Structure and expression of a human gene coding for a 71 kd heat shock ''cognate'' protein.

In all eukaryotes examined so far, hsp70 gene families include cognate genes (hsc70) encoding proteins of about 70 Kd which are expressed constitutively during normal growth and development. We have investigated the structural relationship of heat-inducible and cognate members of the human hsp70 gene family. Among several human genomic clones isolated using Drosophila hsp/hsc70 probes, one contained an hsc70 gene. Its complete sequence is reported here. It is split by eight introns and encodes a predicted protein of 70899 d that would be 81% homologous to hsp70. Structural comparisons with corresponding genes from other species provide one of the most striking examples of gene conservation. Isolation of a corresponding cDNA clone, RNA-mapping and in vitro translation data demonstrate that the gene is expressed constitutively and directs the synthesis of a 71 kd protein. The latter is very likely to be identical to a clathrin uncoating ATPase recently identified as a member of the hsp70-like protein family.     

Mutations in cognate genes of Saccharomyces cerevisiae hsp70 result in reduced growth rates at low temperatures.

Expression of two Saccharomyces cerevisiae genes (YG101 and YG103) that are related to the gene encoding inducible 70K protein (hsp70) is repressed upon heat shock. Mutations of the two genes were constructed in vitro and substituted into the yeast genome in place of the wild-type alleles. No phenotypic effect of single mutations of either gene was detected. However, cells containing both YG101 and YG103 mutations showed altered growth properties; double-mutation cells possess an optimal growth temperature of 37 degrees C rather than 30 degrees C and grow increasingly poorly as the temperature is lowered. Mutations of two other members of this hsp70-related multigene family, YG100 and YG102, have been analyzed (E. A. Craig and K. Jacobsen, Cell 38:841-849, 1984). Cells containing both YG100 and YG102 mutations cannot form colonies at 37 degrees C. Fusions between the YG101 and YG102 promoter regions and the YG100 and YG101 structural genes, respectively, were constructed. The YG101 promoter-YG100 structural gene fusion was not able to restore normal growth properties to the yg101- yg103- mutant. Also, yg100- yg102- cells containing the YG102 promoter-YG101 structural gene fusion were unable to grow at 37 degrees C. Failure of the protein products of related genes to rescue the relative cold sensitivity of growth suggests that members of the hsp70 multigene family are functionally distinct.     

Expression and localization of Drosophila melanogaster hsp70 cognate proteins.

Monoclonal antibodies have been used to identify three proteins in Drosophila melanogaster that share antigenic determinants with the major heat shock proteins hsp70 and hsp68. While two of the proteins are major proteins at all developmental stages, one heat shock cognate protein, hsc70, is especially enriched in embryos. hsc70 is shown to be the product of a previously identified gene, Hsc4. We have examined the levels of hsp70-related proteins in adult flies and larvae during heat shock and recovery. At maximal induction in vivo, hsp70 and hsp68 never reach the basal levels of the major heat shock cognate proteins. Monoclonal antibodies to hsc70 have been used to localize it to a meshwork of cytoplasmic fibers that are heavily concentrated around the nucleus.     

Analysis of protein binding to heat shock protein 70 in pancreatic islet cells exposed to elevated temperatures or interleukin 1 beta

To elucidate a role for heat shock proteins in islet function, isolated pancreatic islets were labeled with [35S]methionine after control, heat shock, or interleukin 1 beta (IL-1 beta) treatment, extracted in the presence of detergent, and then passed over affinity columns with antibodies against heat shock protein 70 (hsp 70), hsp 70 itself, or ATP conjugated to the columns. In control or IL-1 beta-treated islets, the antibody column efficiently absorbed hsp 70 together with two other proteins of molecular masses 46 and 53 kDa. In extracts from heat-shocked cells, the binding of cellularly synthesized hsp 70 to the antibody column was inefficient but improved by the addition of unlabeled partially purified hsp 70 to the extracts. When assessing the binding of proteins in the extracts to the hsp 70 column, hsp 70 and the 46- and 53-kDa proteins among others all bound to the column. No differences in the patterns of binding to the hsp 70 column between extracts from the different islet exposures were noticed. The 46-kDa protein was identified as actin by immunoblot analysis. ATP-agarose column chromatography revealed a pattern of binding similar to that of the hsp 70 column. It is concluded that hsp 70 contains at least two functional domains, one adjacent to the epitope recognized by the antibody and active in restoring cellular function after heat shock, whereas the other has the ability to bind the 46- and 53-kDa and possibly other proteins. Furthermore, the stress induced by heat shock differs significantly from that after IL-1 beta treatment with respect to the functional behavior of hsp 70.     

Mutations that induce the heat shock response of Drosophila

We have isolated a number of mutations in D. melanogaster that result in the constitutive expression of the heat shock response in a tissue-specific manner. These mutations induce alcohol dehydrogenase (ADH) when the ADH structural gene is fused to the promoter for the 70 kd heat shock protein (hsp70) gene. Flies carrying these mutations, the hsp70-Adh fusion, and a deletion in their endogenous Adh genes are ethanol tolerant and exhibit elevated ADH levels. Several of the tissue-specific mutations have also been shown to induce an hsp26-Adh fusion gene in trans. The mutation Act88FKM75, a G----A transition in the indirect flight muscle-specific actin gene, also exhibits this phenotype. Comparisons with the Act88FKM75 mutation suggest that the tissue-specific mutations induce the heat shock response by disrupting the physiology of the cells in which the variant gene product is expressed.     

The constitutive and stress inducible forms of hsp 70 exhibit functional similarities and interact with one another in an ATP- dependent fashion

Mammalian cells constitutively express a cytosolic and nuclear form of heat shock protein (hsp) 70, referred to here as hsp 73. In response to heat shock or other metabolic insults, increased expression of another cytosolic and nuclear form of hsp 70, hsp 72, is observed. The constitutively expressed hsp 73, and stress-inducible hsp 72, are highly related proteins. Still unclear, however, is exactly why most eukaryotic cells, in contrast to prokaryotic cells, express a novel form of hsp 70 (i.e., hsp 72) after experiencing stress. To address this question, we prepared antibodies specific to either hsp 72 or hsp 73 and have compared a number of biological properties of the two proteins, both in vivo and in vitro. Using metabolic pulse-chase labeling and immunoprecipitation analysis, both the hsp 72 and hsp 73 specific antibodies were found to coprecipitate a significant number of newly synthesized proteins. Such interactions appeared transient and sensitive to ATP. Consequently, we suspect that both hsp 72 and hsp 73 function as molecular chaperones, interacting transiently with nascent polypeptides. During the course of these studies, we routinely observed that antibodies specific to hsp 73 resulted in the coprecipitation of hsp 72. Similarly, antibodies specific to hsp 72 were capable of coprecipitating hsp 73. Using a number of different approaches, we show that the constitutively expressed, pre-existing hsp 73 rapidly forms a stable complex with the newly synthesized stress inducible hsp 72. As is demonstrated by double-label indirect immunofluorescence, both proteins exhibit a coincident locale within the cell. Moreover, injection of antibodies specific to hsp 73 into living cells effectively blocks the ability of both hsp 73 and hsp 72 to redistribute from the cytoplasm into the nucleus and nucleolus after heat shock. These results are discussed as they relate to the possible structure and function of the constitutive (hsp 73) and highly stress inducible (hsp 72) forms of hsp 70, both within the normal cell as well as in the cell experiencing stress.     

Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains three heat-inducible hsp70 genes. We have characterized the promoter region of the hsp70 heat shock gene YG100, that also displays a basal level of expression. Deletion of the distal region of the promoter resulted in an 80% drop in the basal level of expression without affecting expression after heat shock. Progressive-deletion analysis suggested that sequences necessary for heat-inducible expression are more proximal, within 233 base pairs of the initiation region. The promoter region of YG100 contains multiple elements related to the Drosophila melanogaster heat shock element (HSE; CnnGAAnnT TCnnG). Deletion of a proximal promoter region containing one element, HSE2, eliminated most of the heat-inducible expression of YG100. The upstream activation site (UAS) of the yeast cytochrome c gene (CYC1) can be substituted by a single copy of HSE2 plus its adjoining nucleotides (UASHS). This hybrid promoter displayed a substantial level of expression before heat shock, and the level of expression was elevated eightfold by heat shock. YG100 sequences that flank UASHS inhibited basal expression of UASHS in the hybrid promoter but not its heat-inducible expression. This inhibition of basal UASHS activity suggests that negative regulation is involved in modulating expression of this yeast heat shock gene.     

Biochemical and antigenic characterization of the Mycobacterium tuberculosis 71 kD antigen, a member of the 70 kD heat-shock protein family

A 71 kiloDalton antigen from Mycobacterium tuberculosis is recognized by antibodies and by T lymphocytes during infection (Britton et al., 1986a). Partial sequence analysis indicates a relationship between this antigen and the highly conserved family of 70-kiloDalton heat shock proteins (hsp70) (Young et al., 1988). Biochemical and serological characterization of the protein confirms its membership of the hsp70 gene family, and metabolic labelling demonstrates that it is a major component of the mycobacterial response to heat stress. The role of stress proteins as antigens during infection is discussed.     

The heat shock response in hydra: immunological relationship of hsp60, the major heat shock protein of Hydra vulgaris,to the ubiquitous hsp70 family

The major heat shock protein (hsp) of Hydra vulgaris has recently been found to be a 60 kDa protein. Since in all organisms studied so far, the major heat shock protein is a 70 kDa protein, we have analyzed the relationship of hydra hsp60 to the highly conserved 70 kDa heat shock protein family. Genes and proteins related to the 70 kDa class of stress proteins are present in hydra. However, antibodies known to cross-react with hsp70 proteins in several different organisms do not cross-react with hydra hsp60 suggesting that hsp60 is not related to the conserved hsp70 proteins.     

The ATPase core of a clathrin uncoating protein

Chymotryptic digestion of bovine brain uncoating ATPase produced a 60-kDa fragment that was subsequently proteolyzed to 44 kDa. Loss of clathrin cage uncoating activity paralleled the conversion of the intact 70-kDa enzyme to the 60-kDa fragment, while clathrin binding activity was lost as the 60-kDa fragment was degraded to 44 kDa. This 44-kDa fragment has been purified to homogeneity and characterized as a clathrin-independent ATPase. The 44-kDa ATPase domain has been localized within the intact enzyme by the use of amino-terminal specific antibodies. This localization relates to the conserved nature of the 70-kDa heat shock protein family, of which bovine brain uncoating ATPase is a constitutively expressed member.     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

Heat shock proteins affect RNA processing during the heat shock response of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the splicing of mRNA precursors is disrupted by a severe heat shock. Mild heat treatments prior to severe heat shock protect splicing from disruption, as was previously reported for Drosophila melanogaster. In contrast to D. melanogaster, protein synthesis during the pretreatment is not required to protect splicing in yeast cells. However, protein synthesis is required for the rapid recovery of splicing once it has been disrupted by a sudden severe heat shock. Mutations in two classes of yeast hsp genes affect the pattern of RNA splicing during the heat shock response. First, certain hsp70 mutants, which overproduce other heat shock proteins at normal temperatures, show constitutive protection of splicing at high temperatures and do not require pretreatment. Second, in hsp104 mutants, the recovery of RNA splicing after a severe heat shock is delayed compared with wild-type cells. These results indicate a greater degree of specialization in the protective functions of hsps than has previously been suspected. Some of the proteins (e.g., members of the hsp70 and hsp82 gene families) help to maintain normal cellular processes at higher temperatures. The particular function of hsp104, at least in splicing, is to facilitate recovery of the process once it has been disrupted.     

Mitochondrial GrpE is present in a complex with hsp70 and preproteins in transit across membranes.

We characterized a 24-kDa protein associated with matrix hsp70 (mt-hsp70) of Neurospora crassa and Saccharomyces cerevisiae mitochondria. By using specific antibodies, the protein was identified as MGE, a mitochondrial homolog of the prokaryotic heat shock protein GrpE. MGE extracted from mitochondria was quantitatively bound to hsp70. It was efficiently released from hsp70 by the addition of Mg-ATP but not by nonhydrolyzable ATP analogs or high salt. A mutant mt-hsp70, which was impaired in release of bound precursor proteins, released MGE in an ATP-dependent manner, indicating that precursor proteins and MGE bind to different sites of hsp70. A preprotein accumulated in transit across the mitochondrial membranes was specifically coprecipitated by either antibodies directed against MGE or antibodies directed against mt-hsp70. The preprotein accumulated at the outer membrane was not coprecipitated by either antibody preparation. After being imported into the matrix, the preprotein could be coprecipitated only by antibodies against mt-hsp70. We propose that mt-hsp70 and MGE cooperate in membrane translocation of preproteins.     

嗜热四膜虫五个hsp70基因的表达分析

鉴定得到嗜热四膜虫13个含有完整保守结构域的hsp70基因,对其中5个高度相似且无内含子的hsp70基因进行表达分析。在37、39和41℃热激条件下,实时荧光定量PCR结果表明,hsp70-2基因对热激响应最敏感。在四膜虫生长、饥饿和接合生殖这3种生理或发育状态下,Microarray结果显示,hsp70-4基因恒定且高表达;在热激条件下,hsp70-4基因的表达水平随着温度的升高而略微增加,证实hsp70-4基因为热休克相关蛋白hsc70基因;克隆的hsp70-4基因全长2208bp,开放阅读框长1959bp,编码653个氨基酸。Microarray结果提示,hsp70-3可能参与四膜虫饥饿早期(0～12h)的耐受和接合生殖后期(6～10h)的新大小核形成,老大核凋亡等事件;hsp70-5可能参与四膜虫饥饿晚期(12～15h)的耐受和接合生殖早期(0～6h)的小核减数分裂、小核交换和原核(pronuclear)融合事件。Blast2GO分析表明,与hsp70-3和hsp70-5共表达的基因分别参与不同的生物学过程,进一步反映了hsp70-3和hsp70-5这两个基因在功能上是存在差异的。