UNIPROT:P11021 - FACTA Search

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

A procedure was developed for the isolation of reticuloplasm, the luminal material of the endoplasmic reticulum (ER). A reticuloplasm-rich extract was prepared from a murine plasmacytoma cell line that contains large amounts of ER, by first extracting the cytoplasmic contents using hypotonic lysis to yield ER-rich 'shells' followed by mechanical lysis to release the ER contents. The extract contains five major proteins with apparent molecular weights of 100, 75, 60, 58 and 55 (X 10(3] Mr by SDS-polyacrylamide gel electrophoresis. The 100, 75 and 58 (X 10(3] Mr species were identified as the known ER proteins endoplasmin, BiP and PD1, respectively. The ER association of the 60 and 55 (X 10(3] Mr proteins was confirmed by confocal fluorescence microscopy with affinity-purified antibodies. Equilibrium dialysis with isolated reticuloplasm gave a calcium-binding capacity of 300 nmoles calcium per mg protein with half-maximal binding at 3 mM-Ca2+. Purified endoplasmin bound 280 nmoles calcium per mg protein at a calcium concentration of 5 mM-Ca2+. A calcium overlay test revealed that, in addition to endoplasmin, reticuloplasm contained at least three other calcium-binding proteins: i.e. BiP, PDI and the 55 X 10(3) Mr protein, respectively, with endoplasmin and the 55 X 10(3) Mr protein (CRP55) accounting for the major proportion of the calcium-binding activity. Treatment of cells with calcium ionophore led to the specific over-expression of the major calcium-binding reticuloplasmins endoplasmin, BiP and CRP55. These studies show that the lumen of the ER contains a family of proteins with the capacity to bind significant amounts of calcium in the millimolar range and thereby to confer upon the ER the ability to perform a calcium storage function analogous to that of the sarcoplasmic reticulum in muscle cells.
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PMID:Identification of a set of calcium-binding proteins in reticuloplasm, the luminal content of the endoplasmic reticulum. 325 4

Folding and assembly of polypeptides translocated into the rough endoplasmic reticulum (RER) is facilitated by a set of resident proteins in the lumen of the RER. We studied the regulation of synthesis of the RER luminal proteins immunoglobulin heavy chain binding protein (BiP) and protein disulfide isomerase (PDI), and of the cytosolic stress 70 protein (hsc70) after hormonal stimulation of the pancreatic exocrine secretory pathway. Their rate of synthesis was assessed at both mRNA and protein levels and under two experimental conditions that are associated with large increases in exocrine production. After in vivo stimulation of the pancreas by either endogenous release of cholecystokinin (CCK) following proteinase inhibitor feeding (FOY-305) or by in vivo infusion of the pancreatic secretagogue cerulein, the relative rates of synthesis detected for BiP and PDI were enhanced 2.5 to 4-fold compared to control. Interestingly, the kinetics and the degree of hsc70 mRNA induction were almost identical to those of BiP and PDI, suggesting coordinated hormonal regulation of BiP, PDI as hormonal stimulation was even twice that following heat shock treatment. The mRNA levels of calreticulin (CaBP3) increased up to 2.3-fold with a kinetic comparable to that of BiP, PDI and hsc 70, while CaBP1 and the RER membrane proteins, ribophorin I and the signal recognition particle receptor did not show any changes in their relative mRNA amounts after hormonal stimulation. The increase in the rates of PDI and chaperone biosynthesis exceeds the associated increase in total protein biosynthesis. In vitro experiments, using transformed rat acinar cells (AR4-2J) in which pancreatic enzyme synthesis can be induced by glycocorticoid hormones, demonstrated that induction of PDI and chaperone mRNA synthesis preceded extensive mRNA expression of secretory proteins.
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PMID:Hormonal regulation of protein disulfide isomerase and chaperone synthesis in the rat exocrine pancreas. 791 86

We have developed a single purification procedure for the four major resident endoplasmic reticulum (ER) proteins: protein disulfide isomerase (PDI), BiP, endoplasmin, and calreticulin. Three of these proteins are thought to play a role in protein folding in vivo, whereas calreticulin is thought to be the major calcium binding protein in the ER. The proteins were purified from fresh bovine liver by taking advantage of individual characteristics of the proteins. Liver microsomes were prepared and then premeabilized to release the lumenal contents. After ammonium sulfate precipitation, the proteins were purified by chromatography; BiP was purified by affinity chromatography on ATP-agarose, and both endoplasmin and calreticulin were purified by affinity chromatography on Con A-Sepharose. PDI was purified by anionic ion exchange chromatography.
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PMID:A single purification procedure for the major resident proteins of the ER lumen: endoplasmin, BiP, calreticulin and protein disulfide isomerase. 795 Mar 79

Intracellular distribution of selected reticuloplasmins, soluble proteins of the endoplasmic reticulum lumen, in rat mammary gland was investigated during pregnancy, lactation, and involution. During lactation the levels of the calcium binding protein calreticulin, and of protein disulfide isomerase, were elevated. Endoplasmic reticulum was as efficient as Golgi apparatus in sequestration and accumulation of Ca2+ from surrounding medium, as suggested from in vitro experiments with isolated cell fractions. Both protein disulfide isomerase and calreticulin were present in cytosol from homogenates of mammary gland prepared under mild conditions. Protein disulfide isomerase was abundant in intracellular lipid droplet precursors of milk lipid globules. Calreticulin and immunoglobulin binding protein (BiP, GRP 78) were associated with lipid droplets. Glucose-regulated protein (GRP 94) was not detected in association with intracellular lipid droplets. Milk lipid globule membrane lacked more than barely detectable quantities of protein disulfide isomerase, calreticulin, and immunoglobulin binding protein, suggesting that these proteins are lost from intracellular lipid droplets before or during their secretion as milk lipid globules. Immunocytochemical localization confirmed the presence of protein disulfide isomerase or calreticulin on intracellular lipid droplets and in non-endoplasmic reticulum regions of cells.
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PMID:Endoplasmic reticulum lumenal proteins of rat mammary gland. Potential involvement in lipid droplet assembly during lactation. 803 38

The postnatal differentiation of sarcoplasmic reticulum (SR) of rabbit skeletal muscles (the slow-twitch soleus and the fast-twitch adductor muscles) was monitored between Days 1 and 12 by following on Western blots the expression and accumulation of molecular markers specific not only for the muscle endomembrane system, i.e., calsequestrin (CS) and the ryanodine-sensitive Ca2+ release channel, but also for the endoplasmic reticulum (ER) at large, i.e., BiP, calnexin (CN) and calreticulin. Our results demonstrate that SR development, documented by the increase of the SR fractional volume, terminal cisternae proliferation, and reorientation of triads, is accompanied by the accumulation of the SR-specific proteins and also of CN, with no change of the other ER general markers. Moreover, the distribution of two of the markers, BiP and CS, was investigated by immunocytochemistry at both the light and the electron microscope level. At Day 1 CS was found to be concentrated both within the few recognizable triad terminal cisternae and within the lumen of numerous, apparently discrete cisternae and tubules, widely scattered throughout both the contractile and the subplasmalemmal areas of the cytoplasm. These structures remain evident until Day 12, when most triad junctions have acquired proper configuration, composition and orientation. BiP, on the other hand, appears widely distributed within the ER/SR of the fibers. From the early stages of postnatal development it does colocalize with the Ca2+ binding protein in the lumen of the CS-rich structures and appears also within the longitudinal SR and the conventional ER cisternae.
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PMID:The endoplasmic reticulum-sarcoplasmic reticulum connection. II. Postnatal differentiation of the sarcoplasmic reticulum in skeletal muscle fibers. 822 98

The major proteins in the lumen of the endoplasmic reticulum (ER) are thought to function in Ca2+ sequestration or as "molecular chaperones" in the folding and assembly of membrane or secreted proteins. Based on the ability of many chaperones to bind selectively to unfolded proteins and to dissociate from them upon ATP hydrolysis, we developed an affinity chromatography method to isolate proteins with these characteristics from pancreatic or liver ER. Seven ER proteins bound selectively to denatured protein columns and were specifically eluted by ATP (10(-6) M) but not by a nonhydrolyzable ATP analog. These proteins were identified with antibodies and microsequencing as the ER chaperone BiP (grp78), grp94, calreticulin, a novel 46-kDa protein that binds azido-ATP, as well as three members of the thioredoxin superfamily: protein-disulfide isomerase, ERp72, and a previously reported 50-kDa protein (p50). This set of seven proteins bound to and was eluted with ATP from a variety of denatured proteins, including histone, gelatin, alpha fetoprotein, thyroglobulin, lysozyme, casein, and IgG. The release of grp94, protein-disulfide isomerase, ERp72, calreticulin, and p50 was stimulated by Ca2+ in the presence of ATP. These proteins thus appear to function as Ca(2+)-dependent chaperones, which may account for the Ca2+ and ATP requirement for protein folding in the ER.
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PMID:A set of endoplasmic reticulum proteins possessing properties of molecular chaperones includes Ca(2+)-binding proteins and members of the thioredoxin superfamily. 829 23

Human cytomegalovirus glycoprotein B (gB) plays a role in the fusion of the virion envelope with the host cell membrane and in syncytium formation in infected cells. Hydrophobic sequences at the carboxyl terminus, amino acids (aa) 714 to 771, anchor gB in the lipid bilayer, but the unusual length of this domain suggests that it may serve another role in gB structure. To explore the function(s) of this region, we deleted aa 717 to 747 (gB deltaI mutation), aa 751 to 771 (gB deltaII mutation), and aa 717 to 772 (gB deltaI-II mutation) and constructed a substitution mutation, Lys-748 to Val (Lys748Val)-Asn749Ala-Pro750Ile (gB KNPm). Mutated forms of gB were expressed in U373 glioblastoma cells and subjected to analysis by flow cytometry, confocal microscopy, and immunoprecipitation. Mutations gB deltaI-II and gB deltaII alone caused secretion of gB into the medium, confirming that aa 751 to 771 function as a membrane anchor. In contrast, mutations gB deltaI and gB KNPm blocked cell surface expression and arrested gB transport in the endoplasmic reticulum (ER). Detailed examination of gB deltaI and gB KNPm with a panel of monoclonal antibodies showed that the mutated forms were indistinguishable from wild-type gB in conformation and formed oligomers; however, they remained sensitive to endoglycosidase H and did not undergo endoproteolytic cleavage. Analysis of protein complexes formed by gB and molecular chaperones in the ER showed that calnexin and calreticulin, lectin-like chaperones, bound equal amounts of uncleaved wild-type gB, gB deltaI, and gB KNPm, but the glucose-regulated proteins 78 (BiP) and 94 formed stable complexes only with the mutated forms, causing their retention in the ER. Our studies show that aa 714 to 750 are key residues in the architecture of gB molecules and that the ER chaperones, which facilitate gB folding and monitor the quality of glycoproteins, detect subtle changes in folding intermediates that are conferred by mutations in this region.
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PMID:Mutations in the carboxyl-terminal hydrophobic sequence of human cytomegalovirus glycoprotein B alter transport and protein chaperone binding. 889 27

Calreticulin is a ubiquitously expressed Ca2+-binding protein of the endoplasmic reticulum (ER), which inhibits DNA binding in vitro and transcriptional activation in vivo by steroid hormone receptors. Transient transfection assays were carried out to investigate the effects of different intracellular targeting of calreticulin on transactivation mediated by glucocorticoid receptor. BSC40 cells were transfected with either calreticulin expression vector (ER form of calreticulin) or calreticulin expression vector encoding calreticulin minus leader peptide, resulting in cytoplasmic localization of the recombinant protein. Transfection of BSC40 cells with calreticulin expression vector encoding the ER form of the protein led to 40-50% inhibition of the dexamethasone-sensitive stimulation of luciferase expression. However, in a similar experiment, but using the calreticulin expression vector encoding cytoplasmic calreticulin, dexamethasone-stimulated activation of the luciferase reporter gene was inhibited by only 10%. We conclude that the ER, but not cytosolic, form of calreticulin is responsible for inhibition of glucocorticoid receptor-mediated gene expression. These effects are specific to calreticulin, since overexpression of the ER lumenal proteins (BiP, ERp72, or calsequestrin) has no effect on glucocorticoid-sensitive gene expression. The N domain of calreticulin binds to the DNA binding domain of the glucocorticoid receptor in vitro; however, we show that the N+P domain of calreticulin, when synthesized without the ER signal sequence, does not inhibit glucocorticoid receptor function in vivo. Furthermore, expression of the N domain of calreticulin and the DNA binding domain of glucocorticoid receptor as fusion proteins with GAL4 in the yeast two-hybrid system revealed that calreticulin does not interact with glucocorticoid receptor under these conditions. We conclude that calreticulin and glucocorticoid receptor may not interact in vivo and that the calreticulin-dependent modulation of the glucocorticoid receptor function may therefore be due to a calreticulin-dependent signaling from the ER.
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PMID:Endoplasmic reticulum form of calreticulin modulates glucocorticoid-sensitive gene expression. 891 Jun 10

Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.
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PMID:A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum. 900 69

Calnexin is a membrane-bound lectin and a molecular chaperone that binds newly synthesized glycoproteins in the endoplasmic reticulum (ER). To analyze the oligomeric properties of calnexin and calnexin-substrate complexes, sucrose velocity gradient centrifugation and chemical cross-linking were used. After CHAPS solubilization of Chinese Hamster Ovary cells, the unoccupied calnexin behaved as a monomer sedimenting at 3.5 S20,W. For calnexin-substrate complexes the S-values ranged between 3.5-8 S20,W, the size increasing with the molecular weight of the substrate. Influenza hemagglutinin, a well-characterized substrate associated with calnexin in complexes that sedimented at 5-5.5 S20,W. The majority of stable complexes extracted from cells, appeared to contain a single calnexin and a single substrate molecule, with about one third of the calnexin in the cell being unoccupied or present in weak associations. However, when chemical cross-linking was performed in intact cells, the calnexin-substrate complexes and calnexin itself was found to be part of a much larger heterogeneous protein network that included other ER proteins. Pulse-chase analysis of influenza-infected cells combined with chemical cross-linking showed that HA was part of large, heterogeneous, cross-linked entities during the early phases of folding, but no longer after homotrimer assembly. The network of weakly associated resident ER chaperones which included BiP, GRP94, calreticulin, calnexin, and other proteins, may serve as a matrix that binds early folding and assembly intermediates and restricts their exit from the ER.
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PMID:Interactions between newly synthesized glycoproteins, calnexin and a network of resident chaperones in the endoplasmic reticulum. 902 87

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