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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

Incubation of Swiss 3T3 cells with [2-3H]adenine, as in other cell types, reveals the ADP-ribosylation of GRP78 (the 78-kDa glucose-regulated protein, also known as BiP, the immunoglobulin heavy chain-binding protein), a resident endoplasmic reticulum protein that assists in the processing of proteins destined for secretion or cell surface expression. Here we show that Pasteurella multocida toxin, a potent growth factor for cultured fibroblasts, decreased the ADP-ribosylation of GRP78/BiP to 16 +/- 6% of the control value (n = 23). The action of the toxin occurred after a lag period, was blocked by lysosomotrophic agents, and potentiated by increased incubation time (ED50 4 ng/ml and 1 ng/ml in 4 and 8 h, respectively), thus indicating that the toxin enters the cells to act. Bombesin and platelet-derived growth factor (PDGF) similarly decreased the ADP-ribosylation of GRP78/BiP (ED50 0.5 nM and 2.5 ng/ml, respectively) but acted more rapidly than the toxin. Signaling pathways activated by the toxin, bombesin, and PDGF had effects on the ADP-ribosylation of GRP78/BiP. Thus, activation of protein kinase C alone by phorbol 12,13-dibutyrate was partially effective, and down-regulation of protein kinase C attenuated but did not block the action of the toxin, bombesin, and PDGF. Agents that mobilize Ca2+ from the endoplasmic reticulum (A23187, ionomycin, and thapsigargin) caused a decrease in the ADP-ribosylation of GRP78/BiP that was similar in magnitude to that achieved by the toxin, bombesin, and PDGF, implicating a role for inositol 1,4,5-trisphosphate-mediated Ca2+ mobilization in the action of the mitogenic agents. The growth factor-induced decrease in the ADP-ribosylation of GRP78/BiP may represent its conversion from an inactive to an active state.
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PMID:Interconversion of GRP78/BiP. A novel event in the action of Pasteurella multocida toxin, bombesin, and platelet-derived growth factor. 146 24

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.
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PMID:Interconversion of three differentially modified and assembled forms of BiP. 174 Jan 16

Starvation of Mouse hepatoma cells for essential amino acids or glucose results in the mono-ADP-ribosylation of the 78 kDa glucose-regulated protein, GRP78. Here we show that the ADP-ribosylated and non-ADP-ribosylated forms of GRP78 are interconvertible during tryptophan starvation and refeeding. In addition, the ADP-ribosylation of GRP78 was shown to be reversible even during nutritional stress. The overexpressed pool of non-ADP-ribosylated GRP78 synthesized during tunicamycin treatment was available for ADP-ribosylation during subsequent amino acid starvation, especially in the absence of tunicamycin. The reversible ADP-ribosylation of GRP78 could be part of a metabolic control mechanism in operation during nutritional stress.
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PMID:Reversible ADP-ribosylation of the 78 kDa glucose-regulated protein. 226 6

Immunoglobulin heavy chain binding protein (BiP/GRP78) is a resident endoplasmic reticulum protein that binds tightly to a number of incompletely assembled or aberrant proteins. BiP also binds ATP and can be purified by ATP affinity chromatography. Here we show that an ATPase activity co-purifies with BiP prepared from canine pancreas. The BiP-associated ATPase has a high affinity for ATP but a low turnover number, suggesting a regulatory, rather than an enzymatic role. We also show that submicromolar levels of ATP or ADP decrease the rate of adsorption of [125I]BiP to nitrocellulose filters coated with protein or non-ionic detergents. In contrast, micromolar levels of AMP increase the rate of adsorption. Furthermore, ATP and ADP decrease the susceptibility of BiP to proteolytic degradation, whereas AMP was found to enhance degradation slightly. Adenine nucleotides may therefore induce or stabilize different conformations of BiP even when ATP hydrolysis does not occur.
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PMID:Interaction of heavy chain binding protein (BiP/GRP78) with adenine nucleotides. 267 May 54

BiP is a member of the Hsp70 heat shock protein family found in the lumen of the endoplasmic reticulum, that binds to a variety of proteins destined to be secreted. Substance P (SP) has been used as a model peptide to study the interaction of BiP with protein substrates. SP stimulates BiP ATPase activity and forms a stable complex with BiP that is dissociated in the presence of levels of ATP > 50 microM. At lower concentrations of ATP, the SP remains bound to BiP, and the results are consistent with the view that a BiP-ATP complex is initially formed that reacts with SP to form a ternary complex, SP-BiP-ATP. Hydrolysis of ATP in this complex yields a SP-BiP-ADP complex. An exchange of ATP with ADP bound to BiP has also been demonstrated, and the results suggest that the interactions of BiP with ATP resemble those seen with GTP-binding proteins and GTP.
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PMID:Similarity of nucleotide interactions of BiP and GTP-binding proteins. 752 51

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

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.
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PMID:In vitro dissociation of BiP-peptide complexes requires a conformational change in BiP after ATP binding but does not require ATP hydrolysis. 759 94

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.
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PMID:ADP-ribosylation of the molecular chaperone GRP78/BiP. 789 57

We have developed two independent assays to study the integration, folding, and intracellular transport of the polytopic plasma membrane H(+)-ATPase in yeast. To follow folding, controlled trypsinolysis was used to distinguish between the E1 conformation of the ATPase (favored in the presence of ADP) and the E2 conformation (favored in the presence of vanadate). By this criterion, wild-type ATPase appears to recognize its ligands and assume distinct conformations within a short time after its biosynthesis. To follow intracellular transport, we have exploited the fact that export of newly synthesized ATPase from the endoplasmic reticulum is accompanied by kinase-mediated phosphorylation, leading to a shift in electrophoretic mobility. Because proper folding is required for transport from the endoplasmic reticulum, the mobility shift also serves as a convenient bioassay for correct folding. As a first step toward identifying cell components important in folding of the nascent ATPase, we have used the dual assays to examine the role of KAR2, encoding the yeast homolog of immunoglobulin heavy chain binding protein/78-kDa glucose-regulated protein, and SEC65, encoding a subunit of the yeast signal recognition particle. Although mutation of KAR2 caused defective translocation of several secretory precursors into the endoplasmic reticulum lumen, ATPase folding and intracellular transport were unperturbed. By contrast, in a sec65 mutant, the folding and intracellular transport of newly synthesized ATPase were delayed. Our data suggest that conformational maturation of the ATPase is a rapid process in wild-type cells and that membrane integration mediated by signal recognition peptide is important for the proper folding of this polytopic protein.
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PMID:Folding and intracellular transport of the yeast plasma-membrane H(+)-ATPase: effects of mutations in KAR2 and SEC65. 851 33


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