Localization of small heat shock proteins to the higher plant endomembrane system.

Three related gene families of low-molecular-weight (LMW) heat shock proteins (HSPs) have been characterized in plants. We describe a fourth LMW HSP family, represented by PsHSP22.7 from Pisum sativum and GmHSP22.0 from Glycine max, and demonstrate that this family of proteins is endomembrane localized. PsHSP22.7 and GmHSP22.0 are 76.7% identical at the amino acid level. Both proteins have amino-terminal signal peptides and carboxyl-terminal sequences characteristic of endoplasmic reticulum (ER) retention signals. The two proteins closely resemble class I cytoplasmic LMW HSPs, suggesting that they evolved from the cytoplasmic proteins through the addition of the signal peptide and ER retention motif. The endomembrane localization of these proteins was confirmed by cell fractionation. The polypeptide product of PsHSP22.7 mRNA was processed to a smaller-M(r) form by canine pancreatic microsomes; in vivo, GmHSP22.0 polysomal mRNA was found to be predominantly membrane bound. In vitro-processed PsHSP22.7 corresponded in mass and pI to one of two proteins detected in ER fractions from heat-stressed plants by using anti-PsHSP22.7 antibodies. Like other LMW HSPs, PsHSP22.7 was observed in higher-molecular-weight structures with apparent masses of between 80 and 240 kDa. The results reported here indicate that members of this new class of LMW HSPs are most likely resident ER proteins and may be similar in function to related LMW HSPs in the cytoplasm. Along with the HSP90 and HSP70 classes of HSPs, this is the third category of HSPs localized to the ER.     

Analysis of conserved domains identifies a unique structural feature of a chloroplast heat shock protein

Summary A low molecular weight heat shock protein which localizes to chloroplasts has been identified in several plant species. This protein belongs to a eukaryotic superfamily of small HSPs, all of which contain a conserved carboxyl-terminal domain. To investigate further the structure of this HSP, we isolated and sequenced cDNA clones for the chloroplast LMW HSPs from Petunia hybrida and Arabidopsis thaliana. The cloning of chloroplast HSPs from these two species enabled us to compare the amino acid sequences of this protein from plant species (petunia, Arabidopsis, pea, soybean and maize) that represent evolutionarily divergent taxonomic subclasses. Three conserved regions were identified, which are designated as regions I, II and III. Regions I and II are also shared by cytoplasmic LMW HSPs and therefore are likely to have functional roles common to all eukaryotic LMW HSPs. In contrast, consensus region III is not found in other LMW HSPs. Secondary structure analysis predicts that this region forms an amphipathic -helix with high conservation of methionine residues on the hydrophobic face and 100% conservation of residues on the hydrophilic face. This structure is similar to three helices, termed methionine bristles, which are found in a methionine-rich domain of a 54 kDa protein component of signal recognition particle (SRP54). The conservation of regions I and II among LMW cytoplasmic and chloroplast HSPs suggests that these HSPs perform related functions in different cellular compartments. However, identification of the methionine bristle domain suggests that chloroplast HSPs also have unique functions or substrates within the special environment of the chloroplast or other plastids.Abbreviations HS heat shock - HSP heat shock protein - LMW low molecular weight     

Expression of a Conserved Family of Cytoplasmic Low Molecular Weight Heat Shock Proteins during Heat Stress and Recovery

Plants synthesize several families of low molecular weight (LMW) heat shock proteins (HSPs) in response to elevated temperatures. We have characterized two cDNAs, HSP18.1 and HSP17.9, that encode members of the class I family of LMW HSPs from pea (Pisum sativum). In addition, we investigated the expression of these HSPs at the mRNA and protein levels during heat stress and recovery. HSP18.1 and HSP17.9 are 82.1% identical at the amino acid level and are 80.8 to 92.9% identical to class I LMW HSPs of other angiosperms. Heat stress experiments were performed using intact seedlings subjected to a gradual temperature increase and held at a maximum temperature of 30 to 42 degrees Celsius for 4 hours. HSP18.1 and HSP17.9 mRNA levels peaked at the beginning of the maximum temperature period and declined rapidly after the stress period. Antiserum against a HSP18.1 fusion protein recognized both HSP18.1 and HSP17.9 but not members of other families of LMW HSPs. The accumulation of HSP18.1-immunodetected protein was proportional to the severity of the heat stress, and the protein had a half-life of 37.7 ± 8 hours. The long half-life of these proteins supports the hypothesis that they are involved in establishing thermotolerance.     

Water relations and osmotic adjustment in Apium graveolens during long‐term NaCl stress and subsequent relief

Pea plants ( Pisum sativum L. cv. Feltham First) exposed to a heat stress of 37°C for 6 h accumulated two low molecular weight (LMW) heat shock proteins (HSPs) of molecular mass 22 kDa. The two LMW HSPs were associated with purified mitochondria. N‐terminal amino acid sequencing analysis indicates that the more basic of these proteins is a novel protein. The response of other cultivars of P. sativum to heat shock revealed that up to three 22‐kDa HSPs were expressed in a cultivar‐specific manner. Evidence presented suggests that the different 22‐kDa HSPs arise as a result of there being multiple 22‐kDa HSP genes. The expression of the most basic novel HSP was studied in the Feltham First cultivar using two dimensional SDS‐PAGE. Treatment of intact plants with chloramphenicol and cycloheximide prior to heat stress treatment indicated that the LMW HSPs were nuclear encoded and de novo synthesised. The response to heat shock was rapid with protein expression detected within 45 min and the protein remained in excess of 6 days following removal of the stress. The protein accumulated to very high levels with maximal expression being 2% of the total mitochondrial protein. The results are discussed in relation to the likely role of LMW HSPs in thermotolerance.     

Expression of sunflower low-molecular-weight heat-shock proteins during embryogenesis and persistence after germination: localization and possible functional implications

We isolated and sequenced Ha hsp 17.9, a DNA complementary (cDNA) of dry-seed stored mRNA that encodes a low-molecular-weight heat-shock protein (LMW HSP). Sequence analysis identified Ha hsp17.9, and the previously reported Ha hsp17.6, as cDNAs encoding proteins (HSP17.6 and HSP17.9) which belong to different families of cytoplasmic LMW HSPs. Using specific antibodies we observed differential expression of both proteins during zygotic embryogenesis under controlled environment, and a remarkable persistence of these LMW HSPs during germination. Immuno-blot analysis of HSP17.9 proteins in two-dimensional gels revealed that the polypeptides expressed in embryos were indistinguishable from LMW HSPs expressed in vegetative tissues in response to water deficit; but they appeared different from homologeous proteins expressed in response to thermal-stress. Tissue-print immunolocalization experiments showed that HSP17.9 and HSP17.6 were homogeneously distributed in every tissue of desiccation-tolerant dry seeds and young seedlings under non-stress conditions. These results demonstrate developmental regulation of specific, cytoplasmic, plant LMW HSPs, suggesting also their involvement in water-stress tolerance.     

Molecular cloning and characterization of rat estrogen receptor cDNA.

A cDNA clone of rat uterus estrogen receptor (ER) has been isolated and sequenced. This clone contains a complete open reading frame encoding 600 amino acid residues which is 5 and 11 amino acids larger than the corresponding molecules of human and chicken, respectively. The molecular weight of this protein is calculated to be 67,029. When this clone was ligated to the pSV2 vector and transfected into COS7 cells, a protein was produced that had the same affinity to estrogen as rat uterus ER. This sequence shows 88% homology with human ER; 528 amino acids are identical and 14 amino acids are conservative substitutions. The comparison of rat, human and chicken ER sequences indicate the presence of three highly conserved regions suggesting that these regions play important roles in ER function. The putative DNA-binding domain is completely identical in rat, human and chicken. The C-terminal half region which is thought to be the estrogen binding domain is also highly conserved and is rich in hydrophobic amino acid residues. Southern blot analysis of genomic DNA with ER cDNA as a probe has shown that related sequences are present in the genome.     

A heat shock protein localized to chloroplasts is a member of a eukaryotic superfamily of heat shock proteins.

We have isolated cDNA clones from soybean and pea that specify nuclear-encoded heat shock proteins (HSPs) which localize to chloroplasts. The mRNAs for these HSPs are undetectable at control temperatures, but increase approximately 150-fold during a 2-h heat shock. Hybridization-selection followed by in vitro translation demonstrates that these HSPs are synthesized as precursor proteins which are processed by the removal of 5-6.5 kd during import into isolated chloroplasts. The nucleotide sequence of the cDNAs shows the derived amino acid sequences of the mature pea and soybean proteins are 79% identical. While the predicted transit peptide encoded by the pea cDNA has some characteristics typical of transit sequences, including high Ser content, multiple basic residues and no acidic residues, it lacks two domains proposed to be important for import and maturation of other chloroplast proteins. The carboxy-terminal region of the chloroplast HSP has significant homology to cytoplasmic HSPs from soybean and other eukaryotes. We hypothesize that the chloroplast HSP shares a common structural and functional domain with low mol. wt HSPs which localize to other parts of the cell, and may have evolved from a nuclear gene.     

Characterization of a cDNA clone, encoding a 70 kDa heat shock protein from the dermatophyte pathogen Trichophyton rubrum

Trichophyton rubrum is an anthropophilic fungus causing up to 90% of chronic cases of dermatophytosis. To characterize T. rubrum proteins at the molecular level, we established a cDNA library of this pathogen. Here we describe a recombinant cDNA clone identical to eukaryotic 70kDa heat-shock proteins (HSPs). Western blot analysis using an anti HSP70 monoclonal antibody detected a recombinant fusion protein in Escherichia coli transformed with the expression vector containing the cloned cDNA insert. Southern blot analysis of T. rubrum genomic DNA detected no other members of the HSP70 gene family. Further analysis revealed the presence of two introns within the ORF of the HSP70 gene. In Northern blot analysis, the cDNA clone was hybridized to a RNA species of about 3.5kb which was constitutively expressed by cells cultured at 27 degrees C and was strongly up-regulated after culture at 37 degrees C. In summary, we have cloned the first member of the HSP family of dermatophytes and characterized it as a member of the Dnak subfamily of 70kDa HSPs.     

Cloning and sequencing of cDNAs encoding a pathogen-induced putative peroxidase of wheat (Triticum aestivum L.)

We report here the complete amino acid sequence of a pathogen-induced putative peroxidase from wheat (Triticum aestivum L.) as deduced from cDNA clones representing mRNA from leaves infected with the powdery mildew fungus Erysiphe graminis. The protein consists of 312 amino acids, of which the first 22 form a putative signal sequence, and has a calculated pI of 5.7. Sequence comparison revealed that the putative wheat peroxidase is most similar to the turnip (Brassica rapa) peroxidase, with which it shares 57% identical and 13% conserved amino acids.     

Cloning and characterization of the trout perforin

The pore-forming protein, perforin is one of the effectors of cell-mediated killing. A perforin cDNA clone was isolated from rainbow trout (Oncorhynchus mykiss) after screening of a spleen cDNA library. The full-length cDNA is 2070 bp in size, encoding for a polypeptide of 589 amino acids. The predicted amino acid sequence of the trout perforin is 64, 58 and 40% identical to those of Japanese flounder, zebrafish and human perforins, respectively. Although its membrane attack complex/perforin (MACPF) domain is conserved, trout perforin shows low homology to human and trout terminal complement components (C6, C7, C8 and C9), ranging from 19 to 26% identity. Expression analysis reveals that the trout perforin gene is expressed in the blood, brain, heart, kidney, intestine and spleen. Phylogenetic analysis of proteins which belong to the MACPF superfamily clusters the trout perforin in the same group with other known perforins.     

Accumulation, stability, and localization of a major chloroplast heat-shock protein

Diverse higher plant species synthesize low molecular weight (LMW) heat shock proteins (HSPs) which localize to chloroplasts. These proteins are homologous to LMW HSPs found in the cytoplasm of all eukaryotes, a class of HSPs whose molecular mode of action is not understood. To obtain basic information concerning the role of chloroplast HSPs, we examined the accumulation, stability, tissue specificity, and intra-chloroplast localization of HSP21, the major LMW chloroplast HSP in pea. Intact pea plants were subjected to heat stress conditions which would be encountered in the natural environment and HSP21 mRNA and protein levels were measured in leaves and roots. HSP21 was not detected in leaves or roots before stress, but the mature, 21-kD protein accumulated in direct proportion to temperature and HSP21 mRNA levels in both tissues. All of the HSP21 in leaves was localized to chloroplasts; there was no evidence for its transport into other organelles. In chloroplast fractionation experiments, greater than 80% of HSP21 was recovered in the soluble chloroplast protein fraction. The half-life of HSP21 at control temperatures was 52 +/- 12 h, suggesting the protein's function is critical during recovery as well as during stress. We hypothesize that HSP21 functions in a catalytic fashion in both photosynthetic and nonphotosynthetic plastids.     

Molecular characterization of a low-molecular-weight glutenin cDNA clone from Triticum durum

Summary A full-length, low-molecular-weight (LMW) glutenin cDNA clone, pTdUCD1, has been isolated from a Triticum durum cv Mexicali wheat cDNA library. The complete sequence was determined and compared to the LMW glutenin genes that have been isolated from hexaploid wheat, Triticum aestivum. This cDNA codes for a protein of 295 amino acids (33,414 daltons) including a 20-amino acid signal peptide as deduced from the DNA sequence. Northern analysis showed that this cDNA hybridizes to a family of related sequences ranging in length from 1,200 to 1,000 nucleotides. This gene is similar but not identical to previously published LMW glutenin gene sequences. The most striking characteristic of all cloned LMW glutenin genes is the conservation of eight cysteine residues, which could be involved in potential secondary or tertiary structure, disulfide bond interactions. This paper presents a structural map defining distinct regions of the LMW glutenin gene family.     

Sequence of carbonic anhydrase II cDNA from chick retina

Sequences of three cDNA clones for carbonic anhydrase II (CA-II) from chick retina are presented. The longest cDNA clone encodes all but the first three amino acids of CA-II, and the encoded sequence generally agrees with published fragments of CA-II sequence from chick red blood cells. It is 70% identical to human CA-II; the active-site residues are conserved, but the chick protein has six extra cysteines. There is a long 3'-untranslated region which contains a second open reading frame, but this is not conserved. There appears to be a single CA-II gene in the chick. Some anomalies in cDNA synthesis and in Bal31 deletion are noted.     

Expression of Low Molecular Weight Heat-Shock Proteins under Field Conditions

Heat-shock proteins (HSPs) are known to be expressed in plants experiencing high-temperature stress. We have examined the expression of class I cytoplasmic low molecular weight (LMW) HSPs and find that these HSPs also frequently accumulate in seeds, seed pods, and flowers during a normal growing season. We first examined the expression of class I cytoplasmic LMW HSPs by western blot analysis in a range of seed samples from both commercially grown and wild legumes. LMW HSPs were present in all seed samples, indicating that these HSPs are regularly expressed in these tissues. To examine more specifically conditions under which LMW HSPs were produced during an average growing season, additional studies of Medicago sativa were carried out during the fall season in Tucson, AZ. Plants were irrigated to avoid conditions of water stress, and canopy temperature was monitored throughout the study period. LMW HSP expression in leaves, flowers, and developing seed pods was analyzed by western blotting. Results show that in the field HSPs are frequently produced in flowers and seed pods, even in plants that show no HSP expression in leaves. Parallel greenhouse studies indicate that HSP expression in seeds is in part developmentally regulated. In total our data suggest a more widespread occurrence of HSPs in optimal growth environments and emphasize their potential role during reproduction.