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

The folding of influenza hemagglutinin (HA0) in the ER was analyzed in tissue culture cells by following the formation of intrachain disulfides after short (1 min) radioactive pulses. While some disulfide bonds were already formed on the nascent chains, the subunits acquired their final disulfide composition and antigenic epitopes posttranslationally. Two posttranslational folding intermediates were identified. In CHO cells constitutively expressing HA0, mature HA0 subunits were formed with a half time of 3 min and their folding reached completion at 22 min. The rate of folding was highly dependent on cell type and expression system, and thus regulated by factors other than the sequence of the protein alone. Exposure of cells to stress conditions increased the level of glucose regulated proteins, including BiP, and decreased the folding rate. The efficiency of folding and subsequent trimerization was not dependent on the rate of translation, nor on temperature between 37 and 15 degrees C; however, the rates of folding and trimerization decreased with decreasing temperature. Whereas the rate of folding was independent of expression level, trimerization was accelerated at higher levels of expression.
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PMID:Folding of influenza hemagglutinin in the endoplasmic reticulum. 165 Mar 70

GRP78/BiP resides in the lumen of the endoplasmic reticulum (ER), a major site of Ca2+ sequestration and early protein processing. Agents, such as ionophore A23187, that mobilize sequestered ER Ca2+ suppress translational initiation within minutes and induce GRP78 within 1-3 h accompanied by development of translational tolerance to the inhibitor. Accommodation is prevented by actinomycin D and reduced by antisense oligonucleotides directed against GRP78 mRNA. In GH3 cells, optimal induction of GRP78 and translational accommodation depended on cAMP elevation and phorbol ester. GRP78 mRNA was induced 3-6-fold with A23187 alone as compared with 12-20-fold with ionophore plus cAMP-elevating agent and phorbol ester, but was not markedly induced without A23187. GRP78 gene transcription in nuclei isolated from A23187-treated cells was increased 2-4-fold by cAMP and phorbol ester. A nucleotide sequence homologous to the cAMP-responsive element consensus potentially exists in the promoter region of the GRP78 gene. GRP78 mRNA in ionophore-treated cells was largely associated with mono- and polysomal fractions rather than ribonuclear protein particles, a distribution different from actin and tubulin mRNAs. While polysomal content increased in cells undergoing translational recovery, cAMP and phorbol esters did not affect GRP78 mRNA stability. Translational accommodation in ionophore-treated GH3 cells is proposed to involve enhanced transcription of GRP78 mRNA promoted by cAMP/phorbol ester in conjunction with preferential polysomal loading of the message.
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PMID:Stimulation of GRP78 gene transcription by phorbol ester and cAMP in GH3 pituitary cells. The accommodation of protein synthesis to chronic deprivation of intracellular sequestered calcium. 165 95

Parallel experiments in living cells and in vitro were undertaken to characterize the mechanism by which misfolded and unassembled glycoproteins are retained in the ER. A thermoreversible folding mutant of vesicular stomatitis virus (VSV) G protein called ts045 was analyzed. At 39 degrees C, newly synthesized G failed to fold correctly according to several criteria: intrachain disulfide bonds were incomplete; the B2 epitope was absent; and the protein was associated with immunoglobulin heavy chain binding protein (BiP), a heat shock-related, ER protein. When the temperature was lowered to 32 degrees C, these properties were reversed, and the protein was transported to the cell surface. Upon the shift up from 32 degrees C back to 39 degrees C, G protein in the ER returned to the misfolded form and was retained, while the protein that had reached a pre-Golgi compartment or beyond was thermostable and remained transport competent. The misfolding reaction could be reconstituted in a cell free system using ts045 virus particles and protein extracts from microsomes. Taken together, the results showed that ER is unique among the organelles of the secretory pathway in containing specific factors capable of misfolding G protein at the nonpermissive temperature and thus participating in its retention.
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PMID:Quality control in the endoplasmic reticulum: folding and misfolding of vesicular stomatitis virus G protein in cells and in vitro. 169 99

During the first cell cycle, the vegetal cortex of the fertilized frog egg is translocated over the cytoplasm. This process of cortical rotation creates regional cytoplasmic differences important in later development, and appears to involve an array of aligned microtubules that forms transiently beneath the vegetal cortex. We have investigated how these microtubules might be involved in generating movement by analyzing isolated cortices and sections of Xenopus laevis and Rana pipiens eggs. First, the polarity of the cortical microtubules was determined using the "hook" assay. Almost all microtubules had their plus ends pointing in the direction of cortical rotation. Secondly, the association of microtubules with other cytoplasmic elements was examined. Immunofluorescence revealed that cytokeratin filaments coalign with the microtubules. The timing of their appearance and their position on the cytoplasmic side of the microtubules suggested that they are not involved directly in generating movement. ER was visualized with the dye DiIC16(3) and by immunofluorescence with anti-BiP (Bole, D. G., L. M. Hendershot, and J. F. Kearney, 1986. J. Cell Biol. 102:1558-1566). One layer of ER was found closely underlying the plasma membrane at all times. An additional, deeper layer formed in association with the microtubules of the array. Antibodies to sea urchin kinesin (Ingold, A. L., S. A. Cohn, and J. M. Scholey. 1988. J. Cell Biol. 107:2657-2667) detected antigens associated with both the ER and microtubules. On immunoblots they recognized microtubule associated polypeptide(s) of approximately 115 kD from Xenopus eggs. These observations are consistent with a role for kinesin in creating movement between the microtubules and ER, which leads in turn to the cortical rotation.
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PMID:Evidence for the involvement of microtubules, ER, and kinesin in the cortical rotation of fertilized frog eggs. 171 12

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

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

Recombinant human insulin-like growth factor-1 (IGF1) secreted from yeast contains only 10-15% of the active monomer. A majority of the IGF1-like molecules are disulfide bonded dimers. These dimers are not formed in an IGF1 mutant where Lys27 has been replaced by glutamic acid. However, increased levels of secreted BiP (the yeast KAR2 gene product) are seen in cells expressing the mutant. These results imply that by preventing ionic interactions between two IGF1 molecules, intermolecular disulfide bonds do not form in yeast, and that in the mutant there is a structural change which induces BiP, allowing its secretion.
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PMID:A Lys27-to-Glu27 mutation in the human insulin-like growth factor-1 prevents disulfide linked dimerization and allows secretion of BiP when expressed in yeast. 175 63

A hybrid protein consisting of the Escherichia coli lipoprotein signal sequence attached to the mature sequence of the B subunit of heat-labile enterotoxin (Lipo-EtxB) was expressed in yeast and E. coli. Analyses of cell lysates from Saccharomyces cerevisiae and E. coli expressing the protein revealed that both organisms were able to assemble Lipo-EtxB into oligomers that were (i) stable in the presence of sodium dodecyl sulphate, (ii) resistant to proteinase K degradation, and (iii) able to bind to GM1-ganglioside receptors. Each of these properties are characteristic of the wild-type B subunit pentamer produced in E. coli. Assembly of Lipo-EtxB was found to be unaffected in a sec18 mutant of S. cerevisiae, which possesses a temperature-sensitive defect in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, but was found not to assemble in a sec53 mutant, which causes the misfolding of proteins targeted to the ER. A kar2-1 mutation with a defect in the yeast homologue of BiP caused an 18-fold reduction in Lipo-EtxB assembly at the non-permissive temperature in S. cerevisiae. However, introduction of the wild-type KAR2 gene on a plasmid into the kar2-1 mutant completely suppressed the inhibition of Lipo-EtxB assembly. This provides the first evidence that KAR2 facilitates the assembly of an oligomeric protein in yeast and thus implicates KAR2 as a 'molecular chaperone'. The possible mechanisms of enterotoxoid assembly in E. coli and S. cerevisiae are discussed.
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PMID:Targeting and assembly of an oligomeric bacterial enterotoxoid in the endoplasmic reticulum of Saccharomyces cerevisiae. 177 57

cDNAs of fibrinogen A alpha and gamma chains were individually subcloned into a eukaryotic expression vector by using the polymerase chain reaction. Triple cotransfection into COS cells of the two plasmids together with a B beta chain expression plasmid, constructed as described previously (Danishefsky, K.J., Hartwig, R., Banerjee, D., and Redman, C. (1990) Biochim. Biophys. Acta 1048, 202-208), resulted in the secretion of complete fibrinogen into the media and the formation of four additional intracellular complexes which we also showed to be present in the hepatocyte cell line Hep 3B. The complexes, which have Mr = 232, 150, 135, and 128 (x 10(-3) conform with the Mr expected for A alpha B beta gamma 2, B beta gamma 2 and gamma 3, respectively. A A mechanism of assembly is proposed based on the assumption that all these complexes are precursors of complete fibrinogen. Each of the expressed fibrinogen chains in transfected COS cells interacts noncovalently with binding protein (BiP, GRP 78), but not to the same extent; gamma chain binds less BiP than the A alpha and B beta chains. Assembly of fibrinogen is not absolutely required for its secretion. In addition to complete fibrinogen, the conditioned media of hepatocytes and of transfected COS cells contained free A alpha, free gamma, and two of the above-mentioned complexes, A alpha gamma 2 and A alpha B beta gamma 2.
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PMID:Studies on the assembly and secretion of fibrinogen. 182 7

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


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