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)

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

The RER retains a specific subset of ER proteins, many of which have been shown to participate in the translocation of nascent secretory and membrane proteins. The mechanism of retention of RER specific membrane proteins is unknown. To study this phenomenon in yeast, where no RER-specific membrane proteins have yet been identified, we expressed the human RER-specific protein, ribophorin I. In all mammalian cell types examined, ribophorin I has been shown to be restricted to the membrane of the RER. Here we ascertain that yeast cells correctly target, assemble, and retain ribophorin I in their RER. Floatation experiments demonstrated that human ribophorin I, expressed in yeast, was membrane associated. Carbonate (pH = 11) washing and Triton X-114 cloud-point precipitations of yeast microsomes indicated that ribophorin I was integrated into the membrane bilayer. Both chromatography on Con A and digestion with endoglycosidase H were used to prove that ribophorin I was glycosylated once, consistent with its expression in mammalian cells. Proteolysis of microsomal membranes and subsequent immunoblotting showed ribophorin I to have assumed the correct transmembrane topology. Sucrose gradient centrifugation studies found ribophorin I to be included only in fractions containing rough membranes and excluded from smooth ones that, on the basis of the distribution of BiP, included smooth ER. Ribosome removal from rough membranes and subsequent isopycnic centrifugation resulted in a shift in the buoyant density of the ribophorin I-containing membranes. Furthermore, the rough and density-shifted fractions were the exclusive location of protein translocation activity. Based on these studies we conclude that sequestration of membrane proteins to rough domains of ER probably occurs in a like manner in yeast and mammalian cells.
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PMID:Protein retention in yeast rough endoplasmic reticulum: expression and assembly of human ribophorin I. 226 58

Folding and assembly of polypeptides translocated into the rough endoplasmic reticulum (RER) is facilitated by a set of resident proteins in the lumen of the RER. We studied the regulation of synthesis of the RER luminal proteins immunoglobulin heavy chain binding protein (BiP) and protein disulfide isomerase (PDI), and of the cytosolic stress 70 protein (hsc70) after hormonal stimulation of the pancreatic exocrine secretory pathway. Their rate of synthesis was assessed at both mRNA and protein levels and under two experimental conditions that are associated with large increases in exocrine production. After in vivo stimulation of the pancreas by either endogenous release of cholecystokinin (CCK) following proteinase inhibitor feeding (FOY-305) or by in vivo infusion of the pancreatic secretagogue cerulein, the relative rates of synthesis detected for BiP and PDI were enhanced 2.5 to 4-fold compared to control. Interestingly, the kinetics and the degree of hsc70 mRNA induction were almost identical to those of BiP and PDI, suggesting coordinated hormonal regulation of BiP, PDI as hormonal stimulation was even twice that following heat shock treatment. The mRNA levels of calreticulin (CaBP3) increased up to 2.3-fold with a kinetic comparable to that of BiP, PDI and hsc 70, while CaBP1 and the RER membrane proteins, ribophorin I and the signal recognition particle receptor did not show any changes in their relative mRNA amounts after hormonal stimulation. The increase in the rates of PDI and chaperone biosynthesis exceeds the associated increase in total protein biosynthesis. In vitro experiments, using transformed rat acinar cells (AR4-2J) in which pancreatic enzyme synthesis can be induced by glycocorticoid hormones, demonstrated that induction of PDI and chaperone mRNA synthesis preceded extensive mRNA expression of secretory proteins.
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PMID:Hormonal regulation of protein disulfide isomerase and chaperone synthesis in the rat exocrine pancreas. 791 86

Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.
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PMID:A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum. 900 69

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.
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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