The Drosophila melanogaster homologue of the hsp60 gene is encoded by the essential locus l(1)10Ac and is differentially expressed during fly development

 The hsp60 (heat-shock protein 60) gene family of molecular chaperones has been a subject of study in numerous systems due to its important role in the correct folding of non-native proteins in development as well as after heat-shock treatment. Here we present the characterization of the first Drosophila hsp60 homologue. Drosophila HSP60 is most closely related (72% identity across the entire protein sequence) to the mouse mitochondrial HSP60. Western blot experiments indicate that Drosophila HSP60 is enriched in the mitochondrial fraction. The distribution of HSP60 protein is dynamic during fly embryogenesis, suggesting that various cell types might have different HSP60 requirements. The molecular analysis of a P-element-induced mutation that affects the l(1)10Ac locus shows that the transposon is inserted in a 3-kb intron present in the hsp60 gene. By genetic rescue experiments we prove that Drosophila HSP60 is encoded by the essential locus l(1)10Ac opening the possibility for detailed genetic analysis of HSP60 functions in the fly. Received: 24 March 1997 / Accepted: 16 June 1997     

Differential gene expression of HSC70/HSP70 in yellowtail cells in response to chaperone-mediated autophagy

A cell line derived from the tailfin of the marine teleost yellowtail fish Seriola quinqueradiata was established to examine cellular temperature regulation in an ectothermic animal. Three cytosolic members of the HSP70 family, heat-shock cognate proteins HSC70-1, HSC70-2 and heat-shock protein HSP70, were isolated from cultured yellowtail cells as stress-responsive biomarkers. Expression of hsp70 was heat-inducible, in contrast to the hsc70-1 gene product, which was expressed constitutively. In addition, expression of hsc70-2 was only induced under severe heat-shock conditions. Subcellular fractionation and immunocytochemistry showed localization of HSC70/HSP70 in the lysosomes, indicating that chaperone-mediated autophagy is induced by heat shock. Thus, chaperone-mediated autophagy is assisted by HSC70/HSP70, and heat-inducible expression of the genes encoding these proteins may be responsible for survival and adaptation under heat-shock conditions in fish cells.     

A Low Molecular Mass Heat-Shock Protein Is Localized to Higher Plant Mitochondria

When pea (Pisum sativum L. var Douce Provence) plants are shifted from a normal growth temperature of 25[deg] C up to 40[deg] C for 3 h, a novel 22-kD protein is produced and accumulates in the matrix compartment of green leaf mitochondria. HSP22 was purified and used as antigen to prepare guinea pig antiserum. The expression of HSP22 was studied using immunodetection methods. HSP22 is a nuclear-encoded protein de novo synthesized in heat-stressed pea plants. The heat-shock response is rapid and can be detected as early as 30 min after the temperature is raised. On the other hand, HSP22 declines very slowly after pea leaves have been transferred back to 25[deg] C. After 100 h at 25[deg] C, the heat-shock pattern was undetectable. The precise localization of HSP22 was investigated and we demonstrated that HSP22 was found only in mitochondria, where it represents 1 to 2% of total matrix proteins. However, the induction of HSP22 does not seem to be tissue specific, since the protein was detected in green or etiolated pea leaves as well as in pea roots. Finally, examination of matrix extracts by nondenaturing polyacrylamide gel electrophoresis and immunoblotting with anti-HSP22 serum revealed a high-molecular mass heat-shock protein complex of 230 kD, which contains HSP22.     

Characterization of HSP27 and three immunologically related polypeptides during Drosophila development

The low-molecular-weight heat-shock protein HSP27 is made in the absence of heat shock during Drosophila melanogaster development. An analysis of the accumulation of HSP27 during specific stages of development is presented using an antiserum recognizing this protein. Whereas HSP27 is abundant during embryogenesis, the level of this protein begins to decrease in the 20-h old embryo and is no longer detectable in second instar larvae. A high level of HSP27 is again observed in third instar larvae and reaches a maximal level in late pupae. While still abundant in young adult flies of both sexes, a greater amount of HSP27 is found in females with the protein being highly concentrated within the ovaries. Following lysis of whole pupae, about 60% of HSP27 is found in the soluble lysate fraction in a form which sediments between 5 and 20 S. Anti-HSP27 serum also recognizes three other developmentally regulated polypeptides with apparent MW of 33, 85 and 120 kDa. The 33 kDa protein accumulates in pupae while those of 85 and 120 kDa are more abundant in third instar larvae. Unlike HSP27, these proteins are not detected in embryos or ovaries. Immunoblot analysis of V8 proteolytic fragments suggests that HSP 27 and 33 kDa are related polypeptides. Exposure of the developing insect to heat-shock treatment results in increased level of HSP27. In larvae, a small amount of the 33 kDa protein accumulates following heat shock, while in pupae and adult flies a decrease in the concentration of this protein is observed after heat shock. Finally, different cellular localizations and distributions within the pupal body have been found for these developmentally regulated polypeptides.     

Changes in synthesis and localization of members of the 70-kDa class of heat-shock proteins accompany the induction of embryogenesis inBrassica napus L. microspores

Elevation of the culture temperature to 32°C for approximately 8 h can irreversibly change the developmental fate of isolatedBrassica napus microspores from pollen development to embryogenesis. This stress treatment was accompanied by de-novo synthesis of a number of heat-shock proteins (HSPs) of the 70-kDa class: HSP68 and HSP70. A detailed biochemical and cytological analysis was performed of the HSP68 and HSP70 isoforms. Eight HSP68 isoforms, one of which was induced three fold by the stress treatment, were detected on two-dimensional immunoblots. Immunocytochemistry revealed a co-distribution of HSP68 with DNA-containing organelles, presumably mitochondria. Six HSP70 isoforms were detected, one of which was induced six fold under embryogenic culture conditions. During normal pollen development, HSP70 was localized in the nucleoplasm during the S phase of the cell cycle, and predominantly in the cytoplasm during the remainder. Induction of embryogenic development in late unicellular microspores was accompanied by an intense anti-HSP70 labeling of the nucleoplasm during an elongated S phase. In early bicellular pollen the nucleus of the vegetative cell, which normally does not divide and never expresses HSP70, showed intense labeling of the nucleoplasm with anti-HSP70 after 8 h of culture under embryogenic conditions. These results demonstrate a strong correlation between the phase of the cell cycle, the nuclear localization of HSP70 and the induction of embryogenesis. As temperature stress alone is responsible for the induction of embryogenic development, and causes an altered pattern of cell division, there might be a direct involvement of HSP70 in this process.Abbreviations HSP heat-shock protein - 2-D two-dimensional - DAPI 4,6-diamidino-2-phenylindole. 1-D = one-dimensional - pI isoelectric point     

Structural organization of the spinach endoplasmic reticulum-luminal 70-kilodalton heat-shock cognate gene and expression of 70-kilodalton heat-shock genes during cold acclimation.

The 70-kD heat-shock proteins (HSP70s) are encoded by a multigene family in eukaryotes. In plants, the 70-kD heat-shock cognate (HSC70) proteins are located in organellar and cytosolic compartments of cells in most tissues. Previous work has indicated that HSC70 proteins of spinach (Spinacia oleracea) are actively synthesized during cold-acclimating conditions. We have isolated, sequenced, and characterized cDNA and genomic clones for the endoplasmic reticulum (ER) luminal HSC70 protein (immunoglobulin heavy chain-binding protein; BiP) of spinach. The spinach ER-luminal HSC70 is a constitutively expressed gene consisting of eight exons. Spinach BiP mRNA appears to be up-regulated during cold acclimation but is not expressed during water stress or heat shock. In contrast to the differential regulation of mRNA, the ER-luminal HSC70 protein levels remain constant in response to various environmental stresses. Two other members of the spinach 70-kD heat-shock (HS70) multigene family also show differential expression in response to a variety of environmental stresses. A constitutively expressed cytosolic HSC70 protein in spinach appears also to be up-regulated in response to both cold-acclimating and heat-shock treatments. Spinach also contains a cold-shock-induced HS70 gene that is not expressed during heat shock or water stress. Since HSP70s are considered to be involved with the chaperoning and folding of proteins, the data further support the concept that they may be important for maintaining cellular homeostasis and proper protein biogenesis during cold acclimation of spinach.     

The human heat-shock protein family. Expression of a novel heat-inducible HSP70 (HSP70B'') and isolation of its cDNA and genomic DNA.

The human heat-shock protein multigene family comprises several highly conserved proteins with structural and functional properties in common, but which vary in the extent of their inducibility in response to metabolic stress. We have isolated and characterized a novel human HSP70 cDNA, HSP70B' cDNA, and its corresponding gene sequence. HSP70B' cDNA hybrid-selected an mRNA encoding a more basic 70 kDa heat-shock protein that both the major stress-inducible HSP70 and constitutively expressed HSC70 heat-shock proteins, which in common with other heat-shock 70 kDa proteins bound ATP. The complete HSP70B' gene was sequenced and, like the major inducible HSP70 gene, is devoid of introns. The HSP70B' gene has 77% sequence similarity to the HSP70 gene and 70% similarity to HSC70 cDNA, with greatest sequence divergence towards the 3'-terminus. The HSP70B' gene represents a functional gene, as indicated by Northern-blot analysis with specific oligonucleotides, hybrid-selected translation with a specific 3' cDNA sequence and S1 nuclease protection experiments. In contrast with HSP70 mRNA, which is present at low concentrations in HeLa cells and readily induced by heat or CdCl2 treatment in both fibroblasts and HeLa cells, HSP70B' mRNA was induced only at higher temperature and showed no basal expression. The differences in patterns of induction may be due to the special features of the promoter region of the HSP70B' gene.     

Analysis by Confocal Microscopy of the Behavior of Heat Shock Protein 70 within the Nucleus and of a Nuclear Matrix Polypeptide during Prolonged Heat Shock Response in HeLa Cells

By means of confocal laser scanning microscopy and indirect fluorescence experiments we have examined the behavior of heat-shock protein 70 (HSP70) within the nucleus as well as of a nuclear matrix protein (M_r = 125 kDa) during a prolonged heat-shock response (up to 24 h at 42°C) in HeLa cells. In control cells HSP70 was mainly located in the cytoplasm. The protein translocated within the nucleus upon cell exposure to hyperthermia. The fluorescent pattern revealed by monoclonal antibody to HSP70 exhibited several changes during the 24-h-long incubation. The nuclear matrix protein showed changes in its location that were evident as early as 1 h after initiation of heat shock. After 7 h of treatment, the protein regained its original distribution. However, in the late stages of the hyperthermic treatment (17-24 h) the fluorescent pattern due to 125-kDa protein changed again and its original distribution was never observed again. These results show that HSP70 changes its localization within the nucleus conceivably because it is involved in solubilizing aggregated polypeptides present in different nuclear regions. Our data also strengthen the contention that proteins of the insoluble nucleoskeleton are involved in nuclear structure changes that occur during heat-shock response.     

Cellular localization of HSP23 during Drosophila development and following subsequent heat shock

The low-molecular-weight heat-shock protein HSP23 is synthesized in the absence of heat shock during Drosophila development. Here, I present a quantitative analysis of this phenomenon and describe the cellular localization of this protein during normal development and after a subsequent heat shock. HSP23 is first detected in the late third instar larvae and continues to accumulate reaching a maximum level in late pupae. In a 1-week-old adult, HSP23 can no longer be detected. Following lysis of whole pupae, HSP23 is found in the soluble lysate fraction in a form which sediments between 10 and 20 S. Exposure of larvae, pupae, and the adult fly to heat stress (37 degrees C) results in an increased amount of HSP23 which, however, is recovered in an insoluble particulate form following insect lysis. During recovery from heat shock, HSP23 is again found in the soluble 10- to 20-S lysate fraction. In pupae which are exposed to a severe heat stress (41 degrees C) HSP23 remains in the pellet fraction after the heat stress and no pupae are able to emerge as adult flies. However, when pupae are first exposed to a mild heat-shock treatment prior to the 41 degrees C stress, the thermotolerance process is induced and HSP23 is again rapidly found in the soluble lysate fraction during the recovery from heat shock. These observations suggest a possible correlation between the survival of pupae after heat shock and the recovery of HSP23 in the soluble lysate fraction as 10- to 20-S structures after the heat shock.     

Identification of a novel Drosophila melanogaster heat-shock gene,lethal(2)denticleless [l(2)dtl], coding for an 83-kDa protein

In this study, we describe the identification of a novel Drosophila melanogaster (Dm) gene, l(2)dtl, characterized by elevated expression under heat-shock (HS) conditions. It encodes a protein of 83 kDa with no homology to known members of the HSP90 family and other proteins. Gene l(2)dtl is located on the right arm of the second chromosome at locus 59F5, close to the tumor suppressor gene l(2)tid, a homolog of the dnaJ encoding a chaperone strongly conserved in evolution. In the following, we present the sequence of l(2)dtl, the putative protein it encodes, and its molecular localization in a closely interspaced gene cluster consisting of at least four nested genes spanning an approximately 10-kb genomic interval. Furthermore, we present the temporal expression of l(2)dtl in the wild type under normal and HS conditions, and describe the isolation and the phenotype of eight embryonic lethal l(2)dtl mutants.     

Spontaneous apoptosis and expression of cell surface heat-shock proteins in cultured EL-4 lymphoma cells

Abstract. The expression of heat-shock proteins (HSPs) is enhanced in stressed cells and can protect cells from stress-induced injury. However, existing data about the relationship between apoptosis and HSP expression is contradictory. In this paper, a mouse lymphoma cell death model system is used to detect simultaneously both the process of apoptosis and the level of HSP expression. The model was established after discovering that spontaneous apoptosis and spontaneous cell surface HSP expression occurs in EL-4 mouse lymphoma cells during normal optimal culture conditions. The data show that apoptotic EL-4 cells had higher levels of hsp25, hsp60, hsp70 and hsp90 exposed on the plasma membrane surface than viable cells. The level of surface HSPs was found to increase through several stages of early and late apoptotic death as measured by flow cytometry, with the highest levels observed during the loss of cell membrane phospholipid asymmetry. Heat shock and actinomycin D significantly increased the proportion of apoptotic cells in culture. However, hyperthermia only stimulated a weak and temporary increase in surface HSP expression, whereas actinomycin D strongly elevated the level of surface and intracellular HSPs, particularly in live cells. These results show an associative relationship between apoptosis and HSP expression. The relationship between the progression of cell death and HSP expression suggests a role for membrane HSP expression in programmed cell death.     

Seasonal variation in expression pattern of genes under HSP70

Heat shock protein 70 (HSP70) is one of the most abundant and best characterized heat shock protein family that consists of highly conserved stress proteins, expressed in response to stress, and plays crucial roles in environmental stress tolerance and adaptation. The present study was conducted to identify major types of genes under the HSP70 family and to quantify their expression pattern in heat- and cold-adapted Indian goats (Capra hircus) with respect to different seasons. Five HSP70 gene homologues to HSPA8, HSPA6, HSPA1A, HSPA1L, and HSPA2 were identified by gene-specific primers. The cDNA sequences showed high similarity to other mammals, and proteins have an estimated molecular weight of around 70 kDa. The expression of HSP70 genes was observed during summer and winter. During summer, the higher expression of HSPA8, HSPA6, and HSPA1A was observed, whereas the expression levels of HSPA1L and HSPA2 were found to be lower. It was also observed that the expression of HSPA1A and HSPA8 was higher during winter in both heat- and cold-adapted goats but downregulates in case of other HSPs. Therefore, both heat and cold stress induced the overexpression of HSP70 genes. An interesting finding that emerged from the study is the higher expression of HSP70 genes in cold-adapted goats during summer and in heat-adapted goats during winter. Altogether, the results indicate that the expression pattern of HSP70 genes is species- and breed-specific, most likely due to variations in thermal tolerance and adaptation to different climatic conditions.     

The effect of ts-mutation on expression of genes induced by heat shock in Drosophila melanogaster. III. Synthesis of proteins cognate to HSP70

2D-electrophoresis performed as described by O'-Farrel has revealed clear-cut differences in the pattern of proteins synthesized in the cells of wild-type flies and in the cells of ts-lethal. The cells of the mutant studied after heat-shock exhibit not only the lack of HSP83 and HSP35 but also the absence of a few of the heat-shock proteins belonging to the HSP70 group. Moreover, in the cells of the mutant an intensive synthesis of a protein with mol. weight 72 KDa was observed after heat-shock. This protein belongs to highly abundant heat-shock cognate proteins (HSCP) which are usually not induced by temperature elevation.     

The role of oxidative stress in the induction of Drosophila heat-shock proteins

The role of oxidative stress in the induction of heat-shock proteins (HSPs) was studied in Drosophila Kc cells by comparing the effects of two different inducers, temperature stress and reoxygenation following a period of anoxia, on cellular respiration, thiol status, and the accumulation of HSPs. A heat shock from 25 to 37 degrees C caused a 60% increase in the rate of O2 uptake but caused little oxidative stress as indicated by a constant level of reduced glutathione, a slight increase in oxidized glutathione, and no change in protein sulfhydryls. Heat shock resulted in a pronounced accumulation of HSPs which was not inhibited by anoxic conditions. A different HSP inducer, reoxygenation following anoxia, resulted in an overall inhibition of respiration, the appearance of CN -insensitive O2 uptake, a 50% decrease in the level of reduced glutathione and a fourfold increase in the ratio of oxidized to reduced glutathione. Despite these indicators of oxidative stress, HSP synthesis was less pronounced than observed during heat shock and was not affected by antioxidants. Oxidative stress may induce HSP synthesis in some cases but is not responsible for HSP synthesis during a heat shock.     

Genetic and molecular characterization of a variegating hsp70-acZ fusion gene in the euchromatic 31 B region of Drosophila melanogaster.

A hsp70-lacZ fusion gene introduced into Drosophila melanogaster at the euchromatic 31B region by Pelement transformation displayed a variegated expression with respect to the lacZ fusion protein in the salivary gland cells under heat-shock conditions. The variegation is also reflected by the chromosome puffing pattern. Subsequent transposition of the 31B P element to other euchromatic positions restored wild-type activity, that is, a nonvariegated phenotype. A lower developmental temperature reduced the amount of expression under heat-shock conditions, similar to genes undergoing position-effect variegation (PEV). However, other modifiers of PEV did not affect the expression pattern of the gene. These results show a novel euchromatic tissue-specific variegation that is not associated with classical heterochromatic PEV.