Expression of the endoplasmic reticulum chaperone GRP94 gene in ischemic gerbil brain

GRP94 (glucose regulated protein 94) gene expression in the ischemic-hippocampus of gerbils, which was induced by a temporary occlusion of the bilateral common carotid arteries (CCAs), was tested by Northern blot analysis. The maximum GRP94 gene expression level was detected at the occipital lobe 10 min after the induction of ischemia. In the hippocampus, GRP94 gene expression reached a maximum 15 min after inducing ischemia. Following reperfusion, the maximum expression level was shown at 12 h and continuing thereafter.     

Enhanced secretion of heterologous proteins in Pichia pastoris following overexpression of Saccharomyces cerevisiae chaperone proteins

In Pichia pastoris, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). However, upon introduction of foreign proteins, heterologous proteins are often retained in the cytoplasm or in the ER as a result of suboptimal folding conditions, leading to protein aggregation. The Hsp70 and Hsp40 chaperone families in the cytoplasm or in ER importantly regulate the folding and secretion of heterologous proteins. However, it is not clear which single chaperone is most important or which combination optimally cooperates in this process. In the present study we evaluated the role of the chaperones Kar2p, Sec63, YDJ1p, Ssa1p, and PDI from Saccharomyces cerevisiae. We found that the introduction of Kar2p, Ssa1p, or PDI improves protein secretion 4-7 times. In addition, we found that the combination chaperones of YDJ1p/PDI, YDJ1p/Sec63, and Kar2p/PDI synergistically increase secretion levels 8.7, 7.6, and 6.5 times, respectively. Therefore, additional integration of chaperone genes can improve the secretory expression of the heterologous protein. Western blot experiments revealed that the chaperones partly relieved the secretion bottleneck resulting from foreign protein introduction in P. pastoris. Therefore, the findings from the present study demonstrate the presence of a network of chaperones in vivo, which may act synergistically to increase recombinant protein yields.     

Analysis of the role of mitochondrial and endoplasmic reticulum localized small heat shock proteins in tomato

This communication examines the role of small heat shock proteins (sHsps) targeted to mitochondria (Mt) and endoplasmic reticulum (ER) in tomato plants (Lycopersicon esculentum Mill.) under heat stress. Genetic response of transgenic and wild type plants varied under optimum, moderately elevated and elevated temperature. In optimum temperature higher biomass was recorded in wild type than the transgenic lines, whereas in moderately elevated temperature biomass increased in Mt-sHsp line. Also, net photosynthetic rate (P_N) increased in Mt-sHsp line in both the elevated temperatures, though higher in moderately elevated. Cell membrane stability (CMS) improved in all the lines after exposure to elevated temperatures, but always remained higher in transgenic lines. Transgenic lines expressed sHsps in different temperature regimes in both vegetative and reproductive parts, while wild type expressed such proteins only after 1 h of heat shock.     

The p88 molecular chaperone is identical to the endoplasmic reticulum membrane protein, calnexin.

We previously described a novel molecular chaperone (designated p88) that participates in the assembly of murine class I histocompatibility molecules (Degen, E., and Williams, D. B. (1991) J. Cell Biol. 112, 1099-1115). Our findings suggest that p88 may either promote proper assembly of class I molecules or retain them, probably within the endoplasmic reticulum (ER), until assembly of the ternary complex of heavy chain, beta 2-microglobulin, and peptide ligand is complete. In this report, we compare p88 to calnexin, a calcium-binding 90-kDa phosphoprotein of the ER membrane (Wada, I., Rindress, D., Cameron, P. H., Ou, W.-J., Doherty, J.-J., II, Louvard, D., Bell, A.W., Dignard, D., Thomas, D. Y., and Bergeron, J. J. M. (1991) J. Biol. Chem. 266, 19599-19610). We show that p88 and calnexin share antigenic epitopes defined by a polyclonal anti-calnexin antiserum. Furthermore, both proteins were immunoprecipitated in association with an intracellularly retained variant of the class I H-2Kb molecule. Since p88 and calnexin were also indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, were resistant to digestion with endoglycosidase H, and exhibited virtually identical patterns of peptide fragments following digestion with either V8 protease or trypsin, we conclude that p88 and calnexin represent the same protein. The identification of the p88 chaperone as a phosphorylated, calcium-binding protein of the ER membrane suggests possible means whereby its interaction with class I molecules may be regulated.     

TPR-containing proteins control protein organization and homeostasis for the endoplasmic reticulum

The endoplasmic reticulum (ER) is a complex, multifunctional organelle comprised of a continuous membrane and lumen that is organized into a number of functional regions. It plays various roles including protein translocation, folding, quality control, secretion, calcium signaling, and lipid biogenesis. Cellular protein homeostasis is maintained by a complicated chaperone network, and the largest functional family within this network consists of proteins containing tetratricopeptide repeats (TPRs). TPRs are well-studied structural motifs that mediate intermolecular protein–protein interactions, supporting interactions with a wide range of ligands or substrates. Seven TPR-containing proteins have thus far been shown to localize to the ER and control protein organization and homeostasis within this multifunctional organelle. Here, we discuss the roles of these proteins in controlling ER processes and organization. The crucial roles that TPR-containing proteins play in the ER are highlighted by diseases or defects associated with their mutation or disruption.     

Calreticulin, a Ca2+-binding chaperone of the endoplasmic reticulum

Calreticulin is a 46-kDa Ca2+-binding chaperone found across a diverse range of species. The protein is involved in the regulation of intracellular Ca2+ homeostasis and endoplasmic reticulum (ER) Ca2+ storage capacity. Calreticulin is also an important molecular chaperone involved in "quality control" within secretory pathways. The protein contains structurally and functionally unique domains with specialized functions. Studies on calreticulin knockout mice indicate that the protein is essential in early cardiac development. The protein also plays an important role in autoimmunity and cancer.     

Cell surface expression of calnexin, a molecular chaperone in the endoplasmic reticulum

The folding and assembly of nascent proteins in the endoplasmic reticulum are assisted by the molecular chaperone calnexin, which is itself retained within the endoplasmic reticulum. It was up to now assumed that calnexin was selectively expressed on the surface of immature thymocytes because of a particular characteristic of the protein sorting machinery in these cells. We now report that a small fraction of calnexin is normally expressed on the surface of various cells such as mastocytoma cells, murine splenocytes, fibroblast cells, and human HeLa cells. Surface biotinylation followed by chase culture of living cells revealed that calnexin is continuously delivered to the cell surface and then internalized for lysosomal degradation. These results suggest that there is continuous exocytosis and endocytosis of calnexin, and the amount of calnexin on the plasma membrane results from the balance of the rates of these two events. To study the structural requirement of calnexin for surface expression, we created deletion mutants of calnexin and found that the luminal domain, particularly the glycoprotein binding domain, is necessary. These findings suggest that the surface expression of calnexin depends on the association with glycoproteins and that calnexin may play a certain role as a chaperone on the plasma membrane as well.     

Structure and assembly of the endoplasmic reticulum. Biosynthetic sorting of endoplasmic reticulum proteins

We have studied the post-translational processing and the biosynthetic sorting of three protein components of murine endoplasmic reticulum (ER), ERp60, ERp72, and ERp99. In pulse-labeled MOPC-315 (where MOPC-315 represents mineral oil-induced plasmacytoma cells) plasmacytoma cells, no precursor forms of these proteins were detected and only ERp99 was sensitive to endoglycosidase H. The ERp99 oligosaccharide remained endoglycosidase H sensitive during a 3-h chase, and analysis by high performance liquid chromatography showed the predominant structure to be Man8GlcNAc2. We have used a sucrose gradient analysis of pulse-labeled MOPC-315 plasmacytoma cells in order to directly study the biosynthetic sorting of both glycosylated and nonglycosylated ERps and have found no strong evidence to suggest these proteins ever leave the endoplasmic reticulum. In spite of their common sorting pathway, these proteins differ in their membrane orientation. Both ERp60 and ERp72 are entirely protected by the endoplasmic reticulum membrane while ERp99 appears to have a large domain exposed on the cytoplasmic face of the endoplasmic reticulum.     

Structure and assembly of the endoplasmic reticulum. The synthesis of three major endoplasmic reticulum proteins during lipopolysaccharide-induced differentiation of murine lymphocytes

Monospecific rabbit antibodies have been prepared against ERp72, ERp99, and ERp60, major protein components of a detergent-solubilized extract of endoplasmic reticulum purified from mineral oil-induced plasmacytoma 315 tissue. When subcellular fractions of mineral oil-induced plasmacytoma 315 tissue were assayed by an immunoprecipitation procedure, all three endoplasmic reticulum proteins (ERps) were found to be enriched in the rough endoplasmic reticulum. In murine lymphoid cells, the three ERps represent two major structural classes of protein. Both ERp72 and ERp60 contain no endoglycosidase H-sensitive, N-linked oligosaccharides. On the other hand, ERp99 is glycoprotein containing, in all likelihood, one endoglycosidase H-sensitive oligosaccharide. Immunologically cross-reacting proteins of similar molecular weight have also been detected in other eukaryotic cell lines. The anti-ERp antibodies were used to quantitate the synthesis and accumulation of the three ERps in splenic lymphocytes cultured in the presence and absence of bacterial lipopolysaccharide (Escherichia coli serotype B5:055) (LPS). In the presence of LPS, lymphocytes differentiate from resting cells into actively secreting cells. The synthesis of ERp72 and ERp99 increased 3- and 10-fold, respectively, in response to LPS. The synthesis of ERp60 does not change significantly. The turnover rates for these three proteins are similar in both control and LPS-treated lymphocytes. As a result, membranes isolated from LPS-treated cells are enriched in ERp72 and ERp99.     

High capacity of the endoplasmic reticulum to prevent secretion and aggregation of amyloidogenic proteins

Protein aggregation is associated with neurodegeneration and various other pathologies. How specific cellular environments modulate the aggregation of disease proteins is not well understood. Here, we investigated how the endoplasmic reticulum (ER) quality control system handles β‐sheet proteins that were designed de novo to form amyloid‐like fibrils. While these proteins undergo toxic aggregation in the cytosol, we find that targeting them to the ER (ER‐β) strongly reduces their toxicity. ER‐β is retained within the ER in a soluble, polymeric state, despite reaching very high concentrations exceeding those of ER‐resident molecular chaperones. ER‐β is not removed by ER‐associated degradation (ERAD) but interferes with ERAD of other proteins. These findings demonstrate a remarkable capacity of the ER to prevent the formation of insoluble β‐aggregates and the secretion of potentially toxic protein species. Our results also suggest a generic mechanism by which proteins with exposed β‐sheet structure in the ER interfere with proteostasis.     

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum

The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.     

Vacuole membrane protein 1 is an endoplasmic reticulum protein required for organelle biogenesis, protein secretion, and development

Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1(-) Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1(-) cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised.     

BiP,an endoplasmic reticulum chaperone,modulates the development of morphine antinociceptive tolerance

Morphine is a potent analgesic, but the molecular mechanism for tolerance formation after repeated use is not fully understood. Binding immunoglobulin protein (BiP) is an endoplasmic reticulum (ER) chaperone that is central to ER function. We examined knock‐in mice expressing a mutant BiP with the retrieval sequence deleted in order to elucidate physiological processes that are sensitive to BiP functions. We tested the thermal antinociceptive effect of morphine in heterozygous mutant BiP mice in a hot plate test. Paw withdrawal latencies before and after a single administration of morphine were not significantly different between the wild‐type and mutant BiP mice. Repeated morphine administration caused the development of morphine tolerance in the wild‐type mice. The activation of glycogen synthase kinase 3b (GSK‐3b) was associated with morphine tolerance, because an inhibitor of GSK‐3β prevented it. On the other hand, the mutant BiP mice showed less morphine tolerance, and the activation of GSK‐3b was suppressed in their brain. These results suggest that BiP may play an important role in the development of morphine tolerance. Furthermore, we found that a chemical chaperone which improves ER protein folding capacity also attenuated the development of morphine tolerance in wild‐type mice, suggesting a possible clinical application of chemical chaperones in preventing morphine tolerance.     

The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.