Structural similarities between corresponding heat-shock proteins from different eucaryotic cells.

Effects of heat treatments on chick embryo fibroblasts, Drosophila embryonic cells, and human lymphoblastoid cells have been compared. Cells from all three species synthesize large heat-shock proteins (hsps) with Mr = 70,000 and 84,000-85,000. Different small hsps with Mr between 22,000 and 27,000 are made at high rates in heat-treated chicken and Drosophila cells but could not be observed in human cells. The structural features of the large hsps from cells of the different organisms were compared by three methods of peptide mapping, namely the examination of tryptic digests by two-dimensional thin layer chromatography or by high pressure liquid chromatography and of incomplete V8 digests by polyacrylamide gel electrophoresis. The Mr = 84,000-85,000 polypeptides from all three organisms are closely related, the chicken and human polypeptides having many peptides in common. The relationship between the Mr = 70,000 polypeptides of the different organisms appears to be less close; possible explanations for this latter result are discussed. Rates of synthesis of total as well as poly(A)+ RNA are much lower in heat-treated than in untreated cells of all three organisms. Heat treatments induce dramatic changes in the shape of chick embryo fibroblasts as seen by microscopic examination. Human lymphoblastoid cells do not show changes in shape.

Effects of heat treatments on chick embryo fibroblasts, Drosophila embryonic cells, and human lymphoblastoid cells have been compared. Cells from all three species synthesize large heat-shock proteins (hsps) with M, = 70,000 and 84,000-85,000. Different small hsps with M, between 22,000 and 27,000 are made at high rates in heat-treated chicken and Drosophila ceUs but could not be observed in human cells. The structural features of the large hsps from cells of the different organisms were compared by three methods of peptide mapping, namely the examination of tryptic digests by two-dimensional thin layer chromatography o r by high pressure liquid chromatography and of incomplete V8 digests by polyacrylamide gel electrophoresis. The M, E: 84,000-85,000 polypeptides from all three organisms are closely related, the chicken and human polypeptides having many peptides in common. The relationship between the M, = 70,000 polypeptides of the different organisms appears to be less close; possible explanations for this latter result are discussed. Rates of synthesis of total as well as poly(A)+ RNA are much lower in heat-treated than in untreated cells of all three organisms. Heat treatments induce dramatic changes in the shape of chick embryo fibroblasts as seen by microscopic examination. Human lymphoblastoid cells do not show changes in shape.
The main consequences of heat treatments of Drosophila cells or salivary glands are a substantial reduction in the overall rate of total and poly(A)+ RNA synthesis (1, 2) and a dramatic stimulation of transcription of a small number of genes (2) including those coding for the so-called heat-shock polypeptides. These polypeptides have characteristic M, = 22,000-84,000 and are abbreviated here as hsp'22, 23, 26, 27, 68, 70, and 84.
Increased synthesis of specific sets of polypeptides following heat treatment has also been observed in chick embryo fibroblasts, mouse L cells, and baby hamster kidney cells (3). Chick embryo fibroblasts produce three different hsps. The molecular weights that have been assigned to these proteins range from 83,000-100,000, 68,000-73,000, and 22,000-27,000 (3-6). Hsps with sizes very similar to those of the two largest chick embryo fibroblast hsps are also found in cultured human foreskin cells (5). Several hsps with Mr between 70,000 and 100,000 are synthesized in Chinese hamster ovary cells (7), in the slime mold Polysphondylium pallidurn (8), in Dictyostelium discoideum (9), Tetrahymenapyriformis (lo), HeLa cells (ll), plant cells (12,13) and even in Saccharomyces cereuisiae (14).
So far, little is known about the structure and function of hsps. Drosophila larvae make large amounts of hsps at 35-37 "C (15) but not at 25 "C, and late chicken embryos make hsps at 43-44 "C but not at 37 "C (16). These experiments suggest that hsp synthesis is a physiological response occurring in intact animals at temperatures which are tolerated and which may be encountered by them in their natural environment. In endothermic animals, such elevated temperatures (44 "C in chickens) (17) may be reached during periods of fever caused by bacterial or viral infections. Mild heat treatments that allow the rapid synthesis of hsps greatly increase the ability of Drosophila melanogaster and D. discoideum cells to survive subsequent heat treatments at temperatures which are normally lethal (9,15). Whether or not hsps are involved in the acquisition of thermal resistance is not known. Recently, it has been reported that one of the chick embryo fibroblast hsps is associated with the Rous sarcoma virus transforming protein pp60"" (hsp89, Ref. 18). It remains to be shown, however, whether this association is of physiological importance.
Synthesis of large hsps appears to be universal in eucaryotic cells. The attractive possibility therefore exists that the large hsps may play identical roles in all eucaryotic cells. If these proteins were indeed to serve the same function in different cell types, their structural features should have been conserved throughout evolution. In order to test this possibility, we attempted to examine and compare the structures of corresponding hsps from three organisms which are not closely related species by various methods of peptide analysis. Since the rates of total and poly(A)+ RNA synthesis are considerably lower in heat-treated than in untreated Drosophila cells (1,2) and since similar observations concerning the accumulation of total RNA have been made also in HeLa cells (19), the generality of these heat effects on RNA synthesis was also examined.

RESULTS
Induction of Hsp Synthesis-In preliminary experiments, the incubation temperatures and the lengths of incubation required for strong hsp production were determined. In agreement with previous findings (3), chick embryo fibroblasts were found to synthesize hsps either only in small amounts or not at all at 41 "C ( Fig. 1 in Miniprint). The rates of synthesis of hsps gradually increased with temperature. At 44 "C, the hsps are the predominant protein products made. Hsp formation occurs at a low rate in human lymphoblastoid cells at 37 "C.
Cultured chick, Drosophila, and human cells all synthesize major hsps of M , = 70,000 and 84,000-85,000 ( Fig. 1 in Miniprint). Small hsps are made at relatively high rates in Drosophila cells (2) and in chick embryo fibroblasts (3), but could not be detected in human lymphoblastoid cells.

Structural Relationships between Hsps from Different Organisms-
The sizes of corresponding hsps from chicken and human cells were found to be identical (Mr = 70,000 and 85,000). The largest major Drosophila hsp, however, appeared to be somewhat smaller than the corresponding vertebrate hsps ( Fig. 1 in Miniprint; Drosophila hsp84 and chicken hsp85). This minor size difference was still seen when hsps purified by electroelution from acrylamide gels were compared. During such purifications, small fractions of the proteins were found to be partially degraded. This effect was most obvious with Drosophila hsp84. Small quantities of a M, = 70,000 polypeptide were usually seen in hsp85 preparations from chicken and human cells and a M, = 6 8 , 0 0 0 polypeptide with Drosophila hsp84 (see top arrow on the left in Fig. 4 in Miniprint, lane 1).
The tryptic peptides of corresponding hsps from the three different organisms (see "Experimental Procedures" for preparation of proteins and peptides) were compared by twodimensional thin layer chromatography and by high pressure liquid chromatography in two different solvent systems. In addition, protease V8 digests of the various hsps were analyzed.
Typical maps obtained by two-dimensional chromatography of tryptic peptides from human hsp85, chicken hsp85, and Drosophila hsp84 are shown in Fig. 2 (a, b, and d). Major peptides are indicated and characterized by numbers. To decide whether or not two individual peptides from different digests co-migrate, mixtures containing equal amounts of radioactivity of chicken and human hsp85 digests (c) or of Drosophila hsp84 and chicken hsp85 digests (e) were also chromatographed. All chromatograms exhibit peptide spot 1 which has a very characteristic shape and does not show in the peptide maps of several other proteins including hsp70. The peptides of the three different digests which are found in spot 1 have very similar properties but do not co-migrate exactly (Fig. 2, c and e). The chicken and human peptides in spots 2,3, 4, and 5 have identical properties (Fig. 2, a, b, and  c ) . The chicken and human hsp85 maps differ in some of the minor peptide spots (see for example 6-11 in Fig. 2, a, b, and  c). The Drosophila hsp&i map contains the major peptide spots 3 and 4 in addition to the characteristic spot 1. Spot 7 is found in the Drosophila hsp84 and in the human hsp85 map (Fig. 2, a and d). Drosophila peptide 12 migrates similarly to chicken and human peptide 2. The major peptide 15 is only found in the Drosophila hsp84 map. Thus, the chicken and human hsp85 digests contain the same major tryptic peptides.
Spot 1 and two of the four other major peptide spots are also present in the Drosophila hsp84 map. These results suggest that chicken and human hsp85 are very similar. Drosophila hsp84 has important structural features in common with chicken and human hsp85.
Analogous results were obtained when the same digests were compared by high pressure liquid chromatography. All digests were analyzed with two different solvent systems (Fig.  3 in Miniprint). Peptides were eluted from the chromatography column by increasing linearly the hydrophobicity of the solvent mixture. Major fractions of all digests did not bind to the column and appeared in column fractions 1-20 (see profile obtained with chicken hsp85 in Fig. 3 in Miniprint, left). Gradient elution started at column fraction 21. Very similar elution patterns were obtained with tryptic digests of hsp85 from chicken and human cells. Using solvent system 1, five of the six major peak fractions were found in identical positions (peaks 1-5 in Fig. 3, Miniprint, left). The peptides in peak fractions 1 and 5 are also present in the Drosophila hsp84 pattern, while peptide 8 is only found in the Drosophila profile. Chicken peptide 7 co-migrates with Drosophila peptide 9. In chromatography runs with solvent system 2, eight of the 10 major chicken and human hsp85 peptides eluted at identical positions ( Fig. 3 in Miniprint, right). Peak fraction 2 is clearly separated from fraction 1 in the chicken pattern but can only be seen as a shoulder to fraction 1 in the human profile. Drosophila hsp84 digests also contain the peptide fractions 1-3 and 5 ( Fig. 3 in Miniprint, right). The peptides in Drosophila peak fractions 11-14 ( Fig. 3 in Miniprint, right) do not co-migrate with any of the major chicken or human peptides. These results are in agreement with the ones obtained by two-dimensional chromatography and show again that chicken and human hsp85 are indeed very similar and that Drosophila hsp84 closely resembles the corresponding chicken and human proteins.
Further evidence for the similarity of the M , = 84,000-85,000 polypeptides from the three different organisms was provided by the examination of the polypeptide patterns produced by partial digestion of the different hsps with Staphylococcus aureus protease V8. The individual hsps were digested with different amounts of V8, and the digests were analyzed on 15% polyacrylamide gels (Fig. 4 in Miniprint). The partial digests of chicken and human hsp85 contain several characteristic high molecular weight degradation products with identical molecular weights (see arrows in Fig.  4, Miniprint). Drosophila hsp84 digests also gave similar patterns.
A similar analysis as with the M , = 84,000-85,000 polypeptides was performed with the M, = 70,000 polypeptides from the three organisms. The two-dimensional tryptic peptide maps of the different M , = 70,000 polypeptides revealed some similarities. A group of characteristic major peptides with low mobilities was found to be present in the maps of all three hsp70 digests (data not shown).
The same tryptic digests were also analyzed by high pressure liquid chromatography. Large fractions of the hsp70 digests did not bind to the column. When this nonadsorbed material was concentrated, desalted and examined by twodimensional chromatography, it was found to contain mainly the characteristic low mobility peptides described above. The adsorbed portions of all hsp70 digests contained several peptides with similar elution properties. Corresponding peptides in the different digests, however, greatly differed in their relative quantities. That many of the chicken and human hsp70 peptides indeed co-migrate was shown by additional experiments in which equal amounts of the individual digests were mixed before chromatography.
V8 digestions of the M , = 70,000 polypeptides from the three organisms gave complicated gel patterns. Several polypeptide bands with identical sues but of very different relative FIG. 5. The shapes of heat-treated and untreated chick embryo fibroblasts. Cells in secondary culture were incubated at 41 "C intensities were found with the three h~p70 digests. Many additional bands were only seen with one or two but not with all hsp7O digests (data not shown).
RNA Synthesis Occurs at Reduced Rates in Heat-shocked Cells-Heat treatments reduce the rates of accumulation of total and poly(A)+ RNA in Drosophila cells (1,2). Heattreated HeLa cells have also been reported to synthesize total RNA at lower rates than untreated cells (19). To learn more about the generality of these heat effects on RNA synthesis, rates of total and poly(A)+ RNA accumulation were measured in chick embryo fibroblasts and human lymphoblastoid cells before and during heat treatment. To label RNA, cells were exposed to r3H]uridine for 1 h. Total RNA was extracted from the different cell samples (see "Experimental Procedures") and purified by gel filtration on Sephadex G-75. Since the cells had been exposed to the radioactivity for only a short period, incorporation of [3H]uridine into RNA was calculated per unit of total RNA. This method of calculation allowed us to eliminate errors caused by losses of cells during heat treatment or of RNA during extraction.
Chick embryo fibroblasts which had been heat-treated for 3 h synthesized 36% less RNA/h than untreated cells (Table  IA, Miniprint). Even lower rates of RNA synthesis were observed after 5 h of heat treatment. The effects of 3-h heat exposures on RNA accumulation in chick embryo fibroblasts could be reversed by incubating the heat-treated cells overnight at normal temperature (41 "C). Longer heat treatments appeared to damage the chick cells irreversibly (see Table IA, Miniprint, and below). Analogous results were obtained with human lymphoblastoid cells (Table IB, Miniprint).
To estimate the relative rates of accumulation of poly(A)+ RNA in heat-treated and untreated cells, aliquots of the different total RNA samples were fractionated on oligo(dT)cellulose. The relative amounts of radioactivity in poly(A)+ and poly@-RNA were then determined for each sample. Identical fractions of the total radioactivity incorporated into RNA were found to be associated with poly(A)+ RNA in heattreated and untreated cells (Table I, A and B, Miniprint). These results suggest that heat treatments affect the rates of total and poly(A)+ RNA synthesis similarly.
Heat Treatments Induce Changes in the Appearance of Chick Embryo Fibroblasts-The shapes of chick embryo fibroblasts which had been heat-treated at 44 "C for 2 ( Fig.   5b), 4 (4, or 6 h (f) were found to differ from those of untreated cells (e). Almost all cells which had been heattreated for 2 h regained their original shapes after overnight incubation at 41 "C (a). Already incomplete reversion was observed with cells which had been heat-treated for 4 h (c).

Six-h heat treatments led to the death of many cells (f).
Analogous experiments were also performed with human lymphoblastoid cells. No similarly obvious changes in the cell shapes could be seen. Our observations with chick embryo fibroblasts suggest that heat treatments induce changes in the cytoskeleton structure.

DISCUSSION
We have compared several aspects of the response to heat treatment of cultured cells from three distantly related organisms: Drosophila, chicken, and man. Several apparently independent parts of this response have been conserved throughout evolution. Rates of total and poly(A)+ RNA synthesis are reduced upon heat treatment in all three cell types (Refs. 1 and 2 and this paper).
Heat treatments have also been noted to induce changes in the cytoskeleton structure of Drosophila cells. Falkner et al. (32) have been able to demonstrate, using immunofluorescent techniques, that cytoskeletal proteins accumulate at the nu-clear membrane of heat-treated cells. Our observation, that the shapes of heat-treated chick embryo fibroblasts are visibly different from those of untreated cells, suggests that analogous changes of the cytoskeleton architecture also occur in these cells upon heat treatment. That similar morphologic changes had never been reported for cultured Drosophila cells and that we were equally unable in this study to find them in human lymphoblastoid cells may be explained by the fact that the latter cell types, unlike chick embryo fibroblasts, grow in suspension, where such observations may be difficult to realize.
Analysis of tryptic digests of isolated hsp84/85 by twodimensional chromatography or high pressure liquid chromatography and of protease V8 digests by sodium dodecyl sulfate-acrylamide gel electrophoresis showed that the polypeptides from chicken and human cells are structurally closely related. As expected (33), the similarities between Drosophila hsp84 and chicken or human hsp85 are less striking than the ones between the two vertebrate polypeptides. Still, considerable homology between Drosophila hsp84 and the corresponding vertebrate hsps clearly exists and could be demonstrated by all three methods of peptide analysis. These results indicate that the structure or at least parts of the structure of hsp84/85 have been conserved well throughout evolution. This hsp may therefore play an identical role in all eucaryotic organisms.
Even though the similarities are much less obvious than in the case of hsp84/85, Drosophila, chicken, and human hsp70 also appear to be related. Two-dimensional chromatography revealed that a group of major hydrophilic peptides is common to the tryptic digests of all three M , = 70,000 proteins. Furthermore, high pressure liquid chromatography indicated that in addition to the common characteristic peptides other peptides with similar properties are present in all hsp70 digests, although in different quantities.
Both hsp84/85 and hsp7O are ubiquitous polypeptides (3,5,(7)(8)(9)(10)(11)(12)(13)(14). One therefore wonders why the structures of the two polypeptide species apparently have not been conserved equally well. In Drosophila cells, the hsp70 gene is present in 5-9 copies/haploid genome (34). In contrast, Drosophila hsp84 is encoded by a single gene (27). The sequences of some of the hsp7O gene copies have been determined (26,35,36). From these data, it can be deduced that polypeptides made from different gene copies may exhibit as many as 16 amino acid differences (26). If multiple hsp7O genes were also present in chicken and human cells, our finding of a larger apparent diversity among the M, = 70,000 than among the 84,000-85,000 hsps could be explained by differences in the relative rates of expression of the various hsp70 genes in the three different organisms. This explanation is consistent with the finding that different hsp7O digests do indeed contain peptides with similar properties but in different relative amounts.
Our observations of structural similarities between Drosophila hsp84 and vertebrate hsp85 and also between different M, = 70,000 polypeptides led us to investigate whether cloned Drosophila heat-shock genes (25,27,37,38) hybridize with RNAs from heat-treated chicken or human cells. In several independent experiments using relatively stringent conditions, however, no specific hybridization could be observed between the Drosophila genes and vertebrate heat-shock RNAs. In addition it has also been shown recently that Drosophila hsp70 genes do not cross-hybridize efficiently with the corresponding mouse (39) or Xenopus genes (40) under stringent conditions. These results are not too surprising. Selective pressure acts on maintaining protein structure and funct.ion. Many third base substitutions do not affect the protein sequence (silent substitutions). Thus, identical or very similar proteins can be encoded by gene sequences which have diverged considerably.