Heat-shock protein synthesis in Chlamydomonas reinhardi. Translational control at the level of initiation of a poly(A)-rich-RNA coded 22-KDa protein in a cell-free system

The effect of temperature on the in vitro translation of control and heat-shock poly(A)-rich RNA, obtained from Chlamydomonas reinhardi cells, incubated for 2 h at 25 degrees C respectively, was studied using the wheat-germ translation system. Incubation of the cells at 42 degrees C induces the synthesis of RNAs coding for several heat-shock proteins, including a 22-kDa major polypeptide as well as several proteins of 45-94 kDa, as demonstrated by run-off translation of polyribosomes isolated from intact cells. However, the high-molecular-mass heat-shock proteins are poorly translated in the wheat-germ system. The poly(A)-rich RNA coding for the 22-kDa heat-induced polypeptide has an apparent sedimentation coefficient higher than that expected from the molecular mass of its translation product, and was preferentially translated in vitro at temperatures above 31 degrees C as compared with pre-existing RNAs. Raising the temperature of translation, slightly inhibited (10%) the runoff translation of polyribosomes isolated from intact cells. However, when initiation was carried out in vitro for a short time at increasing temperatures and translation continued at 25 degrees C in the presence of aurintricarboxylic acid, the 22-kDa heat-shock polypeptides was preferentially translated. Aurintricarboxylic acid did not significantly inhibit incorporation of [35S]methionine when added to polyribosomes isolated from control or heat-shocked cells. From the above data we conclude that the translation of the 22-kDa heat-shock protein is controlled in vitro at the initiation level.     

The nuclear-coded chloroplast 22-kDa heat-shock protein of Chlamydomonas. Evidence for translocation into the organelle without a processing step

A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlamydomonas reinhardtii cells. The clone has a length of 1.1 kb and codes for the complete heat-shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat-shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat-shock protein of 22 kDa synthesized in vivo during heat-shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)-rich RNA from heat-treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5' region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non-coding 3' region of 600 nucleotides. Northern hybridization indicates a length of 1.7 kb for the messenger RNA of heat-shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat-shock proteins demonstrates that this protein belongs to the small-molecular-mass plant heat-shock protein family and also shows similarities with animal heat-shock proteins including the presence of a short region possessing similarity with bovine alpha-crystalline as reported for other heat-shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.     

Translational control and target recognition by Escherichia coli small RNAs in vivo

Small non-coding RNAs (sRNAs) are an emerging class of regulators of bacterial gene expression. Most of the regulatory Escherichia coli sRNAs known to date modulate translation of trans-encoded target mRNAs. We studied the specificity of sRNA target interactions using gene fusions to green fluorescent protein (GFP) as a novel reporter of translational control by bacterial sRNAs in vivo. Target sequences were selected from both monocistronic and polycistronic mRNAs. Upon expression of the cognate sRNA (DsrA, GcvB, MicA, MicC, MicF, RprA, RyhB, SgrS and Spot42), we observed highly specific translation repression/activation of target fusions under various growth conditions. Target regulation was also tested in mutants that lacked Hfq or RNase III, or which expressed a truncated RNase E (rne701). We found that translational regulation by these sRNAs was largely independent of full-length RNase E, e.g. despite the fact that ompA fusion mRNA decay could no longer be promoted by MicA. This is the first study in which multiple well-defined E.coli sRNA target pairs have been studied in a uniform manner in vivo. We expect our GFP fusion approach to be applicable to sRNA targets of other bacteria, and also demonstrate that Vibrio RyhB sRNA represses a Vibrio sodB fusion when co-expressed in E.coli.     

Evidence for the localization of the nuclear-coded 22-kDa heat-shock protein in a subfraction of thylakoid membranes.

The precursor to the nuclear-coded 22-kDa heat-shock protein of chloroplasts (HSP 22) has been transported into isolated intact chloroplasts from heat-shocked plants. The localization of the mature protein in the chloroplast membrane was investigated. We have shown that the processed HSP 22 of pea was not bound to envelopes and found predominantly in thylakoid membranes. The binding of HSP 22 was stable in the presence of high salt concentrations. Solubilization of thylakoid membranes with Triton X-100 and phase partitioning with Triton X-114 indicate an intrinsic localization of HSP 22 or, alternatively, a non-covalent association with integral membrane protein(s). After fractionation into grana and stroma lamellae, HSP 22 was found mostly in the grana-membrane subfraction.     

Functional mode of NtHSP17.6, a cytosolic small heat-shock protein fromNicotiana tabacum

Small heat-shock proteins (sHsps) are ubiquitous stress proteins with molecular chaperone activity. They share characteristic homology with the α-crystallin protein of the mammalian eye lens as well as being ATP-independent in their chaperone activity. We isolated a clone for a cytosolic class I sHsp,NtHSP17.6, fromNicotiana tabacum, and analyzed its functional mode for such activity. Following its transformation intoEscherichia coli and its over-expression, NtHSPI 7.6 was purified and examinedin vitro. This purified NtHSPI 7.6 exhibited typical chaperone activity in a light-scattering test. It was enable to protect a model substrate, firefly luciferase, from heat-induced aggregation. Non-denaturing PAGE showed that NtHSP17.6 formed a dodecamer in its native conformation, and was bound to its substrate under heat stress. A labeling test with bis-ANS indicated that this binding might be linked to newly exposed hydrophobic sites of the NtHSPI 7.6 complexes during heat shock. Based on these data, we suggest that NtHSP17.6 is a molecular chaperone that functions as a dodecamer in a heat-induced manner.     

The small heat-shock chaperone protein, alpha-crystallin, does not recognize stable molten globule states of cytosolic proteins

The small heat-shock protein (sHsp), alpha-crystallin, acts as a molecular chaperone by interacting with destabilized 'substrate' proteins to prevent their precipitation from solution under conditions of stress. alpha-Crystallin and all sHsps are intracellular proteins. Similarly to other chaperones, the 'substrate' protein is in an intermediately folded, partly structured molten globule state when it interacts and complexes with alpha-crystallin. In this study, stable molten globule states of the cytosolic proteins, gamma-crystallin and myoglobin, have been prepared. Within the lens, gamma-crystallin naturally interacts with alpha-crystallin and myoglobin and alpha-crystallin are present together in muscle tissue. The molten globule states of gamma-crystallin and myoglobin were prepared by reacting gamma-crystallin with glucose 6-phosphate and by removing the haem group of myoglobin. Following spectroscopic characterisation of these modified proteins, their interaction with alpha-crystallin was examined by a variety of spectroscopic and protein chemical techniques. In both cases, there was no interaction with alpha-crystallin that led to complexation. It is concluded that alpha-crystallin does not recognise stable molten globule states of cytosolic 'substrate' proteins and only interacts with molten globule states of proteins that are on the irreversible pathway towards an aggregated and precipitated form.     

Translational control by neuroguidin, a eukaryotic initiation factor 4E and CPEB binding protein

CPEB-mediated translation is important in early development and neuronal synaptic plasticity. Here, we describe a new eukaryotic initiation factor 4E (eIF4E) binding protein, Neuroguidin (Ngd), and its interaction with CPEB. In the mammalian nervous system, Ngd is detected as puncta in axons and dendrites and in growth cones and filopodia. Ngd contains three motifs that resemble those present in eIF4G, 4EBP, Cup, and Maskin, all of which are eIF4E binding proteins. Ngd binds eIF4E directly, and all three motifs must be deleted to abrogate the interaction between these two proteins. In injected Xenopus oocytes, Ngd binds CPEB and, most importantly, represses translation in a cytoplasmic polyadenylation element (CPE)-dependent manner. In Xenopus embryos, Ngd is found in both neural tube and neural crest cells. The injection of morpholino-containing antisense oligonucleotides directed against ngd mRNA disrupts neural tube closure and neural crest migration; however, the wild-type phenotype is restored by the injection of a rescuing ngd mRNA. These data suggest that Ngd guides neural development by regulating the translation of CPE-containing mRNAs.     

Different expression time of the 105-kDa protein and 90-kDa heat-shock protein in rat testis

To understand the physiological functions of the 105-kDa protein which is testis-specific and HSP90 (90-kDa heat-shock protein) related protein, the appearance of it in the testis has been followed during the development of rat. On immunoblotting analysis, the 105-kDa protein did not appear until after the age of five weeks, while HSP90 could be detected at three weeks. In the spermatozoa, the 105-kDa protein was much abundant but not in the LC-540 cells (a cell line from Leydig cell tumor in rat testis) cytosol. This finding has attracted much attention to the relationship between this protein and sperm functions.     

De novo translation initiation on membrane-bound ribosomes as a mechanism for localization of cytosolic protein mRNAs to the endoplasmic reticulum

The specialized protein synthesis functions of the cytosol and endoplasmic reticulum compartments are conferred by the signal recognition particle (SRP) pathway, which directs the cotranslational trafficking of signal sequence-encoding mRNAs from the cytosol to the endoplasmic reticulum (ER). Although subcellular mRNA distributions largely mirror the binary pattern predicted by the SRP pathway model, studies in mammalian cells, yeast, and Drosophila have also demonstrated that cytosolic protein-encoding mRNAs are broadly represented on ER-bound ribosomes. A mechanism for such noncanonical mRNA localization remains, however, to be identified. Here, we examine the hypothesis that de novo translation initiation on ER-bound ribosomes serves as a mechanism for localizing cytosolic protein-encoding mRNAs to the ER. As a test of this hypothesis, we performed single molecule RNA fluorescence in situ hybridization studies of subcellular mRNA distributions and report that a substantial fraction of mRNAs encoding the cytosolic protein GAPDH resides in close proximity to the ER. Consistent with these data, analyses of subcellular mRNA and ribosome distributions in multiple cell lines demonstrated that cytosolic protein mRNA-ribosome distributions were strongly correlated, whereas signal sequence-encoding mRNA-ribosome distributions were divergent. Ribosome footprinting studies of ER-bound polysomes revealed a substantial initiation codon read density enrichment for cytosolic protein-encoding mRNAs. We also demonstrate that eukaryotic initiation factor 2α is bound to the ER via a salt-sensitive, ribosome-independent mechanism. Combined, these data support ER-localized translation initiation as a mechanism for mRNA recruitment to the ER.     

Associations between interleukin-6 genetic polymorphisms and levels of autoantibodies to 60-kDa heat-shock proteins

AIMS: Previously we reported an association between levels of anti-Hsp60 autoantibodies and interleukin-6 (IL-6) -174 SNP in Finnish population. The aim of this study was to investigate the same association in an independent population and to study four recently described SNP in IL-6. MATERIALS AND METHODS: 313 healthy Hungarian subjects were recruited and genotyped for IL-6 -174(G-->C), -9316(T-->C), -1363(G-->T), +1753(C-->G), +2954(G-->C). IgG antibodies to Hsp60 were measured by ELISA. LD between SNPs was computed by Haploview 3.2 software. RESULTS: A strong association between IL-6 -174 polymorphism and anti-Hsp60 autoantibody levels was observed. Carriers of -174 CC genotype had significantly lower levels of anti-Hsp60 (p = 0.0052). Eight haplotypes were observed with five SNP-s and autoantibody levels in individuals carrying the most common haplotype (containing allele C of -174) were significantly lower than in all other genotype combinations (p = 0.026). CONCLUSIONS: Allele C of -174 promoter polymorphism of the IL-6 gene was repeatedly shown to be associated with low anti-Hsp60 autoantibody levels. Strong linkage in the IL-6 gene was observed and the most frequent haplotype containing the -174 C allele was significantly associated with autoantibody levels. Since the -174 SNP of IL-6 is a functional polymorphism, our results indicate for a direct regulatory effect of IL-6 genotypes in the determination of autoantibody levels.     

Translational thermotolerance provided by small heat shock proteins is limited to cap-dependent initiation and inhibited by 2-aminopurine

Heat shock results in inhibition of general protein synthesis. In thermotolerant cells, protein synthesis is still rapidly inhibited by heat stress, but protein synthesis recovers faster than in naive heat-shocked cells, a phenomenon known as translational thermotolerance. Here we investigate the effect of overexpressing a single heat shock protein on cap-dependent and cap-independent initiation of translation during recovery from a heat shock. When overexpressing alphaB-crystallin or Hsp27, cap-dependent initiation of translation was protected but no effect was seen on cap-independent initiation of translation. When Hsp70 was overexpressed however, both cap-dependent and -independent translation were protected. This finding indicates a difference in the mechanism of protection mediated by small or large heat shock proteins. Phosphorylation of alphaB-crystallin and Hsp27 is known to significantly decrease their chaperone activity; therefore, we tested phosphorylation mutants of these proteins in this system. AlphaB-crystallin needs to be in its non-phosphorylated state to give protection, whereas phosphorylated Hsp27 is more potent in protection than the unphosphorylatable form. This indicates that chaperone activity is not a prerequisite for protection of translation by small heat shock proteins after heat shock. Furthermore, we show that in the presence of 2-aminopurine, an inhibitor of kinases, among which is double-stranded RNA-activated kinase, the protective effect of overexpressing alphaB-crystallin is abolished. The synthesis of the endogenous Hsps induced by the heat shock to test for thermotolerance is also blocked by 2-aminopurine. Most likely the protective effect of alphaB-crystallin requires synthesis of the endogenous heat shock proteins. Translational thermotolerance would then be a co-operative effect of different heat shock proteins.     

Nucleotide sequence of a cDNA for a member of the human 90-kDa heat-shock protein family

This paper describes the isolation and sequence of a human cDNA homologous to a class of proteins commonly referred to as 90-kDa heat-shock proteins. The complete nucleotide sequence of 2563 bp and the deduced amino acid sequence are presented. A single long open reading frame encodes a protein of 83,303 Da, the amino acid composition of which correlates well with that determined for the human 90-kDa heat-shock or 'stress' protein [Welch, W.J. and Feramisco, J.R., J. Biol. Chem. 257 (1982) 14949-14959]. Moreover, sequence analysis of this gene reveals extensive homology with the Drosophila 83-kDa and yeast 90-kDa heat-shock proteins. A comparison of the translated product of the human cDNA to the published yeast 90-kDa heat-shock protein reveals more than 60% homology at both the nucleotide and amino acid levels. Several regions of 50 aa or more show greater than 90% identity. This cDNA also hybridizes with an RNA species which increases upon heat shock of HeLa cells.     

Synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein in the chloroplast membranes of peas and Chlamydomonas reinhardi

The synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein (HSP) in the chloroplast membranes was studied in pea plants and Chlamydomonas reinhardi. HSPs were detected in both systems by in vivo labeling and in vitro translation of poly(A)⁺RNA, using the wheat-germ and reticulocyte lysate systems. Heat-shock treatment of pea plants for 2 h at 42-45°C induces the expression of ˜10 nuclear coded proteins, among which several (18 kd, 19 kd, 22 kd) are predominant. A 22-kd protein is synthesized as a 26-kd precursor protein and is localized in a chloroplast membrane fraction in vivo. Following post-translational transport into intact chloroplasts in vitro of the 26-kd precursor, the protein is processed but the resulting 22-kd mature protein is localized in the chloroplast stroma. If, however, the in vitro transport is carried out with chloroplasts from heat-shocked plants, the 22-kd protein is preferentially transported to the chloroplast membrane fraction. In C. reinhardi the synthesis of poly(A)⁺RNAs coding for several HSPs is progressively and sequentially induced when raising the temperature for 1.5 h from 36°C to 42°C, while that of several preexisting RNAs is reduced. Various pre-existing poly(A)⁺RNAs endure in the cells at 42°C up to 5 h but are no longer translated in vivo, whereas some poly(A)⁻RNAs persist and are translated. As in pea, a poly(A)⁺RNA coded 22-kd HSP is localized in the chloroplast membranes in vivo, although it is translated as a 22-kd protein in vitro. The in vitro translated protein is not transported in isolated pea chloroplast which, however, processes and transports other nuclear coded chloroplast proteins of Chlamydomonas. The poly(A)⁺RNA coding for the 22-kd HSP appears after 1 h at 36°C. Its synthesis increases with the temperature of incubation up to 42°C, although it decreases after ˜2 h of heat treatment and the already synthesized RNA is rapidly degraded. The degradation is faster upon return of the cells to 26°C. None of the heat-induced proteins is identical to the light-inducible proteins of the chloroplast membranes.     

Translational control by a small RNA: dendritic BC1 RNA targets the eukaryotic initiation factor 4A helicase mechanism

Translational repressors, increasing evidence suggests, participate in the regulation of protein synthesis at the synapse, thus providing a basis for the long-term plastic modulation of synaptic strength. Dendritic BC1 RNA is a non-protein-coding RNA that represses translation at the level of initiation. However, the molecular mechanism of BC1 repression has remained unknown. Here we identify the catalytic activity of eukaryotic initiation factor 4A (eIF4A), an ATP-dependent RNA helicase, as a target of BC1-mediated translational control. BC1 RNA specifically blocks the RNA duplex unwinding activity of eIF4A but, at the same time, stimulates its ATPase activity. BC200 RNA, the primate-specific BC1 counterpart, targets eIF4A activity in identical fashion, as a result decoupling ATP hydrolysis from RNA duplex unwinding. In vivo, BC1 RNA represses translation of a reporter mRNA with 5' secondary structure. The eIF4A mechanism places BC RNAs in a central position to modulate protein synthesis in neurons.     

Beta-internexin is a microtubule-associated protein identical to the 70-kDa heat-shock cognate protein and the clathrin uncoating ATPase

We have examined the relationship of the ubiquitous 68-70-kDa cytoskeletal-associated protein beta-internexin (Napolitano, E. W., Pachter, J. S., Chin, S. S. M., and Liem, R. K. H. (1985) J. Cell Biol. 101, 1323-1331) to heat-shock cognate 70 (hsc70), the major constitutive member of the mammalian heat-shock protein 70 (hsp70) family of stress proteins. We purify beta-internexin from rat brain microtubules and confirm its identity with hsc70 and the clathrin-uncoating ATPase by the following criteria: 1) The partial sequence of a cyanogen bromide-derived peptide from beta-internexin matches the inferred amino acid sequence of the cDNA clone pRC62 encoding hsc70 from rat brain (O'Malley, K., Mauron, A., Barchas, J. D., and Kedes, L. (1985) Mol. Cell. Biol. 5, 3476-3483). 2) Mixing experiments followed by two-dimensional gel analyses reveal the precise co-migration of beta-internexin, the clathrin-uncoating ATPase, and the in vitro translation product of cDNA clone pHSP-4 encoding rat brain hsc70. 3) beta-Internexin is recognized by a monoclonal antibody reactive against the class of hsp70 proteins. 4) beta-Internexin purified from a microtubule-associated protein-enriched fraction of rat brain by virtue of high affinity binding to ATP-agarose possesses clathrin cage-specific ATPase activity.     

Expression and cellular localization of the mRNA for the 25-kDa heat-shock protein in the mouse

The 25-kDa heat-shock protein (Hsp25) is a member of the small heat-shock protein family but its function remains largely unknown. In the present study we examined the expression and cellular localization of Hsp25 mRNA in mice under physiological, unstressed conditions using Northern blot and in situ hybridization analyses with specific oligonucleotide probes. At the organ level, high amounts of Hsp25 mRNA were detected in the oesophagus, skin,eye, stomach, lung and urinary bladder, with moderate amounts in the heart, skeletal muscle, aorta, adrenal gland, ovary, testis, uterus, large intestine, and thymus. At the cellular level, intense to moderate signals for Hsp25 mRNA were localized in the muscle cells of smooth, heart and skeletal types, in the epithelial cells of stratified squamous and transitional types and of the oviduct, in the steroid endocrine cells of the adrenal cortex and corpus luteum, as well as in the spermatocytes of the testis. In contrast, the signal was scarcely detectable in the nervous tissues, lymphatic tissues, the columnar epithelial cells of the digestive tract, or the parenchymal cells of the liver, pancreas and kidney. These results suggest some significant role for Hsp25 in distinct populations of mouse cells under physiological conditions.     

Temperature- and pH-dependent accumulation of heat-shock proteins in the acidophilic green alga Chlamydomonas acidophila

Chlamydomonas acidophila, a unicellular green alga, is a dominant phytoplankton species in acidic water bodies, facing severe environmental conditions such as low pH and high heavy metal concentrations. We examined the pH-, and temperature-dependent accumulation of heat-shock proteins in this alga to determine whether heat-shock proteins play a role in adaptation to their environment. Our results show increased heat-shock proteins accumulation at suboptimal pHs, which were not connected with any change in intracellular pH. In comparison to the mesophilic Chlamydomonas reinhardtii, the acidophilic species exhibited significantly higher accumulations of heat-shock proteins under control conditions, indicating an environmental adaptation of increased basal levels of heat-shock proteins. The results suggest that heat-shock proteins might play a role in the adaptation of C. acidophila, and possibly other acidophilic algae, to their extreme environment.     

Identification and characterisation of a Leishmania donovani antigen belonging to the 70-kDa heat-shock protein family

A Leishmania donovani promastigote cDNA library was screened with serum obtained from a patient infected with visceral leishmaniasis. Sequence analysis of a clone obtained from this library revealed that the 600-bp insert corresponded to the carboxy-terminal region of an antigen related to the 70-kDa heat-shock protein family. The full-length sequence of the corresponding gene (1959 nucleotides) was determined after isolation of genomic clones. Genes encoding the antigen are present on a single chromosome as a series of approximately twelve 3.7-kb direct tandem repeats. The antigen can be identified as a 70-kDa heat-shock cognate protein by virtue of its molecular mass, sequence and constitutive expression during heat shock. It is expressed at all stages of the parasite life-cycle. Antibodies against the lambda gt11 fusion protein were detected in more than 50% of serum samples obtained from patients with visceral leishmaniasis, but were not detected in sera from patients with cutaneous leishmaniasis or Chagas' disease.