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

Using specific anti-BiP/Kar2 antibody as the probe, we have developed an efficient purification method of BiP/Kar2 protein from the total cell extract of Saccharomyces cerevisiae. Overproduction of BiP/Kar2 protein was achieved by the cloning of the KAR2 gene on multicopy plasmids and the treatment of cells harboring the cloned KAR2 gene with tunicamycin. Freeze-thaw treatment, hydroxyapatite high pressure liquid chromatography, and ATP-agarose column chromatography of crude extract yielded homogeneous BiP/Kar2 protein (including less than 0.2% of degradative derivative) with a 430-fold purification and 28% recovery. Edman degradation of purified BiP/Kar2 suggests that the mature protein corresponds to a processed product with the removal of a 42-amino acid presequence. It is active as a homodimer and exhibits ATPase activity with a specific activity of 2 pmol/min/micrograms of protein. Protease susceptibility indicated that the ADP form of BiP/Kar2 is more resistant than the ATP form to the chymotrypsin digestion and that BiP/Kar2 required the presence of ATP to avoid the irreversible denaturation. Synthesis of BiP/Kar2 was induced by the inducible expression of an aberrant heterologous protein, yeast killer prepro-signal mouse alpha-amylase fusion protein.
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PMID:Purification and characterization of BiP/Kar2 protein from Saccharomyces cerevisiae. 132 40

Treatment of developing bean cotyledons with the inhibitor of N-glycosylation tunicamycin enhanced the synthesis of at least two polypeptides with molecular mass 78 kDa and 97 kDa. Pulse-chase experiments and subcellular fractionation indicated that these are endoplasmic reticulum (ER) residents. The 78 kDa protein is a major component of the ER protein fraction and, by N-terminal sequencing, was identified as a bean homolog of the mammalian 78 kDa glucose-regulated protein (GRP78). This is a molecular chaperone that is probably involved in the folding and oligomerization of several animal and yeast proteins in the ER. When newly synthesized storage glycoproteins phaseolin, phytohemagglutinin or alpha-amylase inhibitor were immunoprecipitated from an ER preparation of tunicamycin-treated tissue, the GRP78 homolog was always co-precipitated. Bound GRP78 homolog could be released by ATP treatment. These results suggest that, at least when glycosylation is inhibited, this protein plays a role in the early stages of the synthesis of vacuolar storage proteins.
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PMID:Bean homologs of the mammalian glucose-regulated proteins: induction by tunicamycin and interaction with newly synthesized seed storage proteins in the endoplasmic reticulum. 134 85

The proteins DnaK (hsp70) and GroEL (cpn60) from Escherichia coli are prototypes of two classes of molecular chaperones conserved throughout evolution. The analysis of transferred nuclear Overhauser effects in two-dimensional NMR spectra is ideally suited to determine chaperone-bound conformations of peptides. The peptide vsv-C (amino-acid sequence KLIGVLSSLFRPK) stimulates the ATPase of BiP and Hsc70 (ref. 3) and the intrinsic ATPase of DnaK. The affinity of the vsv-C peptide for DnaK is greatly reduced in the presence of ATP. Here we analyse transferred nuclear Overhauser effects and show that the peptide is in an extended conformation while bound to DnaK but is helical when bound to GroEL. NMR also indicates that the mobility of the peptide backbone is reduced more by binding to DnaK than by binding to GroEL, whereas the side chains are less mobile when bound to GroEL.
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PMID:Different conformations for the same polypeptide bound to chaperones DnaK and GroEL. 134 69

The mouse Wnt-1 gene, a target for insertional activation in mouse mammary tumor virus-induced mammary tumors, encodes poorly secreted, cysteine-rich glycoproteins required for proper central nervous system development. We have been analyzing the biosynthesis of Wnt-1 proteins in several cell lines that express Wnt-1 cDNA from heterologous promoters. A protein of 78 kDa was found to be associated with the intracellular forms of Wnt-1 proteins in mammalian and avian cells by using multiple antisera against Wnt-1 proteins. We have identified p78 as the binding protein BiP with anti-BiP antisera and by its release from Wnt-1 immunoprecipitates upon incubation with MgCl2 and ATP. Experiments with a Wnt-1 mutant that lacks the sequence encoding the signal peptide indicates that Wnt-1 proteins must enter the secretory pathway in order to interact with BiP. We demonstrate that Wnt-1 proteins are associated with BiP in cells in which active Wnt-1 proteins are produced, such as a cultured mammary epithelial cell line and Wnt-1 transgenic mouse mammary tumor cells. The association of Wnt-1 proteins with BiP may be a factor in determining the efficiency of secretion of Wnt-1 gene products.
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PMID:Interaction of Wnt-1 proteins with the binding protein BiP. 153 Oct 88

Secretory proteins are segregated from cytosolic proteins by their translocation into the endoplasmic reticulum (ER). A modified secretory protein trapped during translocation across the ER membrane can be crosslinked to two previously identified proteins, Sec61p and BiP (Kar2p). The dependence of this cross-linking upon proteins and small molecules was examined. Mutations in SEC62 and SEC63 decrease the ability of Sec61p to be cross-linked to the secretory polypeptide trapped in translocation. ATP is also required for interaction of Sec61p with the secretory protein. Three kar2 alleles display defective translocation in vitro. Two of these alleles also decrease the ability of Sec61p to be cross-linked to the secretory protein. The third allele, while exhibiting a severe translocation defect, does not affect the interaction of Sec61p with the secretory protein. These results suggest that Sec61p is directly involved in translocation and that BiP acts at two stages of the translocation cycle.
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PMID:Sec61p and BiP directly facilitate polypeptide translocation into the ER. 156 50

Immunoglobulin heavy chain binding protein (BiP, GRP 78) coprecipitates with soluble and membrane-associated variants of the T-cell antigen receptor alpha chain (TCR-alpha) which are stably retained within the ER. Chelation of Ca2+ during solubilization of cells leads to the dissociation of BiP from the TCR-alpha variants, which is dependent upon the availability of Mg2+ and hydrolyzable ATP; this suggests that Ca2+ levels can serve to modulate the association/dissociation of these proteins with BiP. In vivo treatment of cells expressing either the soluble or membrane-anchored TCR-alpha variants with the Ca2+ ionophore, A23187, or an inhibitor of an ER Ca(2+)-ATPase, thapsigargin, or the membrane-permeant Ca2+ chelator BAPTA-AM, results in the redistribution of these proteins out of the ER and their subsequent secretion or cell surface expression. Under the same assay conditions, no movement of BiP out of the ER is observed. Taken together, these observations indicate that decreased Ca2+ levels result in the dissociation of a protein bound to BiP, leading to its release from ER retention. These data suggest that the intracellular fate of newly synthesized proteins stably associated with BiP can be regulated by Ca2+ levels in the ER.
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PMID:Regulating the retention of T-cell receptor alpha chain variants within the endoplasmic reticulum: Ca(2+)-dependent association with BiP. 164 96

Rat liver and canine pancreas rough endoplasmic reticulum-derived vesicles, which were sealed and of the same topographical orientation as in vivo, were used in a system in vitro to demonstrate translocation of ATP into their lumen. Translocation of ATP is saturable (apparent Km: 3-4 microM and Vmax: 3-7 pmol/min/mg of protein) and protein mediated because treatment of intact vesicles with Pronase, N-ethylmaleimide, or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibit transport. The entire ATP molecule is being translocated; this was shown by high performance liquid chromatography analysis and the use of a nonhydrolyzable analog. Control experiments rule out that translocation of ATP attributed to rough endoplasmic reticulum-derived vesicles is due to contamination by mitochondria and Golgi vesicles. Following translocation of ATP into the lumen of the vesicles, binding to luminal proteins including BiP (immunoglobulin heavy chain-binding protein-glucose-regulated protein 78) and glucose-regulated protein 94 was observed. This binding appeared to be specific because similar experiments with GTP were negative. These studies strongly suggest that translocation of ATP into the rough endoplasmic reticulum lumen may serve as a mechanism for making ATP available in proposed energy requiring reactions within the lumen.
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PMID:Translocation of ATP into the lumen of rough endoplasmic reticulum-derived vesicles and its binding to luminal proteins including BiP (GRP 78) and GRP 94. 174 Apr 46

Transport of bovine preprolactin into dog pancreas microsomes involves a microsomal protein which is sensitive to photoaffinity labeling with azido-ATP and which is distinct from the ATP-binding protein, immunoglobulin heavy chain binding protein. Here we addressed the question of what stage of preprolactin transport is affected. Thus a nascent presecretory protein which is related to preprolactin, termed ppl-86mer, was employed. Here we show that the nascent preprolactin did not become associated with the alpha-subunit of the signal sequence receptor complex after photoaffinity labeling of microsomes with azido-ATP. Therefore, we conclude that the microsomal protein which is sensitive to photoaffinity labeling with azido-ATP acts prior to the signal sequence receptor complex.
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PMID:Photoaffinity labeling of dog pancreas microsomes with 8-azido-ATP inhibits association of nascent preprolactin with the signal sequence receptor complex. 186 86

The characteristics of phosphorylation of the 78-kDa glucose-regulated protein (Grp78), also known as the immunoglobulin heavy chain binding protein, were studied in vitro and in vivo. The purified protein from either calf liver or bovine kidney cells (MDBK) could be phosphorylated in vitro with [gamma-32P]ATP, in a reaction that is stimulated by Ca2+ and inhibited by the Ca(2+)-chelator ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA). In the presence of EGTA, excess Ca2+ increased the rate of phosphorylation about 18-fold. Based on EGTA/Ca2+ titrations, the optimal Ca2+ concentration for phosphorylation was estimated to be 10-50 microM. Other divalent cations such as Mg2+, Mn2+, and Zn2+ were found to be inhibitory as was the Ca2+ antagonist lanthanum (La3+). The in vivo phosphorylation of Grp78 was studied in MDBK cells labeled with 32Pi. In the presence of inducers of Grp78 synthesis, such as ionomycin, tunicamycin, or 2-deoxyglucose, there was a large increase in the level of Grp78 in the cells but a decrease in the amount of phosphorylated protein. Two-dimensional gel analysis of Grp78 purified from bovine liver and MDBK cells identified at least four isoforms. After in vivo and in vitro phosphorylation of Grp78 all the acidic isoforms contained radioactivity but not the most basic isoform. Phosphoamino acid analysis of Grp78 showed that serine and threonine were phosphorylated in vivo and only threonine was phosphorylated in vitro.
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PMID:Calcium-dependent autophosphorylation of the glucose-regulated protein, Grp78. 191 Mar 17

We have translated the HLA-B27 heavy chain in vitro and studied its assembly with beta 2-microglobulin and peptide in microsomes from human cells. The assembly process requires ATP. However, the translocation of peptide across the endoplasmic reticulum (ER) membrane does not require ATP, and binding of biotinylated peptide to BiP, an ER luminal protein, occurs after ATP depletion. Proteinase K treatment of the microsomes does not block peptide translocation. Thus, ATP is required in the lumen of the ER for efficient assembly to occur. Microsomes prepared from Raji and T1 cells show similar levels of assembly, whereas assembly in T2 microsomes is 10-fold lower. This difference remains after peptide stimulation of assembly. The inefficient assembly in T2 microsomes is not due to impaired peptide translocation across the ER membrane, as no difference was found compared with microsomes from T1 cells. Instead, the defect seems to reside in the lumen of the ER.
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PMID:ATP is required for in vitro assembly of MHC class I antigens but not for transfer of peptides across the ER membrane. 191 23


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