[31] Purification and properties of BiP

Chevalier, Mathieu S.; King, L.E.; Blond, Sylvie Y.

doi:10.1016/s0076-6879(98)90033-7

Cited by 15 publications

(15 citation statements)

References 39 publications

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“…ATPase Assays-Concentration-dependent assays were carried out as described (49). Time-dependent assays were done as follows: 70 l of total assay buffer supplemented with 100 M ATP, 1 Ci of [␥- , and 75 mM KCl or 50 mM NaCl.…”

Section: Methodsmentioning

confidence: 99%

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Interaction of Murine BiP/GRP78 with the DnaJ Homologue MTJ1

Chevalier

Rhee

Elguindi³

et al. 2000

Journal of Biological Chemistry

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The activity of Hsp70 proteins is regulated by accessory proteins, among which the most studied are the members of the DnaJ-like protein family. BiP/GRP78 chaperones the translocation and maturation of secreted and membrane proteins in the endoplasmic reticulum. No DnaJ-like partner has been described so far to regulate the function of mammalian BiP/GRP78. We show here that murine BiP/GRP78 interacts with the lumenal J domain of the murine transmembrane protein MTJ1 (J-MTJ1). J-MTJ1 stimulates the ATPase activity of BiP/GRP78 at stoichiometric concentrations. The Cterminal tail of BiP/GRP78 is not required for the interaction with J-MTJ1, leaving the function of this portion of the molecule still unclear. Physical interactions between J-MTJ1 and BiP/GRP78 are stable and can be abolished by a single histidine 3 glutamine substitution in the highly conserved HPD motif shared by all DnaJlike proteins. The J-MTJ1 fragment, but not the mutant J-MTJ1:H89Q fragment, stimulates the ATPase activity of Escherichia coli DnaK, although at a higher concentration than its genuine partner DnaJ. Full-length DnaJ does not stimulate BiP over the range of concentrations investigated. These results indicate that the J domain of MTJ1 is sufficient for its interaction with BiP/GRP78 and cannot be substituted by E. coli DnaJ.

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Section: Methodsmentioning

confidence: 99%

“…After incubation at 37°C, 10-l aliquots were quenched with 2 l of 1 N HCl and put on ice. Conversion of ATP into ADP was determined by thin layer chromatography as described (49).…”

Section: Methodsmentioning

confidence: 99%

Interaction of Murine BiP/GRP78 with the DnaJ Homologue MTJ1

Chevalier

Rhee

Elguindi³

et al. 2000

Journal of Biological Chemistry

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“…ATPase assays were performed as described previously (41,42). The purified WT J-ERdj4 or Mu J-ERdj4 was added to recombinant BiP (0.…”

Section: Atpase Assaymentioning

confidence: 99%

Identification and Characterization of a Novel Endoplasmic Reticulum (ER) DnaJ Homologue, Which Stimulates ATPase Activity of BiP in Vitro and Is Induced by ER Stress

Shen

Meunier

Hendershot

2002

Journal of Biological Chemistry

190

183

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The activity of Hsp70 proteins is regulated by accessory proteins, which include members of the DnaJ-like protein family. Characterized by the presence of a highly conserved 70-amino acid J domain, DnaJ homologues activate the ATPase activity of Hsp70 proteins and stabilize their interaction with unfolded substrates. DnaJ homologues have been identified in most organelles where they are involved in nearly all aspects of protein synthesis and folding. Within the endoplasmic reticulum (ER), DnaJ homologues have also been shown to assist in the translocation, secretion, retro-translocation, and ER-associated degradation (ERAD) of secretory pathway proteins. By using bioinformatic methods, we identified a novel mammalian DnaJ homologue, ERdj4. It is the first ER-localized type II DnaJ homologue to be reported. The signal sequence of ERdj4 remains uncleaved and serves as a membrane anchor, orienting its J domain into the ER lumen. ERdj4 colocalized with GRP94 in the ER and associated with BiP in vivo when they were co-expressed in COS-1 cells. In vitro experiments demonstrated that the J domain of ERdj4 stimulated the ATPase activity of BiP in a concentration-dependent manner. However, mutation of the hallmark tripeptide HPD (His 3 Gln) in the J domain totally abolished this activation. ERdj4 mRNA expression was detected in all human tissues examined but showed the highest level of the expression in the liver, kidney, and placenta. We found that ERdj4 was highly induced at both the mRNA and protein level in response to ER stress, indicating that this protein might be involved in either protein folding or ER-associated degradation. The endoplasmic reticulum (ER)1 is the site of synthesis and maturation of secretory pathway proteins, which include resident proteins of the endocytic and exocytic organelles as well as surface and secreted proteins. Approximately one-third of all cellular proteins are translocated into the lumen of ER, which possesses a unique oxidizing and Ca 2ϩ -rich environment, where post-translational modification, folding, and oligomerization of nascent proteins occur. ER molecular chaperones and folding enzymes associate with the newly synthesized proteins to prevent their aggregation and help them fold and assemble correctly. Through a process called ER quality control, proteins that do not mature properly are retained in the ER and are eventually targeted for ER-associated degradation (ERAD) through the action of the chaperones (1).BiP, also known as GRP78, is the mammalian ER member of the Hsp70 family and was the first component of the ER quality control apparatus to be identified (2). Hsp70 family members exist in all organisms and in all organelles, where they aid in the folding and assembly of nascent proteins and prevent their aggregation during conditions of physiological stress (3, 4). Like other Hsp70 proteins, BiP plays an essential role in the biosynthesis of proteins. In addition, BiP maintains the permeability barrier of the ER translocon during early stages of protein translocation ...

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“…All chemicals were purchased from Sigma, with the exception of sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate that was purchased from Pierce and NAP-5 desalting columns from Amersham Pharmacia. Rat liver GT and BiP were purified as described (3,9).…”

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confidence: 99%

UDP-Glc:glycoprotein glucosyltransferase recognizes structured and solvent accessible hydrophobic patches in molten globule-like folding intermediates

Caramelo

Castro²,

Alonso³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

164

129

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Protein folding in the cell involves the action of different molecular chaperones and folding-facilitating enzymes. In the endoplasmic reticulum (ER), the folding status of glycoproteins is stringently controlled by a glucosyltranferase enzyme (GT) that creates monoglucosylated structures recognized by ER resident lectins (calnexin͞ calreticulin, CNX͞CRT). GT serves as a folding sensor because it only glucosylates misfolded or partly folded glycoproteins. Nevertheless, the molecular mechanism behind this recognition process remains largely unknown. In this paper we explore the structural determinants for GT recognition by using a single domain model protein. For this purpose we used a family of chemically glycosylated proteins derived from chymotrypsin inhibitor-2 as GT substrates. T he folding status of glycoproteins is strictly censored in the endoplasmic reticulum (ER), before they attain their final destination to membranes, organelles, or the extracellular environment (1). Proteins that fail to fold properly are initially retained in the ER and eventually degraded in the proteasomes. The quality control system involves the action of UDPGlc:glycoprotein glucosyltranferase (GT) and glucosidase II (GII) enzymes and the ER resident lectins calnexin (CNX) and calreticulin (CRT). Proteins that are incompletely folded are glucosylated by GT, thus generating Glc 1 Man 9 GlcNAc 2 structures. On binding those structures, CNX and͞or CNR retain the monoglucosylated glycoproteins in the ER. Eventually, the Glc units added by GT are removed by GII, causing the release of glycoproteins from the lectin anchors. Glucosylationdeglucosylation cycles catalyzed by the opposing activities of GT and GII continue until native conformations are attained. Glycoproteins then become substrates of GII but not of GT and are thus free to pursue their travel through the secretory pathway.The CNX͞CRT-monoglucosylated glycan interaction is one of the mechanism by which cells retain incompletely folded glycoproteins in the ER and, in addition, it enhances folding efficiency by preventing protein aggregation and allowing intervention of additional ER chaperones and folding accessory proteins. The key element in this mechanism is GT as it is the only component exclusively acting on incompletely folded glycoproteins. This feature is based on the recognition of two structural determinants not exposed in native conformations. One of them is the innermost GlcNAc unit, which is buried within the protein scaffold in native but solvent exposed in nonnative conformations (2). In addition, there is an ill-defined determinant in incompletely folded polypeptides that is also recognized by GT. This protein determinant has eluded identification so far, mainly because of the difficulties intrinsic to the study of partly folded conformations of possible intermediates along the folding pathway of nascent polypeptides. It has been suggested that hydrophobic residues exposed in incompletely folded conformers could be the protein elements recognized by GT as exp...

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[31] Purification and properties of BiP

Cited by 15 publications

References 39 publications

Interaction of Murine BiP/GRP78 with the DnaJ Homologue MTJ1

Interaction of Murine BiP/GRP78 with the DnaJ Homologue MTJ1

Identification and Characterization of a Novel Endoplasmic Reticulum (ER) DnaJ Homologue, Which Stimulates ATPase Activity of BiP in Vitro and Is Induced by ER Stress

UDP-Glc:glycoprotein glucosyltransferase recognizes structured and solvent accessible hydrophobic patches in molten globule-like folding intermediates

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