Steroid hormone regulation of the Achlya ambisexualis 85-kilodalton heat shock protein, a component of the Achlya steroid receptor complex.

The steroid hormone antheridiol regulates sexual development in the fungus Achlya ambisexualis. Analyses of in vivo-labeled proteins from hormone-treated cells revealed that one of the characteristic antheridiol-induced proteins appeared to be very similar to the Achyla 85-kilodalton (kDa) heat shock protein. Analysis of in vitro translation products of RNA isolated from control, heat-shocked, or hormone-treated cells demonstrated an increased accumulation of mRNA encoding a similar 85-kDa protein in both the heat-shocked and hormone-treated cells. Northern (RNA) blot analyses with a Drosophila melanogaster hsp83 probe indicated that a mRNA species of approximately 2.8 kilobases was substantially enriched in both heat-shocked and hormone-treated cells. The monoclonal antibody AC88, which recognizes the non-hormone-binding component of the Achyla steroid receptor, cross-reacted with Achlya hsp85 in cytosols from heat-shocked cells. This monoclonal antibody also recognized both the hormone-induced and heat shock-induced 85-kDa in vitro translation products. Taken together, these data suggest that similar or identical 85-kDa proteins are independently regulated by the steroid hormone antheridiol and by heat shock and that this protein is part of the Achyla steroid receptor complex. Our results demonstrate that the association of hsp90 family proteins with steroid receptors observed in mammals and birds extends also to the eucaryotic microbes and suggest that this association may have evolved early in steroid-responsive systems.     

Heat-shock-induced changes in the phosphorylation of ribosomal and ribosome-associated proteins in the filamentous fungus Achlya ambisexualis

In the filamentous fungus Achlya ambisexualis, heat shock resulted in a rapid reduction in the rate of protein synthesis. This was accompanied by dephosphorylation of a prominent basic 30 kD protein associated with the small subunit of Achlya ribosomes and which may be analogous to ribosomal protein S6 of vertebrates. A large ribosomal subunit protein with a relative molecular weight (MW) of 24,500 exhibited increased phosphorylation during heat shock, while a second large subunit protein having a relative MW of 22,000 was dephosphorylated. Several proteins which could be dissociated from Achlya ribosomes by 0.5 M KCl also exhibited altered patterns of phosphorylation during heat shock. These KCl-soluble proteins included proteins at 50, 21, 20 and 19 kD, which exhibited decreased phosphorylation with heat shock and proteins at 32 and 23.5 kD, which exhibited increased phosphorylation with heat shock. Such alterations in the phosphorylation of components of the Achlya translational apparatus may be involved in the qualitative and quantitative changes in protein synthesis which are observed with heat shock in Achlya.     

Reduced activity of the interferon-induced double-stranded RNA-dependent protein kinase during a heat shock stress

Previous studies have shown that the antiviral response induced by interferon in murine cells could be degraded after a heat shock. Here we have confirmed that a similar effect occurs also in interferon-treated human HeLa cells subjected to a heat shock. In addition, we have investigated the fate of the interferon-induced, double-stranded RNA-dependent protein kinase in heat-shocked cells. This protein kinase is a Mr 68,000 protein (p68 kinase) which, when autophosphorylated, catalyzes phosphorylation of the protein synthesis eukaryotic initiation factor-2, thus mediating inhibition of protein synthesis. After heat shock of interferon-treated HeLa cells, the double-stranded RNA-dependent autophosphorylation of p68 kinase in cytoplasmic extracts is greatly reduced whereas the phosphorylation of other cellular proteins is not affected. In vivo, autophosphorylation of p68 kinase is also reduced in heat-shocked cells whereas there is no apparent effect on the phosphorylation state of other proteins. In such cells, the interferon-mediated antiviral response becomes modified according to the virus challenge, i.e. these cells remain resistant to vesicular stomatitis virus but become partially sensitive to encephalomyocarditis virus (EMCV) infection. The reduction in the activity of p68 kinase is due to its reduced nonionic detergent solubility occurring during the heat shock period. The resultant reduced detergent extractibility of p68 kinase is dependent on the intensity of the thermal stress. In contrast to the effect after a heat shock, arsenite treatment of interferon-treated HeLa cells induces heat shock proteins, but neither modifies the antiviral response nor affects the extractibility of p68 kinase. These results indicate that the degradation of the anti-EMCV response and reduced p68 kinase activity occur in response to heat treatment independently of the induction of heat shock proteins. The role of p68 kinase in the mechanism of the antiviral response against EMCV and vesicular stomatitis virus is discussed.     

Effects of heat shock on neuroblastoma (N1E 115) cell proliferation and differentiation.

Heat shock (44 degrees C) applied for only 15 min induced the development of neurites in neuroblastoma cells 3-6 days later. During the first day after heat shock a transient increase in the rate of cytokinesis together with a synchronizing effect was observed, which led to waves of cytokinesis 14.5 h apart. Individual cell cycles were determined and showed a lengthening in the minimal cell cycle duration and a decrease in the cell cycle variance after shock. Two to 3 days after heat shock the proliferation rate decreased and then recovered. During the 6 days after heat shock, total protein synthesis was lower compared to the untreated cultures. The synthesis of heat shock proteins (100, 90, 84, 70, 68 kDa and some of lower MW) reached a maximum 6 h after heat shock. Parallel changes in the phosphorylation state of proteins were observed in an in vitro assay. Four proteins (100, 89, 67, and 15 kDa) increased and two proteins (97, 73 kDa) decreased their phosphorylation state significantly. Six days after heat shock two proteins (89, 55 kDa) increased their phosphorylation state; the 55-kDa phosphoprotein was identified as tubulin. The effect of heat shock on the intracellular calcium level was determined by measuring Fura 2 fluorescence. Six hours after shock, the Ca2+ level increased to a maximum (about three times the control value) and then dropped during the following days below the control values. We conclude from these results that a decrease in the calcium level may be causally involved in the differentiation process. The calcium effect is probably mediated by changes in the activity of different kinases. This assumption is compatible with the results of experiments with cyclic nucleotides when 10(-5) M cAMP and cGMP were added to in vitro assays of protein phosphorylation. They had different stimulating effects in heat-shocked, differentiating, and growing (control) cells.     

Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress

During stress, the mammalian small heat shock protein Hsp27 enters cell nuclei. The present study examines the requirements for entry of Hsp27 into nuclei of normal rat kidney (NRK) renal epithelial cells, and for its interactions with specific nuclear structures. We find that phosphorylation of Hsp27 is necessary for the efficient entry into nuclei during heat shock but not sufficient for efficient nuclear entry under control conditions. We further report that Hsp27 is recruited to an RNAse sensitive fraction of SC35 positive nuclear speckles, but not other intranuclear structures, in response to heat shock. Intriguingly, Hsp27 phosphorylation, in the absence of stress, is sufficient for recruitment to speckles found in post-anaphase stage mitotic cells. Additionally, pseudophosphorylated Hsp27 fused to a nuclear localization peptide (NLS) is recruited to nuclear speckles in unstressed interphase cells, but wildtype and nonphosphorylatable Hsp27 NLS fusion proteins are not. The expression of NLS-Hsp27 mutants does not enhance colony forming abilities of cells subjected to severe heat shock, but does regulate nuclear speckle morphology. These data demonstrate that phosphorylation, but not stress, mediates Hsp27 recruitment to an RNAse soluble fraction of nuclear speckles and support a site-specific role for Hsp27 within the nucleus.     

Subnuclear Trafficking of Glucocorticoid Receptors In Vitro: Chromatin Recycling and Nuclear Export

We have used digitonin-permeabilized cells to examine in vitro nuclear export of glucocorticoid receptors (GRs). In situ biochemical extractions in this system revealed a distinct subnuclear compartment, which collects GRs that have been released from chromatin and serves as a nuclear export staging area. Unliganded nuclear GRs within this compartment are not restricted in their subnuclear trafficking as they have the capacity to recycle to chromatin upon rebinding hormone. Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality. In addition, chromatin-released receptors export from nuclei of permeabilized cells in an ATP- and cytosol-independent process that is stimulated by sodium molybdate, other group VI-A transition metal oxyanions, and some tyrosine phosphatase inhibitors. The stimulation of in vitro nuclear export by these compounds is not unique to GR, but is restricted to other proteins such as the 70- and 90-kD heat shock proteins, hsp70 and hsp90, respectively, and heterogeneous nuclear RNP (hnRNP) A1. Under analogous conditions, the 56-kD heat shock protein, hsp56, and hnRNP C do not export from nuclei of permeabilized cells. If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited. Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.     

Heat shock impairs the interaction of cap-binding protein complex with 5' mRNA cap

Cell-free protein synthesizing systems prepared from heat-shocked Ehrlich cells retain the inhibition of translation that is seen at the cellular level. Recently, we showed that a highly purified cap-binding protein complex composed of the p220 and p28 subunits of eukaryotic initiation factor 4F, in a 1:1 molar ratio, restores protein synthesis in these cell-free translation systems (Lamphear, B.J., and Panniers, R. (1990) J. Biol. Chem. 265, 5333-5336). Here we have estimated the amount of cap-binding complex in cell extracts that can restore protein synthesis in heat-shocked cells. We find reduced restoring activity in heat-shocked cell extracts. Further, less cap-binding complex can be purified by 7-methyl-guanosine triphosphate Sepharose affinity chromatography from heat-shocked cell extracts, and we conclude that heat shock impairs the binding of complex to 5' mRNA cap. We have ruled out proteolysis and competitive inhibitors as mediators of this impairment. However we cannot distinguish between two possible explanations: (i) reduced association of p220 with p28 or (ii) a non-competitive inhibitor blocks complex binding to cap. We have also examined the affect of heat shock on the phosphorylation state of two forms of p28, p220.p28 complex and p28 free of p220. Both forms have reduced levels of phosphorylation during heat shock. The significance of these changes is discussed.     

Small heat shock protein p26 associates with nuclear lamins and HSP70 in nuclei and nuclear matrix fractions from stressed cells

The small heat shock/alpha-crystallin protein p26 undergoes nuclear translocation in response to stress in encysted embryos of the brine shrimp Artemia franciscana. About 50% of total p26 translocates to nuclei in embryos treated with heat shock or anoxia, and in embryo homogenates incubated at low pH. Nuclear fractionation shows that the majority of nuclear p26 and a nuclear lamin are associated with the nuclear matrix fraction. To further explore the roles of p26 and other HSPs in stabilizing nuclear matrix proteins (NMPs), nuclear matrices from control, and heat-shocked embryos were disassembled in urea and evaluated by one and two-dimensional (2-D) gel electrophoresis and Western immunoblotting after reassembling. Nuclear lamins were present only in reassembled fractions and, in the case of heat shock, p26 and HSP70 were also present. HSP90 was not detected in any nuclear fraction. Confocal microscopy on isolated nuclei and nuclear matrix preparations from control and heat-shocked embryos showed that the majority of p26 and a nuclear lamin share similar nuclear distributions. The combination of microscopy and fractionation results suggests that p26 and HSP70 play a role in the protection of nuclear lamins within the nuclear matrix.     

Excess protein in nuclei isolated from heat-shocked cells results from a reduced extractability of nuclear proteins

An excellent correlation has been established between the quantity of protein associated with nuclei isolated from heat-shocked cells and the level of hyperthermic cell killing. However, controversy remains about whether increases in nuclear-associated protein result from a heat-induced migration of cytoplasmic proteins into the nucleus or because hyperthermia reduces the solubility of nuclear proteins in the detergent buffers commonly used to isolate nuclei. To address this controversy, the nuclear protein content was measured in whole and detergent-extracted cells before and following hyperthermia. It was found that hyperthermia caused no significant change in the nuclear protein content of whole, unextracted cells, and when fluorescently labeled proteins were microinjected into the cytoplasm no gross change in the selective permeability of the nuclear membrane to soluble proteins was observed during or following hyperthermia. Measurements in extracted cells showed that the detergent buffers removed protein from both the nucleus and cytoplasm of control, nonheated cells and that hyperthermia reduced the extractability of both nuclear and cytoplasmic proteins. The amount of protein found in nuclei isolated from heated cells approached that observed in nuclei within nonheated whole cells as the hyperthermic exposure was increased. Thus, the dose-dependent, two- to threefold increase in the protein content of nuclei isolated from heated cells represents a heat-induced reduction in the extractability of proteins normally present within cell nuclei and does not result from a mass migration of cytoplasmic proteins into the nucleus, although some specific proteins (e.g., the 70 KDa heat shock protein) do migrate to the nucleus following heat shock. Differential scanning calorimetry (DSC) measurements of whole cells, isolated nuclei, cytoplasts, and karyoplasts supported these conclusions and suggested that most of the detergent-insoluble proteins remaining in the nuclei and cytoplasm of heated cells are in their native state. Thus, a relatively small amount of denatured protein may be sufficient to initiate and sustain insoluble protein aggregates comprised of mostly native proteins. Analyses of the DSC data also implied that the previously identified critical target proteins, predicted to have a Tm of 46.0°C, are present in both the nucleus and cytoplasm. © 1996 Wiley-Liss, Inc.     

Activation of hemin-regulated initiation factor-2 kinase in heat-shocked HeLa cells

Protein synthesis was drastically inhibited in HeLa cells incubated for 5 min at 42.5 degrees C, but it resumed after 20 min at a rate about 50% that of control cells. After 10 min of heat shock, the binding of Met-tRNAf to 40 S ribosomal subunits was greatly reduced and a polypeptide identified by immunoprecipitation with the alpha subunit of eukaryotic initiation factor-2 (eIF-2) was phosphorylated. Extracts prepared from control and heat-shocked cells were assayed for in vitro protein synthesis. Both extracts were active when supplemented with hemin, but the extract from heat-shocked cells had little initiation activity without this addition. A Mr 90,000 polypeptide and eIF-2 alpha were phosphorylated in this extract, but hemin or an antibody which inhibits the protein kinase designated heme-controlled repressor reduced this phosphorylation. These findings implicated heme-controlled repressor as the kinase at least in part responsible for eIF-2 alpha phosphorylation. Furthermore, the initial inhibition of protein synthesis and eIF-2 alpha phosphorylation after heat shock were reduced by adding hemin to intact HeLa cells. These cells synthesized heat-shock proteins with some delay relative to cells without added hemin. The binding of Met-tRNAf to 40 S ribosomal subunits was inhibited by about 50% in extracts prepared from cells heat-shocked for 40 min, and eIF-2 alpha phosphorylation was increased in these cells. These results suggest that heme-controlled repressor is activated in heat-shocked cells and that eIF-2 alpha phosphorylation limits mRNA translation even after partial recovery of protein synthesis.     

Cellular localization of steroid hormone-regulated proteins during sexual development in Achlya

In the fungus Achlya ambisexualis sexual development in the male strain E87 is controlled by the steroid hormone antheridiol. To investigate the effects of antheridiol on the synthesis and/or accumulation of specific cellular proteins we have analysed [35S]methionine-labeled proteins from control and hormone-treated cells using both one-dimensional (1D) and two-dimensional (2D) PAGE. Since in a total cell extract, hormone-induced changes in specific proteins might not be apparent against a background of more abundant proteins, cells were fractionated prior to protein isolation. It was also necessary to establish a concentration of hormone carrier, in this case methanol, which by itself did not alter the pattern of protein synthesis. Using these approaches the addition of the hormone antheridiol to vegetatively growing cells of Achlya E87 was found to result in changes in the synthesis and/or accumulation of at least 16 specific proteins, which could be localized to the cytoplasmic, nuclear or cell wall/cell membrane fractions. The most prominent changes observed in the hormone-treated cells included the appearance in the cytoplasmic fraction of labeled proteins at 28.4 and 24.3 kD which were not detectable in control cells, and a significant enrichment in the labeling of a 24.3 kD protein in the cell wall/cell membrane fraction. A marked increase in the labeling of 85, 63 and 47 kD proteins in the nuclear fraction from hormone-treated cells was also noted. The molecular weight (MW) and the behavior on 2D gels of the 85 kD hormone-induced protein appeared very similar to that of the 85 kD heat-shock protein reported in Achlya. Quantitive changes in the [35S]methionine labeling of several other proteins were noted in all three cell fractions.     

A normal mitochondrial protein is selectively synthesized and accumulated during heat shock in Tetrahymena thermophila.

We have identified and purified a 58-kilodalton protein of Tetrahymena thermophila whose synthesis during heat shock parallels that of the major heat shock proteins. This protein, hsp58, was found in both non-heat-shocked as well as heat-shocked cells; however, its concentration in the cell increased approximately two- to threefold during heat shock. The majority of hsp58 in both non-heat-shocked and heat-shocked cells was found by both cell fractionation studies and immunocytochemical techniques to be mitochondrially associated. During heat shock, the additional hsp58 was found to selectively accumulate in mitochondria. Nondenatured hsp58 released from mitochondria of non-heat-shocked or heat-shocked cells sedimented in sucrose gradients as a 20S to 25S complex. We suggest that this protein may play a role in mitochondria analogous to the role the major heat shock proteins play in the nucleus and cytosol.     

Cell cycle-dependent association of HSP70 with specific cellular proteins

In asynchronous populations of HeLa cells maintained at control or heat shock temperatures, HSP70 levels and its subcellular distribution exhibit substantial heterogeneity as demonstrated by indirect immunofluorescence with HSP70-specific monoclonal antibodies. Of particular interest is a subpopulation of cells in which the characteristic nuclear accumulation and nucleolar association of HSP70 is not detected after heat shock treatment. This apparent variation in the heat shock response is not observed when synchronized cells are examined. In this study, we demonstrate that three monoclonal antibodies to HSP70, in particular, do not detect nucleolar-localized HSP70 in heat-shocked G2 cells. This is not due to an inability of G2 cells to respond to heat shock as measured by increased HSP70 mRNA and protein synthesis, or due to a lack of accumulation of HSP70 after heat shock in G2. Rather the epitopes recognized by the various antibodies appear to be inaccessible, perhaps due to the association of HSP70 with other proteins. Non-denaturing immunoprecipitations with these HSP70-specific antibodies suggest that HSP70 may interact with other cellular proteins in a cell cycle-dependent manner.     

Changes in nuclear phenotypes following cold shock in Panstrongylus megistus (Burmeister)

The nuclear phenotypes of Malpighian tubule epithelial cells of 5th instar male nymphs of the blood-sucking insect Panstrongylus megistus were studied immediately after a short (1 h) cold shock at 0 degrees C, and 10 and 30 days later. The objective was to compare the responses to a cold shock with those known to occur after hyperthermia in order to provide insight into the cellular effect of cold in this species. Nuclei which usually exhibited a conspicuous Y chromosome chromocenter were the most frequent phenotype in control and treated specimens. Phenotypes in which the heterochromatin was unravelled, or in which there was nuclear fusion or cell death were more abundant in the shocked specimens. Most of the changes detected have also been found in heat-shocked nymphs, except for nuclear fusion which generates giant nuclei and which appeared to be less effective or necessary than that elicited after heat shock. Since other studies showed that a short cold shock does not affect the survival of more than 14% of 5th instar nymphs of P. megistus with domestic habit and can induce tolerance to a prolonged cold shock, heat shock proteins proteins are probably the best candidates for effective protection of the cells and the insects from drastic damage caused by low temperature shocks.     

Involvement of ATP in the nuclear and nucleolar functions of the 70 kd heat shock protein.

The major heat shock protein, hsp70, is an ATP-binding protein which is synthesized in very large amounts in response to stress. In unstressed, or recovered, mammalian cells it is found in both nucleus and cytoplasm. Under these conditions, its interaction with nuclei is weak, and it is readily released from them upon lysis of cells in isotonic buffer. After heat shock, hsp70 binds tightly first to some nuclear component(s) and then to nucleoli. It can be released from these binding sites rapidly and specifically in vitro by as little as 1 microM ATP, but not by non-hydrolysable ATP analogues. Studies of hsp70 deletion mutations show that the ability of mutants to be released by ATP correlates with their ability to migrate to heat-shocked nucleoli and aid their repair in vivo. We propose a model in which ATP-driven cycles of binding and release of hsp70 help to solubilize aggregates of proteins or RNPs that form after heat shock. Cells also contain proteins related to hsp70 that are synthesized in the absence of stress. The most abundant of these shows the same behaviour as hsp70 after heat shock, and thus may perform a related function in both normal and stressed cells.     

Heat shock regulates the respiration of cardiac H9c2 cells through upregulation of nitric oxide synthase

Mild and nonlethal heat shock (i.e., hyperthermia) is known to protect the myocardium and cardiomyocytes against ischemic injury. In the present study, we have shown that heat shock regulates the respiration of cultured neonatal cardiomyocytes (cardiac H9c2 cells) through activation of nitric oxide synthase (NOS). The respiration of cultured cardiac H9c2 cells subjected to mild heat shock at 42 degrees C for 1 h was decreased compared with that of control. The O2 concentration at which the rate of O2 consumption is reduced to 50% was increased in heat-shocked cells, indicating a lowering of O2 affinity in the mitochondria. Western blot analyses showed a fourfold increase in the expression of heat shock protein (HSP) 90 and a twofold increase in endothelial NOS (eNOS) expression in the heat-shocked cells. Immunoblots of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) in the immunoprecipitate of HSP90 of heat-shocked cells showed that there was a sevenfold increase in eNOS and no changes in iNOS and nNOS. Confocal microscopic analysis of cells stained with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate showed higher levels of NO production in the heat-shocked cells than in control cells. The results indicate that heat shock-induced HSP90 forms a complex with eNOS and activates it to increase NO concentration in the cardiac H9c2 cells. The generated NO competitively binds to the complexes of the respiratory chain of the mitochondria to downregulate O2 consumption in heat-shocked cells. On the basis of these results, we conclude that myocardial protection by hyperthermia occurs at least partly by the pathway of HSP90-mediated NO production, leading to subsequent attenuation of cellular respiration.     

Distinct activation mechanisms of protein kinase B by growth-factor stimulation and heat-shock treatment

Protein kinase B (PKB) alpha, having the pleckstrin homology (PH) and catalytic domains in its amino- and carboxyl-terminal regions, respectively, is activated in the signaling pathway of growth factors as a downstream target of phosphatidylinositol 3-kinase and becomes an active form in heat-shocked cells in a manner independent of the lipid kinase. Therefore, the activation mechanisms of PKBalpha were compared in platelet-derived growth factor (PDGF)-stimulated and heat-shocked cells by monitoring the protein kinase activity and phosphorylation of the mutant molecules expressed in COS-7 cells. In heat-shocked cells, PKBalpha was activated to a certain level without phosphorylation on Thr-308 in the activation loop and on Thr-450 and Ser-473 in the carboxyl-terminal end region, which is critical for growth-factor-induced activation of PKBalpha. Metabolic labeling with (32)P-orthophosphate in the transfected cells revealed that there is no major phosphorylation site other than the three residues in PKBalpha. PKBalpha activated by heat shock was more stable than the enzyme stimulated by PDGF in the cells, and PKBalpha recovered from heat-shocked cells was resistant to the protein phosphatase treatment, whereas the enzyme obtained from the growth-factor-stimulated cells was inactivated by dephosphorylation. Heat shock also enhanced the association of the PH-domain fragment to the full-length PKBalpha in the transfected cells. On the other hand, the PH-domain fragment of PKBalpha, which moves from the cytosol to the plasma membrane upon PDGF stimulation by the interaction with the phosphatidylinositol 3-kinase products, did not translocate but stayed in the cytosol in heat-shocked NIH 3T3 cells. Furthermore, PKBalpha was associated with the nuclear region in heat-shocked cells, which is not observed in growth-factor-stimulated cells. These results indicate that heat shock induces the conformational change of PKBalpha that accompanies the protein complex formation and perinuculear/nuclear localization of the enzyme, to generate an active form by a mechanism distinct from that in the growth-factor-signaling pathway.     

Intracellular localization and partial amino acid sequence of a stress-inducible 40-kDa protein in HeLa cells.

We earlier discovered a novel 40-kDa protein (hsp40) induced by heat shock and other stresses in mammalian and avian cells. In this report, we purified the hsp40 in HeLa cells, using modified two-dimensional gel electrophoresis, and determined the amino terminal amino acid sequence of this protein. The hsp40 is homologous to DnaJ, an Escherichia coli heat-shock protein, as well as to DnaJ-homologous proteins in yeast such as SCJ1, Sec63/Np11, YDJ1 and SIS1. Indirect immunofluorescence staining using an anti-hsp40 polyclonal antibody demonstrated that hsp40 was localized faintly throughout the cell in non-heat-shocked cells and was accumulated in nuclei and nucleoli in heat-shocked cells. The intracellular localization of hsp40 was very similar to that of hsp70, suggesting that these two hsps colocalize in heat-shocked HeLa cells.     

Heat shock response in mycoplasmas, genome-limited organisms.

We have measured the effect of heat shock on three mycoplasmas (Acholeplasma laidlawii K2 and JA1 and Mycoplasma capricolum Kid) and demonstrated the induction of mycoplasma heat shock proteins under these conditions. Increased synthesis of at least 5 heat shock proteins in A. laidlawii K2, 11 heat shock proteins in A. laidlawii JA1, and 7 heat shock proteins in M. capricolum was observed by electrophoretic analysis of proteins from heat-shocked cells in sodium dodecyl sulfate-polyacrylamide gels. In all three strains, major heat shock proteins (66 to 68 and 26 to 29 kilodaltons [kDa]) were found. The 66- to 68-kDa protein cross-reacted with antibody to Escherichia coli DnaK protein, suggesting that this heat shock protein has been conserved in spite of major reductions in genetic complexity during mycoplasma evolution. A. laidlawii also contained a 60-kDa protein that cross-reacted with eubacterial GroEL protein and a 40-kDa protein that cross-reacted with E. coli RecA protein. Unlike with coliphages, the mycoplasma virus L2 progeny yield was not increased when virus was plated on heat-shocked A. laidlawii host cells. However, UV-irradiated L2 virus could be host cell reactivated by both A. laidlawii SOS repair and heat shock systems.