Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P11021 (BiP)
 2,049 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previously we found that in rat exocrine pancreatic cells, protein disulfide-isomerase (PDI), one of the major resident proteins in the lumen of the endoplasmic reticulum (ER) of many cells, is localized not only in the ER but also in the Golgi apparatus, secretory granules, plasma membranes, and even in the glandular lumens, despite possessing the ER retention signal KDEL (Lys-Asp-Glu-Leu) at the carboxyl terminus. In this report, we examined whether other ER luminal proteins bearing the KDEL signal at their C-termini, such as BiP/GRP78 and endoplasmin/GRP94 are also exported from the ER. We prepared two kinds of affinity-purified polyclonal antibodies; one against a synthetic peptide with 12 amino acids which is identical to the carboxyl terminus of BiP and another against purified endoplasmin. Immunoblot analysis using these two antibodies showed that BiP and endoplasmin exist in both the plasma membrane and the microsomal fractions, similar to the intracellular distribution of PDI in rat exocrine pancreas. The ratios of the amount of the three proteins in the two fractions, however, were variable, suggesting that the KDEL-bearing proteins such as PDI, BiP, and endoplasmin are exported from the ER with different efficiencies. Postembedding protein A-immunogold electron microscopy revealed that endoplasmin was exported from the ER and secreted to the extracellular space. The secretion of PDI in rat pancreatic lobules was inhibited by Brefeldin A (BFA) and by guanidino acid esters (FOY-305), which are known to be the inhibitors of the intracellular transport. Taken together with the previous immunogold electron microscopic analyses by Akagi et al. (1988), it is strongly suggested that in rat exocrine pancreatic cells PDI and the other KDEL-bearing proteins found in the extracellular space were not artificially released by cell damage during incubation but were secreted via the normal secretory pathway.
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PMID:Heavy chain binding protein (BiP/GRP78) and endoplasmin are exported from the endoplasmic reticulum in rat exocrine pancreatic cells, similar to protein disulfide-isomerase. 131 87

The C-terminal region of the chaperone-like pro-sequence (py) of yeast carboxypeptidase Y (CPY) is suggested to be crucial for the folding of mature CPY. In order to study the influence of hydrophobic residues in this domain, a set of mutations have been introduced in py. Unexpectedly, only small amounts of CPY precursors are expressed when Leu108, at the C-terminal end of py, is substituted for polar residues Lys, Arg or Asp. In contrast, substitution with hydrophobic residues Val, Ile or Ala permit normal expression. Interestingly, the poorly expressed molecules are coreglycosylated, implying that they have failed to leave the endoplasmic reticulum (ER). The ER-retained molecules caused an induction in the levels of BiP, signifying that polar substitutions at position 108 of pre-py-CPY induce misfolding. Quite surprisingly, a reporter gene, linked to concatamerized unfolded-protein-response elements, reveals that py-mediated misfolding of CPY is not really identical in all mutants. This shows that a simple transcriptional assay can assess the subtleties of pro-sequence mediated protein folding in an eukaryotic cell.
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PMID:The unfolded-protein-response element discriminates misfolding induced by different mutant pro-sequences of yeast carboxypeptidase Y. 772 51

Members of the 70-kD heat shock protein family have been found in all free-living organisms investigated and in major compartments of eukaryotic cells where they are essential to a wide range of functions, including protein folding and targeting. We have isolated a mitochondrial homolog (mtHSP70) from rat liver using ATP agarose affinity chromatography. Its identity was confirmed on the basis of immunological analysis and Ca(2+)-dependent autophosphorylation. Using protein sequence obtained from the amino termius and nine endo Lys-C peptide fragments, we have employed oligonucleotides to isolate a full-length cDNA clone. The open reading frame encodes a protein of 679 amino acids and calculated M(r) 73,913 daltons. The sequence has a high degree of identity with other members of the HSP70 family, including Escherichia coli DnaK (51%), Saccharomyces cerevisiae SSC1p (65%), the constitutive cytosolic HSP70 from rat, HSC70 (46%), and the rat endoplasmic reticulum isoform, BiP, (49%). The cDNA encodes a precursor protein with a 46-amino-acid signal peptide that is absent from the protein isolated from rat liver. The protein also shows a high degree of identity (98%) with a protein isolated from mouse and human tissues (PBP74, Domanico et al., 1993; mortalin, Wadhwa et al., 1993a; CSA, Michikawa et al., 1993a); however, the intracellular localization of these proteins is uncertain. We show that the precursor of mtHSP70 is efficiently imported into isolated mitochondria from rat liver and processed from 74 kD to the mature 69-kD protein.
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PMID:cDNA cloning and efficient mitochondrial import of pre-mtHSP70 from rat liver. 781 87

We have studied the binding of synthetic peptides to three hsp70 molecular chaperones, DnaK, BiP, and hsc70, as a model for the interaction of hsp70 proteins with unfolded regions of target polypeptides. We measured the ability of 53 peptides to inhibit the formation of complexes between the hsp70 proteins and denatured lactalbumin. Peptides that bound with highest affinity to all three hsp70 proteins contained stretches of at least 7 residues that included large hydrophobic and basic amino acids, but few or no acidic residues. Amino acid substitutions within one heptameric peptide showed that an important feature for its binding to all three chaperones was a large hydrophobic residue in position 4, while specificity differences between the chaperones were revealed by substitutions at positions 2 and 6. Such specificity differences were frequently observed with other peptides, the most extreme example being a peptide rich in basic residues that bound with high affinity to DnaK, intermediate affinity to hsc70, and negligible affinity to BiP. Substitution of a lysine residue at position 2 in this peptide by tyrosine abolished the specificity difference by increasing the affinities of the DnaK and hsc70 proteins 5- and 20-fold, respectively, and that of BiP by greater than 2 orders of magnitude. Thus, hsp70 proteins can exhibit common or exclusive binding specificities, depending on the peptide sequence.
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PMID:Common and divergent peptide binding specificities of hsp70 molecular chaperones. 798 63

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

A G273D mutation immediately proximal to the first calcium binding domain of platelet GPIIb impairs the export of GPIIb-IIIa heterodimers to the platelet surface. To examine how this mutation might alter the structure of GPIIb, G273 was replaced by other amino acids and the resulting mutants were coexpressed with GPIIIa in COS-1 cells. Although replacement with Ala or Val had no effect on GPIIb-IIIa expression, replacement with Glu, Lys, Pro, or Asn caused intracellular retention of GPIIb-IIIa. Concurrently, the consequences of these replacements were examined by comparative modeling by introducing them into the analogous position of the first helix-loop-helix (HLH) motif of calmodulin, based on homology between the calcium binding domains of GPIIb and the calcium binding loops of HLH-containing proteins. The modeling revealed that as the side chain of the introduced amino acid increased in size, it progressively interfered with hydrophobic interactions between the incoming and outgoing helices of the motif. To test whether this observation also applies to GPIIb, V286, located immediately distal to the first GPIIb calcium binding domain, was replaced by Asp and Phe. Expression of these mutants in COS-1 cells also resulted in the intracellular retention of GPIIb-IIIa, suggesting that interactions between sequences that flank the first calcium binding domain of GPIIb affect its folding. Finally, the endoplasmic reticulum chaperone BiP was detected in immunoprecipitates of GPIIb-IIIa containing GPIIb with Ala, Val, Lys, or Pro, but not Gly, at position 273. This suggests that although BiP binding is a sensitive indication of the fidelity of GPIIb-IIIa folding, it is not sufficient to account for the intracellular retention of the heterodimer.
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PMID:Effect of mutagenesis of GPIIb amino acid 273 on the expression and conformation of the platelet integrin GPIIb-IIIa. 891 16

A constitutively active form of At-ACA8, a plasma membrane Ca(2+)-ATPase from Arabidopsis thaliana (L.) Heynh., from which the first 74 amino acids containing the calmodulin-binding domain (delta74- At-ACA8) had been deleted, was expressed in Saccharomyces cerevisiae strain K616, which lacks the main endogenous active Ca(2+) transport systems. Delta74- At-ACA8 complemented the K616 phenotype, making it able to grow in a calcium-depleted medium. Delta74- At-ACA8 protein, which co-migrated with the endoplasmic reticulum marker BiP in a sucrose-density gradient, catalyzed MgATP-dependent Ca(2+) uptake and Ca(2+)-dependent MgATP hydrolysis, and retained the biochemical characteristics of the native plant plasma membrane Ca(2+)-ATPase (low specificity for nucleoside triphosphate, high sensitivity to inhibition by the fluorescein derivatives erythrosin B and eosin Y), thus confirming that it is correctly folded and functional. Substitution of the (794)HE residues (numbers refer to full-length At-ACA8) following the highly conserved TGDG(TV)NDP(AS)L motif in the cytoplasmic headpiece with two lysine residues generated an hyperactive protein, with a catalytic activity 2-fold higher than that of delta74- At-ACA8. The (794)HE-->KK mutant was also about 6-fold more sensitive than delta74- At-ACA8 to inhibition by vanadate, indicating that the mutation determines an increase in the proportion of enzyme in the E(2) state during the catalytic cycle.
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PMID:Functional expression in yeast of an N-deleted form of At-ACA8, a plasma membrane Ca(2+)-ATPase of Arabidopsis thaliana, and characterization of a hyperactive mutant. 1464 21

Protein targeting mechanisms in flagellated protozoan parasites have received considerable interest because of a huge bias in these organisms toward the glycosylphosphatidylinositol anchor as a mechanism for the membrane attachment of cell surface macromolecules. In this study, the trafficking of invariant surface glycoprotein 65 (ISG65), a family of type I transmembrane proteins, was examined. Analysis of the C-terminal domains of ISG65 family members demonstrated a high level of conservation and, in particular, the presence of three lysine residues contained within the cytoplasmic tails of all ISG65s. ISG65 was expressed on the cell surface, in agreement with earlier work, but an intracellular pool of ISG65 was also detected within a Rab5A early endosome. Transplantation of the C-terminal 74 amino acids of ISG65 (encompassing the 23 C-terminal residues of the extracellular domain, the transmembrane peptide, and the cytoplasmic domain) onto the N-terminal domain of BiP (BiPN) was sufficient to target the chimera to the same internal compartments as native ISG65. Further, site-directed mutagenesis indicated that the cytoplasmic tail was required for endoplasmic reticulum exit and that at least two of the cytoplasmic domain lysine residues are needed for endosomal targeting, as removal of all three led to surface expression. Kinetic measurements demonstrate that the BiPN fusion protein (containing the ISG65 C terminus) has a short half-life, indicating rapid turnover. In contrast, BiPN fusion proteins containing a glycosylphosphatidylinositol anchor instead of the ISG65 C-terminal region are stably expressed on the surface, confirming the requirement for the ISG65 sequence for endosomal targeting. We suggest that the lack of surface expression of the BiPN-ISG65 fusion protein is likely due to more efficient internalization compared with ISG65. Taken together, these data demonstrate the presence of a lysine-dependent endocytosis signal in the ISG65 family.
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PMID:Cytoplasmic targeting signals in transmembrane invariant surface glycoproteins of trypanosomes. 1534 36

In this study, we investigated the antigenic structures and maturation of some C-terminal-deficient derivatives of rabies virus glycoprotein (G). The Gs protein, a soluble form of G protein shed from infected cells, displayed antigenicity to most of our conformational epitope-specific anti-G mAbs, but took the 1-30-44 epitope-deficient conformation (termed G(C) form). (The 1-30-44 epitope was acid-sensitive and dependent on two separate regions, the Lys-202-containing and Asn-336-containing regions; Kankanamge et al., Microbiol. Immunol., 47: 507-519). Intact G proteins took the 1-30-44 epitope-positive form (referred to as G(B) form) on the cell surface, but not inside the cell. A deletion mutant G(1-429) (termed GDeltaTC), lacking the transmembrane (TM) and cytoplasmic domains, was shown to be accumulated in the rough endoplasmic reticulum (rER) with BiP and did not seem to be shed. Another C-terminal-deficient mutant G(1-462) (termed CT1) was deprived of the whole cytoplasmic domain except for a basic amino acid left at the C-terminus, but was transported to the cell surface, where it showed pH-dependent cell fusion activity and almost full antigenicity to most of the anti-G mAbs with the exception of very weak antigenicity to mAb #1-30-44. No Gs protein could be detected in the CT1-producing cultures. Based on these results, we think that the cytoplasmic domain was not necessary for the G protein to be transported to the cell surface, but was necessary to keep its 1-30-44 epitope-positive G(B) conformation. Gs proteins might have lost the C-terminal regions during the maturation process after being exported from the rER.
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PMID:Further studies on the soluble form (gs) of rabies virus glycoprotein (g): molecular structure of gs protein and possible mechanism of the shedding. 1611 2

K28 is a viral A/B toxin that traverses eukaryotic cells by endocytosis and retrograde transport through the secretory pathway. Here we show that toxin retrotranslocation from the endoplasmic reticulum (ER) requires Kar2p/BiP, Pdi1p, Scj1p, Jem1p, and proper maintenance of Ca(2+) homeostasis. Neither cytosolic chaperones nor Cdc48p/Ufd1p/Npl4p complex components or proteasome activity are required for ER exit, indicating that K28 retrotranslocation is mechanistically different from classical ER-associated protein degradation (ERAD). We demonstrate that K28 exits the ER in a heterodimeric but unfolded conformation and dissociates into its subunits as it emerges into the cytosol where beta is ubiquitinated and degraded. ER export and in vivo toxicity were not affected in a lysine-free K28 variant nor under conditions when ubiquitination and proteasome activity was blocked. In contrast, toxin uptake from the plasma membrane required Ubc4p (E2) and Rsp5p (E3) and intoxicated ubc4 and rsp5 mutants accumulate K28 at the cell surface incapable of toxin internalization. We propose a model in which ubiquitination is involved in the endocytic pathway of the toxin, while ER-to-cytosol retrotranslocation is independent of ubiquitination, ERAD and proteasome activity.
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PMID:Retrotranslocation of a viral A/B toxin from the yeast endoplasmic reticulum is independent of ubiquitination and ERAD. 1700 44


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