Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To examine how binding of BiP (a molecular chaperone of the hsp70 family that resides in the endoplasmic reticulum) influences the conformational maturation of thyroglobulin (Tg, the precursor for thyroid hormone synthesis), we have developed a system of recombinant Tg stably expressed in wild-type Chinese hamster ovary (CHO) cells and CHO-B cells genetically manipulated for selectively increased BiP expression. The elevation of immunoreactive BiP in CHO-B cells is comparable to that seen during the unfolded protein response in the thyrocytes of certain human patients and animals suffering from congenital hypothyroid goiter with defective Tg. However, in CHO-B cells, we expressed Tg containing no mutations that induce misfolding (i.e. no unfolded protein response), so that levels of all other endoplasmic reticulum chaperones were normal. Increased availability of BiP did not accelerate Tg secretion; rather, the export of newly synthesized Tg was delayed. Tg detained intracellularly was concentrated in the endoplasmic reticulum. By coimmunoprecipitation, BiP exhibited enhanced binding to Tg in CHO-B cells. Moreover, two-dimensional gel analysis showed that BiP associated especially well with intracellular Tg containing mispaired disulfide bonds, thought to represent early Tg folding intermediates. An endoplasmic reticulum chaperone of the hsp90 family, GRP94, was also associated in Tg-chaperone complexes. The results suggest that increased binding of BiP to Tg leads to its delayed conformational maturation in the endoplasmic reticulum.
Mol Endocrinol 1998 Mar
PMID:Enhanced binding to the molecular chaperone BiP slows thyroglobulin export from the endoplasmic reticulum. 951 62

The evolutionarily conserved Sec61 protein complex mediates the translocation of secretory proteins into the endoplasmic reticulum. To investigate the role of Sec61p, which is the main subunit of this complex, we generated recessive, cold-sensitive alleles of sec61 that encode stably expressed proteins with strong defects in translocation. The stage at which posttranslational translocation was blocked was probed by chemical crosslinking of radiolabeled secretory precursors added to membranes isolated from wild-type and mutant strains. Two classes of sec61 mutants were distinguished. The first class of mutants was defective in preprotein docking onto a receptor site of the translocon that included Sec61p itself. The second class of mutants allowed docking of precursors onto the translocon but was defective in the ATP-dependent release of precursors from this site that in wild-type membranes leads to pore insertion and full translocation. Only mutants of the second class were partially suppressed by overexpression of SEC63, which encodes a subunit of the Sec61 holoenzyme complex responsible for positioning Kar2p (yeast BiP) at the translocation channel. These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane.
Mol Biol Cell 1998 Dec
PMID:Sec61p serves multiple roles in secretory precursor binding and translocation into the endoplasmic reticulum membrane. 984 81

The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST-63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST-63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST-63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP. We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners.
Mol Biol Cell 1998 Dec
PMID:Specific molecular chaperone interactions and an ATP-dependent conformational change are required during posttranslational protein translocation into the yeast ER. 984 86

Proteins of the Hsp70 family of ATPases, such as BiP, function together with J proteins to bind polypeptides in numerous cellular processes. Using a solid phase binding assay, we demonstrate that a conserved segment of the J proteins, the J domain, catalytically activates BiP molecules to bind peptides in its immediate vicinity. The J domain interacts with the ATP form of BiP and stimulates hydrolysis resulting in the rapid trapping of peptides, which are then only slowly released upon nucleotide exchange. Activation by the J domain allows BiP to trap peptides or proteins that it would not bind on its own. These results explain why BiP and probably all other Hsp70s can interact with a wide range of substrates and suggest that the J partner primarily determines the substrate specificity of Hsp70s.
Mol Cell 1998 Nov
PMID:J proteins catalytically activate Hsp70 molecules to trap a wide range of peptide sequences. 984 32

Six new members of the yeast p24 family have been identified and characterized. These six genes, named ERP1-ERP6 (for Emp24p- and Erv25p-related proteins) are not essential, but deletion of ERP1 or ERP2 causes defects in the transport of Gas1p, in the retention of BiP, and deletion of ERP1 results in the suppression of a temperature-sensitive mutation in SEC13 encoding a COPII vesicle coat protein. These phenotypes are similar to those caused by deletion of EMP24 or ERV25, two previously identified genes that encode related p24 proteins. Genetic and biochemical studies demonstrate that Erp1p and Erp2p function in a heteromeric complex with Emp24p and Erv25p.
Mol Biol Cell 1999 Jun
PMID:Erp1p and Erp2p, partners for Emp24p and Erv25p in a yeast p24 complex. 1035 6

The Drosophila melanogaster HSC3 and HSC4 genes encode Hsc70 proteins homologous to the mammalian endoplasmic reticulum (ER) protein BiP and the cytoplasmic clathrin uncoating ATPase, respectively. These proteins possess ATP binding/hydrolysis activities that mediate their ability to aid in protein folding by coordinating the sequential binding and release of misfolded proteins. To investigate the roles of HSC3 (Hsc3p) and HSC4 (Hsc4p) proteins during development, GAL4-targeted gene expression was used to analyze the effects of producing dominant negatively acting Hsc3p (D231S, K97S) and Hsc4p (D206S, K71S) proteins, containing single amino acid substitutions in their ATP-binding domains, in specific tissues of Drosophila throughout development. We show that the production of each mutant protein results in lethality over a range of developmental stages, depending on the levels of protein produced and which tissues are targeted. We demonstrate that the functions of both Hsc3p and Hsc4p are required for proper tissue establishment and maintenance. Production of mutant Hsc4p, but not Hsc3p, results in induction of the stress-inducible Hsp70 at normal temperatures. Evidence is presented that lethality is caused by tissue-specific defects that result from a global accumulation of misfolded protein caused by lack of functional Hsc70. We show that both mutant Hsc3ps are defective in ATP-induced substrate release, although Hsc3p(D231S) does undergo an ATP-induced conformational change. We believe that the amino acid substitutions in Hsc3p interfere with the structural coupling of ATP binding to substrate release, and this defect is the basis for the mutant proteins' dominant negative effects in vivo.
Mol Biol Cell 1999 Jul
PMID:Tissue-specific expression of dominant negative mutant Drosophila HSC70 causes developmental defects and lethality. 1039 52

The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP-heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.
Mol Biol Cell 1999 Jul
PMID:BiP and immunoglobulin light chain cooperate to control the folding of heavy chain and ensure the fidelity of immunoglobulin assembly. 1039 60

The unfolded protein response (UPR) controls the levels of molecular chaperones and enzymes involved in protein folding in the endoplasmic reticulum (ER). We recently isolated ATF6 as a candidate for mammalian UPR-specific transcription factor. We report here that ATF6 constitutively expressed as a 90-kDa protein (p90ATF6) is directly converted to a 50-kDa protein (p50ATF6) in ER-stressed cells. Furthermore, we showed that the most important consequence of this conversion was altered subcellular localization; p90ATF6 is embedded in the ER, whereas p50ATF6 is a nuclear protein. p90ATF6 is a type II transmembrane glycoprotein with a hydrophobic stretch in the middle of the molecule. Thus, the N-terminal half containing a basic leucine zipper motif is oriented facing the cytoplasm. Full-length ATF6 as well as its C-terminal deletion mutant carrying the transmembrane domain is localized in the ER when transfected. In contrast, mutant ATF6 representing the cytoplasmic region translocates into the nucleus and activates transcription of the endogenous GRP78/BiP gene. We propose that ER stress-induced proteolysis of membrane-bound p90ATF6 releases soluble p50ATF6, leading to induced transcription in the nucleus. Unlike yeast UPR, mammalian UPR appears to use a system similar to that reported for cholesterol homeostasis.
Mol Biol Cell 1999 Nov
PMID:Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. 1056 71

We are studying endoplasmic reticulum-associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI(332), containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI(332) was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of approximately 45 min was followed by a second phase of threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI(332) in which the single used N-glycosylation consensus site had been removed (RI(332)-Thr) was monophasic and rapid, implying a role of the N-linked glycan in the first proteolytic phase. RI(332) degradation was enhanced when the binding of glycoproteins to calnexin was prevented. Moreover, the truncated glycoprotein interacted with calnexin preferentially during the first proteolytic phase, which strongly suggests that binding of RI(332) to the lectin-like protein may result in the slow, initial phase of degradation. Additionally, mannose trimming appears to be required for efficient proteolysis of RI(332). After treatment of cells with the inhibitor of N-glycosylation, tunicamycin, destruction of the truncated RI variants was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI(332) and RI(332)-Thr were stabilized, despite the presence or absence of the N-linked glycan. On the other hand, both drugs are known to trigger the unfolded protein response (UPR), resulting in the induction of BiP and other ER-resident proteins. Indeed, only in drug-treated cells could an interaction between BiP and RI(332) and RI(332)-Thr be detected. Induction of BiP was also evident after overexpression of murine Ire1, an ER transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI(332) was observed. Together, these results suggest that binding of the substrate proteins to UPR-induced chaperones affects their half lives.
Mol Biol Cell 1999 Dec
PMID:Degradation of a short-lived glycoprotein from the lumen of the endoplasmic reticulum: the role of N-linked glycans and the unfolded protein response. 1058 43

Unassembled immunoglobulin light chains expressed by the mouse plasmacytoma cell line NS1 (kappa(NS1)) are degraded in vivo with a half-life of 50-60 min in a way that closely resembles endoplasmic reticulum (ER)-associated degradation (). Here we show that the peptide aldehydes MG132 and PS1 and the specific proteasome inhibitor lactacystin effectively increased the half-life of kappa(NS1), arguing for a proteasome-mediated degradation pathway. Subcellular fractionation and protease protection assays have indicated an ER localization of kappa(NS1) upon proteasome inhibition. This was independently confirmed by the analysis of the folding state of kappa(NS1) and size fractionation experiments showing that the immunoglobulin light chain remained bound to the ER chaperone BiP when the activity of the proteasome was blocked. Moreover, kinetic studies performed in lactacystin-treated cells revealed a time-dependent increase in the physical stability of the BiP-kappa(NS1) complex, suggesting that additional proteins are present in the older complex. Together, our data support a model for ER-associated degradation in which both the release of a soluble nonglycosylated protein from BiP and its retrotranslocation out of the ER are tightly coupled with proteasome activity.
Mol Biol Cell 2000 Jan
PMID:Dissociation from BiP and retrotranslocation of unassembled immunoglobulin light chains are tightly coupled to proteasome activity. 1063 3


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>