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

We studied the molecular nature of the interaction between the integral membrane protein Sec63p and the lumenal Hsp70 BiP to elucidate their role in the process of precursor transit into the ER of Saccharomyces cerevisiae. A lumenal stretch of Sec63p with homology to the Escherichia coli protein DnaJ is the likely region of interface between Sec63p and BiP. This domain, purified as a fusion protein (63Jp) with glutathione S-transferase (GST), mediated a stable ATP-dependent binding interaction between 63Jp and BiP and stimulated the ATPase activity of BiP. The interaction was highly selective because only BiP was retained on immobilized 63Jp when detergent-solubilized microsomes were mixed with ATP and the fusion protein. GST alone was inactive in these assays. Additionally, a GST fusion containing a point mutation in the lumenal domain of Sec63p did not interact with BiP. Finally, we found that the soluble Sec63p lumenal domain inhibited efficient precursor import into proteoliposomes reconstituted so as to incorporate both BiP and the fusion protein. We conclude that the lumenal domain of Sec63p is sufficient to mediate enzymatic interaction with BiP and that this interaction positioned at the translocation apparatus or translocon at the lumenal face of the ER is vital for protein translocation into the ER.
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PMID:The lumenal domain of Sec63p stimulates the ATPase activity of BiP and mediates BiP recruitment to the translocon in Saccharomyces cerevisiae. 919 65

We have identified the rat and Caenorhabditis elegans homologues of a 'core ATPase'-encoding Hsp70-like gene, designated Stch. We observed that the human, rat, and C. elegans Stch genes have conserved a stop codon immediately distal to the sequence encoding the Hsp70 ATPase domain. This results in the functional equivalent of an N-terminal, proteolytically cleaved fragment of Hsc70/BiP. Each homologue contains a hydrophobic signal sequence, demonstrates striking identity within the Hsp70 ATPase domain, and retains a similar C-terminal sequence (STCH specific cluster III) that is unique among Hsp70 proteins and which truncates the peptide binding domain. In addition, we have identified an internal 35-aa region that is homologous to the minimal sequence of the Hip chaperone co-factor that is required for direct binding to the ATPase domain of Hsp70. Adjacent to this region, the rat and human STCH protein sequences diverge within a short internal 'insertion' sequence that interrupts the ATPase subdomain between the phosphate-2 and adenosine ATP-binding sites. We have also demonstrated that both human and rat Stch are constitutively produced and are induced by the calcium ionophore A23187, but not by heat shock. The recognition that the truncated 'core ATPase' structure of the STCH molecule is conserved in human, rat, and C. elegans tissues suggests an important role for this unique member of the membrane-bound Hsp70 family.
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PMID:A 'core ATPase', Hsp70-like structure is conserved in human, rat, and C. elegans STCH proteins. 935 68

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament proteins that are expressed in 'simple-type' epithelial cells. They associate with several proteins including the 70 kDa cytoplasmic heat shock proteins (hsp70). We identified the human 78 kDa glucose-regulated protein (grp78) as a keratin-associated protein. Keratin-grp78 association was noted after co-immunoprecipitation of K8/18 from HT29 detergent solubilized cell lysates, and appears to involve non-posttranslationally modified grp78. The grp78-K8/18 association is induced by culturing cells in the presence of tunicamycin or after glucose starvation. K8/18-bound grp78 can be dissociated by Mg-ATP and the association can be reconstituted in vitro using purified grp78, then redissociated again by Mg-ATP. Binding of grp78 occurs preferentially with K8, and when reconstituted does not depend on the posttranslational modification state of K8/18. Co-incubation of K8/18 with hsp70 and grp78 shows preferential association with hsp70. Our results demonstrate a direct association of grp78 with K8 under conditions that induce grp78 expression.
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PMID:Association of glucose-regulated protein (grp78) with human keratin 8. 940 41

BiP is found in association with calreticulin, both in the presence and absence of endoplasmic reticulum stress. Although the BiP-calreticulin complex can be disrupted by ATP, several properties suggest that the calreticulin associated with BiP is neither unfolded nor partially or improperly folded. (1) The complex is stable in vivo and does not dissociate during 8 hr of chase. (2) When present in the complex, calreticulin masks epitopes at the C terminus of BiP that are not masked when BiP is bound to an assembly-defective protein. And (3) overproduction of calreticulin does not lead to the recruitment of more BiP into complexes with calreticulin. The BiP-calreticulin complex can be disrupted by low pH but not by divalent cation chelators. When the endoplasmic reticulum retention signal of BiP is removed, complex formation with calreticulin still occurs, and this explains the poor secretion of the truncated molecule. Gel filtration experiments showed that BiP and calreticulin are present in distinct high molecular weight complexes in which both molecules interact with each other. The possible functions of this complex are discussed.
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PMID:BiP and calreticulin form an abundant complex that is independent of endoplasmic reticulum stress 959 39

To investigate the role of each domain in BiP/GRP78 function, we have used a full-length recombinant BiP engineered to contain two enterokinase sites; one site is located after an N-terminal FLAG epitope, and a second site has been inserted at the junction between the N- and C-terminal domains (FLAG-BiP.ent). FLAG-BiP.ent oligomerizes into multiple species that interconvert with each other in a slow, concentration- and temperature-dependent equilibrium. Binding of ATP or AMP-PNP (adenosine 5'-(beta, gamma-imino)triphosphate), but not ADP, or of a peptidic substrate induces depolymerization of FLAG-BiP.ent and stabilization of monomeric species. Enterokinase cleavage of monomeric, nucleotide-free BiP.ent results in the physical dissociation of the 44-kDa N-terminal ATPase fragment (N44.ent) from the 30-kDa C-terminal substrate binding domain (C30.ent). Upon dissociation, the freed C-terminal substrate binding domain readily undergoes self-association while N44.ent remains monomeric. Enterokinase cleavage performed in the presence of a synthetic peptide prevents oligomerization of the freed C30.ent domain. Addition of ATP during enterokinase cleavage has no effect on C30.ent oligomerization. Our data clearly indicate that binding of a specific peptide onto the C-terminal domain, or ATP onto the N-terminal domain, induces internal conformational change(s) within the C30 domain that result(s) in BiP depolymerization.
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PMID:Substrate binding induces depolymerization of the C-terminal peptide binding domain of murine GRP78/BiP. 975 27

To determine whether mitochondrial hsp70 (mHsp70) could substitute for the endoplasmic retuculum (ER) Hsp70 (BiP) during protein translocation, we assembled ER-derived reconstituted proteoliposomes supplemented with either protein. We found that only BiP restored translocation in kar2 mutant vesicles and stimulated translocation approximately 3-fold in wild type proteoliposomes. mHsp70 associated poorly with both a BiP binding (DnaJ) domain of Sec63p and an ER precursor, and its ATPase activity was poorly enhanced upon incubation with the DnaJ domain. In contrast, BiP bound to the Sec63p-DnaJ domain in an ATP-dependent manner and its ATPase activity was stimulated significantly by this polypeptide. We conclude that mHsp70 is unable to support protein translocation into the ER because it fails to associate productively with Sec63p and a precursor.
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PMID:Mitochondrial Hsp70 cannot replace BiP in driving protein translocation into the yeast endoplasmic reticulum. 976 4

GRP78/BiP, a molecular chaperone in the endoplasmic reticulum, is induced under such adverse conditions for cell survival as glucose starvation. Induction of GRP78 has been shown to coincide with G1 cell cycle arrest, which is an important cellular defense system. In this study, we investigated involvement of GRP78 in the mechanism of growth arrest by using human epidermoid carcinoma A431 cells. Under a chemical stress condition with 2-deoxyglucose, GRP78 was induced 3-4-fold. In the stressed cells, an underglycosylated form of epidermal growth factor receptor (EGFR) was produced and the mature form was decreased. We found that the molecular chaperone GRP78 in the endoplasmic reticulum formed a stable complex with the underglycosylated EGFR but did not with the mature form. This complex formation occurred specifically under the stress conditions, and the complex was dissociated upon removal of the stress. Treatment of the GRP78-underglycosylated EGFR complex with ATP resulted in a release of the underglycosylated EGFR from GRP78, indicating that the complex could be formed through the chaperone function of GRP78. In accordance with the complex formation with endoplasmic reticulum-resident GRP78, the underglycosylated EGFR could not be translocated to the cell surface. As a result, EGF could not induce expression of cyclin D3, a G1 cyclin, in the stressed cells, whereas it did in non-stressed cells. These results indicated that, in the stressed cells, GRP78 participated in down-regulation of EGF-signaling pathway by forming a stable complex with EGFR and inhibiting EGFR translocation to the cell surface.
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PMID:Down-regulation of epidermal growth factor receptor-signaling pathway by binding of GRP78/BiP to the receptor under glucose-starved stress conditions. 976 25

The mechanism by which ATP binding transduces a conformational change in 70-kDa heat shock proteins that results in release of bound peptides remains obscure. Wei and Hendershot demonstrated that mutating Thr37 of hamster BiP to glycine impeded the ATP-induced conformational change, as monitored by proteolysis [(1995) J. Biol. Chem. 270, 26670-26676]. We have mutated the equivalent resitude of the bovine heat shock cognate protein (Hsc70), Thr13, to serine, valine, and glycine. Solution small-angle X-ray scattering experiments on a 60-kDa fragment of Hsc70 show that ATP binding induces a conformational change in the T13S mutant but not the T13V or T13G mutants. The kinetics of ATP-induced tryptophan fluorescence intensity changes in the 60-kDa proteins is biphasic for the T13S mutant but monophasic for T13V or T13G, consistent with a conformational change following initial ATP binding in the T13S mutant but not the other two. Crystallographic structures of the ATPase fragments of the T13S and T13G mutants at 1.7 A resolution show that the mutations do not disrupt the ATP binding site and that the serine hydroxyl mimics the threonine hydroxyl in the wild-type structure. We conclude that the hydroxyl of Thr13 is essential for coupling ATP binding to a conformational change in Hsc70. Molecular modeling suggests this may result from the threonine hydroxyl hydrogen-bonding to a gamma-phosphate oxygen of ATP, thereby inducing a structural shift within the ATPase domain that couples to its interactions with the peptide binding domain.
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PMID:The hydroxyl of threonine 13 of the bovine 70-kDa heat shock cognate protein is essential for transducing the ATP-induced conformational change. 979

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.
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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.
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PMID:Specific molecular chaperone interactions and an ATP-dependent conformational change are required during posttranslational protein translocation into the yeast ER. 984 86


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