation, the Drosophila inducible/secreted expression system, a highly efficient, economical method, was used to produce HPV16 VLPs. Drosophila Schneider-2 cells were cotransfected with pMT/BiP/V5-His expression vector containing the target gene encoding HPV16L1 protein without nucleus localization sequence and the selection vector pCoHygro plasmids at the ratio of 4:1. The stabled hygromycin-resistant cell line was obtained 1 month later, and the protein expression was induced by copper sulfate. The molecular mass of expressed HPV16L1 protein was 66 kDa, as revealed by SDS-PAGE, and confirmed by Western blot analysis. The yield of HPV16L1 protein was 0.554 mg per 1x10(7) cells. The characteristics of HPV16L1 protein were further analyzed by mouse erythrocyte hemagglutination assay, hemagglutination inhibition assay, and transmission electron microscopy. Results showed that the truncated protein was as biologically active as natural HPVL1 protein, inducing murine erythrocyte agglutination and VLP formation. These findings indicate that the Drosophila inducible/secreted expression system is promising as a convenient and economical method for the preparation of HPV16 VLP vaccine.
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PMID:Stable high-level expression of truncated human papillomavirus type 16 L1 protein in Drosophila Schneider-2 cells. 1846 29


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