Maize HSP101 plays important roles in both induced and basal thermotolerance and primary root growth

HSP101 belongs to the ClpB protein subfamily whose members promote the renaturation of protein aggregates and are essential for the induction of thermotolerance. We found that maize HSP101 accumulated in mature kernels in the absence of heat stress. At optimal temperatures, HSP101 disappeared within the first 3 days after imbibition, although its levels increased in response to heat shock. In embryonic cells, HSP101 concentrated in the nucleus and in some nucleoli. Hsp101 maps near the umc132 and npi280 markers on chromosome 6. Five maize hsp101-m-::Mu1 alleles were isolated. Mutants were null for HSP101 and defective in both induced and basal thermotolerance. Moreover, during the first 3 days after imbibition, primary roots grew faster in the mutants at optimal temperature. Thus, HSP101 is a nucleus-localized protein that, in addition to its role in thermotolerance, negatively influences the growth rate of the primary root. HSP101 is dispensable for proper embryo and whole plant development in the absence of heat stress.     

Induction of synthesis of Hsp104 of Saccharomyces cerevisiae in heat shock is controlled by mitochondria

Heat shock protein Hsp104 of Saccharomyces cerevisiae functions as a protector of cells against heat stress. When yeast are grown in media containing nonfermentable carbon sources, the constitutive level of this protein increases, which suggests an association between the expression of Hsp104 and yeast energy metabolism. In this work, it is shown that distortions in the function of mitochondria appearing as a result of mutation petite or after exposure of cells to the mitochondrial inhibitor sodium azide reduce the induction of Hsp104 synthesis during heat shock. Since the addition of sodium azide suppressed the formation of induced thermotolerance in the parent type and in mutant hsp104, the expression of gene HSP104 and other stress genes during heat shock is apparently regulated by mitochondria.     

Characterization of a heat-shock-inducible hsp70 gene of the green alga Volvox carteri

The green alga Volvox carteri possesses several thousand cells, but just two cell types: large reproductive cells called gonidia, and small, biflagellate somatic cells. Gonidia are derived from large precursor cells that are created during embryogenesis by asymmetric cell divisions. The J domain protein GlsA (Gonidialess A) is required for these asymmetric divisions and is believed to function with an Hsp70 partner. As a first step toward identifying this partner, we cloned and characterized V. carteri hsp70A, which is orthologous to HSP70A of the related alga Chlamydomonas reinhardtii. Like HSP70A, V. carteri hsp70A contains multiple heat shock elements (HSEs) and is highly inducible by heat shock. Consistent with these properties, Volvox transformants that harbor a glsA antisense transgene that is driven by an hsp70A promoter fragment express Gls phenotypes that are temperature-dependent. hsp70A appears to be the only gene in the genome that encodes a cytoplasmic Hsp70, so we conclude that Hsp70A is clearly the best candidate to be the chaperone that participates with GlsA in asymmetric cell division.     

A soybean 101-kD heat shock protein complements a yeast HSP104 deletion mutant in acquiring thermotolerance.

A cDNA clone encoding a 101-kD heat shock protein (HSP101) of soybean was isolated and sequenced. Genomic DNA gel blot analysis indicated that the corresponding gene is a member of a multigene family. The mRNA for HSP101 was not detected in 2-day-old etiolated soybean seedlings grown at 28 degrees C but was induced by elevated temperatures. DNA sequence comparison has shown that the corresponding gene belongs to the Clp (caseinolytic protease) (or Hsp100) gene family, which is evolutionarily conserved and found in both prokaryotes and eukaryotes. On the basis of the spacer length between the two conserved ATP binding regions, this gene has been identified as a member of the ClpB subfamily. Unlike other Clp genes previously isolated from higher plants, the expression of this soybean Hsp101 gene is heat inducible, and it does not have an N-terminal signal peptide for targeting to chloroplasts. Transformation of the soybean Hsp101 gene into a yeast HSP104 deletion mutant complemented restoration of acquired thermotolerance, a process in which cells survive an otherwise lethal heat stress after they are given a permissive heat treatment.     

The Induction of Saccharomyces cerevisiae Hsp104 Synthesis by Heat Shock Is Controlled by Mitochondria

Heat shock protein Hsp104 of Saccharomyces cerevisiae functions as a protector of cells against heat stress. When yeast are grown in media containing nonfermentable carbon sources, the constitutive level of this protein increases, which suggests an association between the expression of Hsp104 and yeast energy metabolism. In this work, it is shown that distortions in the function of mitochondria appearing as a result of mutation petite or after exposure of cells to the mitochondrial inhibitor sodium azide reduce the induction of Hsp104 synthesis during heat shock. Since the addition of sodium azide suppressed the formation of induced thermotolerance in the parent type and in mutant hsp104,the expression of gene HSP104 and other stress genes during heat shock is apparently regulated by mitochondria.     

Examination of the expression of the heat shock protein gene, hsp110, in Xenopus laevis cultured cells and embryos

Eukaryotic organisms respond to various stresses with the synthesis of heat shock proteins (HSPs). HSP110 is a large molecular mass HSP that is part of the HSP70/DnaK superfamily. In this study, we have examined, for the first time, the expression of the hsp110 gene in Xenopus laevis cultured cells and embryos. Sequence analysis revealed that the protein encoded by the hsp110 cDNA exhibited 74% identity with its counterparts in mammals and only 27-29% with members of the Xenopus HSP70 family. Hsp110 mRNA and/or protein was detected constitutively in A6 kidney epithelial cells and was inducible by heat shock, sodium arsenite, and cadmium chloride. However, treatment with ethanol or copper sulfate had no detectable effect on hsp110 mRNA levels. Similar results were obtained for hsp70 mRNA except that it was inducible with ethanol. In Xenopus embryos, hsp110 mRNA was present constitutively during development. Heat shock-inducible accumulation of hsp110 mRNA occurred only after the midblastula stage. Whole mount in situ hybridization analysis revealed that hsp110 mRNA accumulation in control and heat shocked embryos was enriched in selected tissues. These studies demonstrate that Xenopus hsp110 gene expression is constitutive and stress inducible in cultured cells and developmentally- and tissue specifically-regulated during early embryogenesis.     

Structure and Expression of a Heat-Shock Protein 83 Gene of Pharbitis nil

Four cDNA clones representing mRNAs whose levels were affected by a photoperiod that induces flowering in Pharbitis nil were isolated by a differential hybridization screening procedure. The level of mRNAs represented by three clones (12L, 15L, and 17L) increased following a photoperiod that induces flowering and that represented by the fourth clone (clone 27) increased under conditions in which flowering was inhibited. DNA sequence analysis showed that one cDNA, clone 17L, is homologous to members of the 83- to 90-kD heat-shock protein (hsp) gene family. The corresponding gene, hsp83A, was isolated and its DNA sequence was determined. hsp83A encodes a protein that exhibits 70% amino acid identity with Drosophila melanogaster HSP83. The P. nil hsp83A gene contains two introns within the coding region. hsp83A mRNA was not detectable in cotyledons of plants grown in continuous light, but its level increased transiently following a 14-h dark period and reached a maximum 2 h after the lights were turned on. A dramatic increase in the level of hsp83A mRNA was also found 2 h after an end-of-day dark treatment. Genomic Southern blot analysis demonstrated that the P. nil hsp83-90 gene family consists of at least six members, one of which appears to be constitutively expressed in the light.     

Small heat-shock protein Hsp9 has dual functions in stress adaptation and stress-induced G2-M checkpoint regulation via Cdc25 inactivation in Schizosaccharomyces pombe

The small heat-shock protein Hsp9 from Schizosaccharomyces pombe was previously reported to be a homologue of Saccharomyces cerevisiae HSP12. Although Hsp9 is expressed in response to heat shock and nutritional limitation, its function is still not completely understood. Here, we explored the biological function of Hsp9 in S. pombe. The hsp9 gene might play a role in stress adaptation; hsp9 deletion caused heat sensitivity and overexpression induced heat tolerance. In addition, Hsp9 also contribute to cell cycle regulation in the nucleus. Δhsp9 cells grew more quickly and were shorter in length than wild-type cells. Moreover, Δhsp9 cells did not achieve checkpoint arrest under stress conditions, leading to cell death, and exhibited a short doubling time and short G2 phase. Overexpression of hsp9 induced cell cycle delay, increased the population of G2 phase cells, and rescued the phenotypes of cdc2-33, cdc25-22, Δrad24, and Δrad25 mutants, suggesting that Hsp9 probably regulates Cdc2 phosphorylation by modulating the Cdc25 activity. Indeed, immunoprecipitation experiments revealed that Hsp9 is associated with 14-3-3 and Cdc25. In Δhsp9 cells, the association of 14-3-3 with Cdc25 was weakened and Cdc2 phosphorylaton was reduced. Together, our data suggest that Hsp9 has dual functions in stress adaptation and regulating a G2-M checkpoint by the Cdc25 inactivation; this differs from S. cerevisiae HSP12, which maintains cell membrane stability under stress conditions.     

Regulation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Plasma membrane H<Superscript>+</Superscript>-ATPase (Pma1) by Dextrose and Hsp30 during Exposure to Thermal Stress

Pma1p is an essential plasma membrane H⁺-pump in Saccharomyces cerevisiae that pumps out H⁺ at the expense of cellular ATP. Its activity is induced by glucose at 30°C and is inhibited by Hsp30 during exposure to heat shock conditions. To further investigate the regulation of Pma1 function by glucose and Hsp30 during exposure to thermal stress, we estimated Pma1 activity, its protein levels and ser-phosphorylation status in membrane fractions isolated from BY4741 and hsp30Δ cells grown in dextrose and sorbitol at 30°C, and following exposure at 40°C for 30 min. Our results demonstrate that Pma1 activity and protein levels were reduced in Hsp30⁺ cells following exposure to thermal stress in dextrose media. The above was not observed in hsp30Δ cells wherein Pma1 activity did not decrease following exposure to similar conditions. Although Pma1p levels decreased in heat-shocked hsp30Δ cells, it was lower compared to that observed in Hsp30⁺ cells. Total ser-phosphorylation of Pma1 also showed a decrease following exposure to heat shock condition in dextrose media in both BY4741 and hsp30Δ cells. Its levels were also reduced in BY4741 cells upon heat shock treatment in sorbitol unlike that observed in hsp30Δ cells wherein it was increased. Taken together the above indicate that heat shock induced reduction in Pma1 activity and protein levels in dextrose media required Hsp30. To examine functional interactions between dextrose utilization, Hsp30 and the regulation of various aspects of Pma1, we determined if dextrose regulated other functions attributed to Hsp30. Results demonstrate that the deletion of HSP30 rendered cells dependent on dextrose utilization for survival during exposure to lethal heat stress. Our study has hence been able to establish a functional relationship between glucose utilization, Hsp30 function and the regulation of Pma1 activity. Finally, since the deletion of HSP30 renders Pma1p levels and its activity unresponsive to thermal stress in dextrose media, we concluded that Hsp30 is necessary to maintain Pma1 in a regulation competent conformation. Hsp30 may thus act as a chaperone in the S. cerevisiae plasma membrane.     

The heat-induced molecular disaggregase Hsp104 of Candida albicans plays a role in biofilm formation and pathogenicity in a worm infection model

The consequences of deprivation of the molecular chaperone Hsp104 in the fungal pathogen Candida albicans were investigated. Mutants lacking HSP104 became hypersusceptible to lethally high temperatures, similarly to the corresponding mutants of Saccharomyces cerevisiae, whereas normal susceptibility was restored upon reintroduction of the gene. By use of a strain whose only copy of HSP104 is an ectopic gene under the control of a tetracycline-regulated promoter, expression of Hsp104 prior to the administration of heat shock could be demonstrated to be sufficient to confer protection from the subsequent temperature increase. This result points to a key role for Hsp104 in orchestrating the cell response to elevated temperatures. Despite their not showing evident growth or morphological defects, biofilm formation by cells lacking HSP104 proved to be defective in two established in vitro models that use polystyrene and polyurethane as the substrates. Biofilms formed by the wild-type and HSP104-reconstituted strains showed patterns of intertwined hyphae in the extracellular matrix. In contrast, biofilm formed by the hsp104Δ/hsp104Δ mutant showed structural defects and appeared patchy and loose. Decreased virulence of the hsp104Δ/hsp104Δ mutant was observed in the Caenorhabditis elegans infection model, in which high in vivo temperature does not play a role. In agreement with the view that stress responses in fungal pathogens may have evolved to provide niche-specific adaptation to environmental conditions, these results provide an indication of a temperature-independent role for Hsp104 in support of Candida albicans virulence, in addition to its key role in governing thermotolerance.     

Analysis of the variation in the hsp70-1 and hsp90alpha mRNA expression in human myocardial tissue that has undergone surgical stress

In the present work we reported a semiquantitative detection of messenger ribonucleic acids (mRNAs) encoding the human heat shock proteins Hsp70-1, the stress inducible member of the HSP70 family, and hsp90alpha, the inducible member of the HSP90 family. We investigated the change in the expression of these mRNAs in tissue samples taken from the right atrium of 48 pediatric patients, soon after the ischemic period during surgery to correct congenital heart diseases, in which a crystalloid cold cardioplegic solution was used. No significant variations were found for either hsp70-1 or hsp90alpha expressions. Moreover, we searched for an association between the hsp70-1 promoter region polymorphism and the expression of the hsp70-1 in a smaller group of these patients (n = 27). The -110AA genotype was on average significantly associated with a decrease in the hsp70-1 mRNA level (P < 0.05), whereas the other genotypes -110AC or -110CC did not seem to be associated with the hsp70-1 expression level. The lack of any observed increase in the hsp70-1 expression level may be due to the high basal level of the Hsp70 protein in the tissues examined.     

Functional and Structural Analysis of Maize Hsp101 IRES

Maize heat shock protein of 101 KDa (HSP101) is essential for thermotolerance induction in this plant. The mRNA encoding this protein harbors an IRES element in the 5′UTR that mediates cap-independent translation initiation. In the current work it is demonstrated that hsp101 IRES comprises the entire 5′UTR sequence (150 nts), since deletion of 17 nucleotides from the 5′ end decreased translation efficiency by 87% compared to the control sequence. RNA structure analysis of maize hsp101 IRES revealed the presence of three stem-loops toward its 5′ end, whereas the remainder sequence contains a great proportion of unpaired nucleotides. Furthermore, HSP90 protein was identified by mass spectrometry as the protein preferentially associated with the maize hsp101 IRES. In addition, it has been found that eIFiso4G rather than eIF4G initiation factor mediates translation of the maize hsp101 mRNA.