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

Hep G2 cells produce surplus A alpha and gamma fibrinogen chains. These excess chains, which are not secreted, exist primarily as free gamma chains and as an A alpha-gamma complex. We have determined the intracellular location and the degradative fate of these polypeptides by treatment with endoglycosidase-H and by inhibiting lysosomal enzyme activity, using NH4Cl, chloroquine, and leupeptin. Free gamma chain and the gamma component of A alpha-gamma are both cleaved by endoglycosidase-H, indicating that the gamma chains accumulate in a pre-Golgi compartment. Lysosomal enzyme inhibitors did not affect the disappearance of free gamma chains but inhibited A alpha-gamma by 50%, suggesting that A alpha-gamma is degraded in lysosomes. The degradative fate of individual chains was determined in transfected COS cells which express but do not secrete single chains. Leupeptin did not affect B beta chain degradation, had very little affect on gamma chain, but markedly inhibited A alpha chain degradation. Antibody to immunoglobulin heavy chain-binding protein (GRP 78) co-immunoprecipitated B beta but not A alpha or gamma chains. Preferential binding of heavy chain-binding protein to B beta was also noted in Hep G2 cells and in chicken hepatocytes. Taken together these studies indicate that B beta and gamma chains are degraded in the endoplasmic reticulum, but only B beta is bound to BiP. By contrast A alpha chains and the A alpha-gamma complex undergo lysosomal degradation.
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PMID:Assembly and secretion of fibrinogen. Degradation of individual chains. 142 62

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

The intracellular fates of membrane and secretory immunoglobulin heavy chains were examined in a pre-B cell line that has switched to the gamma isotype. The membrane form of the heavy chain (gamma m) was rapidly degraded while the secretory form (gamma s) was retained intracellularly in association with BiP. The degradation of gamma m could not be inhibited by ammonium chloride, chloroquine, or monensin suggesting that it occurred in a nonlysosomal compartment. The inability to detect any Endo H-resistant form of gamma m before its degradation suggested that degradation occurs before entry into the Golgi compartment. Degradation of gamma m could be inhibited by incubation at 24 degrees C. In a derivative of this cell line expressing a transfected kappa gene, gamma s formed disulfide linked tetramers with kappa and was secreted, while gamma m, although associated with kappa, continued to be rapidly degraded. These observations suggest that membrane and secretory heavy chain proteins are retained by distinct intracellular mechanisms. Although masking of the CH1 domain abrogates gamma s retention, this domain does not influence the intracellular fate of gamma m.
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PMID:Distinct intracellular fates of membrane and secretory immunoglobulin heavy chains in a pre-B cell line. 191 56

The mechanism by which endoplasmic reticulum (ER) stress proteins are induced by the accumulation of incompletely assembled or malfolded proteins in the ER is poorly understood. The 78-kDa glucose-regulated protein (BiP), one of the ER stress proteins, has often been detected in stable complexes with these accumulated proteins. We have transfected COS cells with an immunoglobulin (Ig) mu heavy chain expression plasmid. Expressed mu-chain accumulated in the cells and formed stable complexes with BiP. As a result, the synthesis of three ER stress proteins, BiP, the 94-kDa glucose-regulated protein (GRP94/ERp99), and ERp72, was increased as were their mRNA levels. In addition, the degradation rate of BiP was increased, possibly because of its interaction with mu-chain. Cotransfection of the mu-chain plasmid with an Ig lambda light chain expression plasmid resulted in the appearance of mu-chain in the media in a covalent complex with lambda-chain. An intracellular consequence of this was a reduction in the levels of BiP.mu-chain complex, and a diminished stimulation in the synthesis of the ER stress proteins. These results suggest that the BiP.mu-chain complex in the ER may be involved in the signaling pathway for the induction of ER stress proteins and may represent one regulatory mechanism operating in differentiating B-lymphocytes.
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PMID:Regulation of endoplasmic reticulum stress proteins in COS cells transfected with immunoglobulin mu heavy chain cDNA. 193 4

Isolation and biochemical analysis of the components involved in protein translocation into the rough endoplasmic reticulum (ER) requires starting material highly enriched in membranes derived from this organelle. We have chosen to study the yeast Saccharomyces cerevisiae in order to profit from the ease of genetic manipulation. To date, however, no efficient scheme has been devised that allows the purification of functional rough ER-derived membranes from yeast, largely because proteins have yet to be identified that are rough ER-specific. In the experiments described here, we expressed the human rough ER marker ribophorin I to facilitate the analysis of subcellular fractionation. We found that the endoplasmic reticulum of yeast could be separated into two distinct domains by fractionation on continuous sucrose gradients. This procedure revealed a bimodal distribution of ER markers. The yeast homologue of the heavy chain-binding protein, BiP (encoded by the KAR2 gene), and the product of the SEC62 gene were present in two fractions having equilibrium densities of 1.146 and 1.192 g/ml, respectively. In contrast, our analysis showed that preprotein translocation activity and retention of the rough ER-specific protein ribophorin I were specific only to the membrane fraction with an equilibrium density of 1.192 g/ml. To prepare fractions highly enriched in translocation competent rough ER-derived membranes for analysis, we developed a density shift fractionation scheme that optimizes the purity of membranes containing human ribophorin I. Membranes obtained by this method were found to possess the majority of the appropriate functional markers, including ATP-independent preprotein binding, ribosome binding, and post-translational translocation. Mitochondria, the major contaminant of the 1.192 g/ml fraction, were significantly depleted in density-shifted membrane populations.
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PMID:Purification and functional characterization of membranes derived from the rough endoplasmic reticulum of Saccharomyces cerevisiae. 207 10

Immunoglobulin light chains are usually secreted from cells when they are synthesized alone or in molar excess of heavy chains, but, there have been reports of nonsecreted light chains. We wished to determine whether immunoglobulin heavy chain binding protein (BiP), which blocks the transport of free heavy chains, might be responsible for the lack of secretion of some light chains. In two murine lymphoid cell lines that synthesize but do not secrete immunoglobulin light chains, the free light chain polymers were found bound to BiP. Examination of 20 other cell lines and hybridomas failed to disclose any cells synthesizing free or excess light chains that associated with BiP, in all cases the free light chains were secreted as dimers. Despite their association with BiP and their blocked secretion, the aberrant light chains could combine with heavy chains and could be secreted as intact Ig molecules. Thus, while light chains do not usually express signals which allow them to bind to BiP, it appears that such signals can be expressed on certain light chains, resulting in their combination with BiP and blocked secretion. When single chain mutant cell lines are isolated from parental lines producing both heavy and light chains, they are almost always light chain producers suggesting that free heavy chains are much more toxic than free light chains. In both PC700 and P3X63Ag cells, however, clones that have lost either heavy chains or transport-defective light chains are present at the same frequency. Our findings that the light chains in both of these lines are associated with BiP raise the possibility that BiP actually contributes to heavy chain toxicity instead of preventing it.
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PMID:Association of transport-defective light chains with immunoglobulin heavy chain binding protein. 211 93

To investigate the function of heavy chain binding protein (BiP, GRP 78) in the endoplasmic reticulum, we have characterized its interaction with a model plasma membrane glycoprotein, the G protein of vesicular stomatitis virus. We used a panel of well characterized mutant G proteins and immunoprecipitation with anti-BiP antibodies to determine if BiP interacted with newly synthesized G protein and/or mutant G proteins retained in the endoplasmic reticulum. We made three major observations: 1) BiP bound transiently to folding intermediates of wild-type G protein which were incompletely disulfide-bonded; 2) BiP did not bind stably to all mutant G proteins which remain in the endoplasmic reticulum; and 3) BiP bound stably only to mutant G proteins which do not form correct intrachain disulfide bonds.
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PMID:Heavy chain binding protein recognizes incompletely disulfide-bonded forms of vesicular stomatitis virus G protein. 215 12

During the process of folding and assembly of antibody molecules in the endoplasmic reticulum, immunoglobulin heavy and light chains associate transiently with BiP, a resident endoplasmic reticulum protein that is a member of the Hsp70 family of molecular chaperones. BiP is thought to recognize unfolded or unassembled polypeptides by binding extended sequences of approximately seven amino acids that include bulky hydrophobic residues not normally exposed on the surface of native proteins. We used a computer algorithm developed to predict BiP binding sites within protein primary sequences to identify sites within immunoglobulin chains that might mediate their association with BiP. Very few of the sequential heptapeptides in the heavy or light chain sequences were potential BiP binding sites. Analysis of the ability of synthetic heptapeptides corresponding to 24 potential sites in heavy chains to stimulate the ATPase activity of BiP indicated that at least half of them were authentic BiP binding sequences. These sequences were not confined to a single domain of the heavy chain but were distributed within both the VH and CH domains. Interestingly, when the BiP binding sequences were mapped onto the three-dimensional structure of the Fd antibody fragment, the majority involve residues that participate in contact sites between the heavy and light chains. Therefore, we suggest that in vivo BiP chaperones the folding and assembly of antibody molecules by binding to hydrophobic surface regions on the isolated immunoglobulin chains that subsequently participate in interchain contacts.
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PMID:BiP binding sequences in antibodies. 749 21

High-level gene expression does not always lead to corresponding high-level secretion of heterologous proteins in yeast. The rate-limiting step in many cases has been shown to exit from the endoplasmic reticulum (ER). Within the ER, the correct folding of secreted proteins is required for export competence; hence, the cellular proteins involved in these events are likely to be important for efficient secretion. We have found that the extractable levels of two ER-resident proteins involved in folding--heavy chain binding protein (BiP) and protein disulfide isomerase (PDI)--are significantly reduced by prolonged constitutive overexpression of human granulocyte colony stimulating factor (GCSF), human erythropoietin, or Schizosaccharomyces pombe acid phosphatase. However, the rate of BiP synthesis measured in pulse--chase radiolabeling experiments is not reduced by GCSF overexpression, and galactose-directed transcription of the BiP gene does not restore normal BiP protein levels once they have been depleted. The observed loss of lumenal resident proteins, either by proteolysis or irreversible aggregation, is expected to contribute significantly to the inefficiency of foreign protein secretion in yeast.
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PMID:Constitutive overexpression of secreted heterologous proteins decreases extractable BiP and protein disulfide isomerase levels in Saccharomyces cerevisiae. 753 23

Two pathways operate to target newly-synthesised proteins to the endoplasmic reticulum. In one, the signal recognition particle attaches to the signal sequences of nascent chains on ribosomes and slows or stops translation until contact is made with the docking protein at the membrane. The second operates via molecular chaperons. The pathways converge at the level of a 43 kDa signal binding protein integrated into the membrane, where translocation through a proteinaceous pore is initiated. In the lumen, proteins fold and disulphide formation is catalysed by the enzyme protein disulphide isomerase. The heavy chain binding protein may attach to unassembled or unfolded proteins and prevent their exit from the ER to the Golgi. Cholecystokinin (CCK) treatment increases the biosynthesis and secretion of pancreatic proteins, increases the levels of PDI and the 43 kDa binding protein, and reduces levels of BiP. These proteins may be possible targets for genetic manipulation to improve processing of heterologous proteins from cultured mammalian cells.
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PMID:Changes in levels of pancreatic endoplasmic reticulum proteins that function in translocation and maturation of secretory proteins in response to cholecystokinin. 776 25


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