Heat-shock proteins of Drosophila are associated with nuclease-resistant, high-salt-resistant nuclear structures.

Abstract

Proteins produced in cultured Drosophila cells during the heat-shock response (HSPs) were recently shown by autoradiography to be confined in large measure to the cell nucleus. We report here that nuclear HSPs are not associated with nucleosomes solubilized by treatment with staphylococcal nuclease at low ionic strength nor are HSPs released by extraction with high salt, which solubilizes most of the remaining histones and DNA. Possible functions of nuclear HSPs are discussed.When Drosophila cells are subjected to a 10°C increase in temperature, synthesis of most normal RNAs and proteins is strongly repressed. Concurrently, one observes vigorous production ofa few new (heat-shock) mRNAs and proteins (HSPs) (1-11) . Many of the HSP genes have been cloned and mapped (12), and the chromatin structure ofsome ofthese loci has been analyzed (13-16). A considerable proportion of the newly synthesized HSPs were recently shown by autoradiography to be transported to the interior of the cell nucleus (1) .We report here the results of a biochemical study which confirms and extends the autoradiographic data (1) . From 30 to 35% of the HSPs synthesized during a 90-min pulse of ["Slmethionine are found in the purified, extensively washed nuclei . Treatment of the nuclei with staphylococcal nuclease, while solubilizing up to 40%., of the chromatin in the form of mono-and oligonucleosomes, leaves most of the HSPs within the insoluble pellet. After further extraction with 2 M NaCl, which removes most ofthe remaining DNA and histones, HSPs continue to sediment with the nuclease-resistant, high saltresistant insoluble fraction .Because transcription is one of the processes most strongly affected by the heat-shock response, it seems likely that nuclear HSPs play a role in protecting the template-active portion of the genome from adverse effects ofheat shock, as suggested by Velazquez et al. (1) . On the basis of our results, it appears that such protection arises from structures which are physically distinct from solubilizable chromatin (nucleosomes) . MATERIALS AND METHODSDrosophila melanogaster cells (Schneider line 2; gift of Dr. M. L. Pardue) were grown in spinner culture at 25°C as described by Lengyel et al. (l7) . Cells were pelleted and resuspended in methionine-free medium, allowed to recover for 15 min at 25°C, and then shifted to 35°C for 30 min. Thereafter, ["S]methionine (400 Ci/mmol; New England Nuclear, Boston, Mass .) was added to 50 ,yCi/ml and incubation at 35°C was continued foran additional 90 min. Cells (from 5-10 x 10' per preparation) were then pelleted at 4°C, washed once with 0.14 M NaCl, 5 mM Tris-HCl, pH 7.5, and lysed by gentle pipetting in 0.25% Nonidet P-40,

Dynamic changes in the structure and intracellular locale of the mammalian low-molecular-weight heat shock protein.

“…In the case of the cells exposed to either the amino acid analog of proline (azetidine [10 mM] for 12 h; panel 5) or to 100 ,uM sodium arsenite for 2 h (panel 6), two other inducers of the stress response, the 28-kDa protein partitioned into both the soluble and insoluble phase. Thus, as the temperature was increased there was a corresponding increased redistribution of the 28-kDa protein into the insoluble fraction, similar to the situation for the low-mw Drosophila hsps (1,3,31,47).…”

“…In heat-shocked cells, most of the 28-kDa protein fractionated within the low-speed pellet regardless of whether the cells were lysed in the presence or absence of detergent. This insolubility of the 28-kDa protein after heat shock is analogous to the situation in Drosophila cells, where all the low-MW HSPs accumulate within the nuclease-resistant and high-salt-insoluble nuclear fraction after heat shock (1,31,47). However, when the cells were first made thermotolerant (i.e., a prior exposure to a mild heat shock treatment), significantly less of the 28-kDa HSP was found in the insoluble phase after a second and more severe heat shock treatment.…”

Expression and localization of Drosophila melanogaster hsp70 cognate proteins.

“…In D. melanogaster, a 46,000-dalton protein that is related to vimentin forms structures similar to the 10-nm filaments of vertebrates, but in tissue culture cells fails to form the thick bundles that give rise to the characteristic vertebrate vimentin-staining pattern (70 In D. melanogaster and vertebrate cells, localization by indirect immunofluorescence and cell fractionation techniques places hsp70 in both the nucleus and the cytoplasm with decreasing nuclear localization on recovery from heat shock (66,67). Nuclear hsp70 has been reported to be chromatin (3,67), nucleolus (45,73), and nuclear matrix associated (32,57). A fraction of the cytoplasmic hsp70 of chicken and vertebrate cells is reported to be cytoskeleton associated (22,71,73).…”

A heat shock-resistant variant of Chinese hamster cell line constitutively expressing heat shock protein of Mr 90,000 at high level.

“…Among these variants, only one has been analyzed for its protein composition : That is a heat shock-resistant variant of Chinese hamster fibroblastic cell line that over-expresses HSP70 to a significant degree (13). The previous studies have shown that HSP70 translocates into nuclei upon exposure to heat shock (14,25) and binds to nucleoli (20,28). It was suggested, therefore, that HSP70 may function in nuclei.…”