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Chromatin-associated heat shock proteins of Dictyostelium
DEVELOPMENTAL
BIOLOGY
90,
412-418 (1982)
Chromatin-Associated WILLIAM Department
of Biology, Received
August
Heat Shock
Proteins
F. LOOMIS AND STEVEN University 20, 1981;
of California accepted
at
of Dic~yostelium
A. WHEELER
San Diego, Lo Jolla, CalZfornia 92099
in revised
fcvw
November
SO, 1981
A heat shock response has been observed in a wide variety of eukaryotic organisms and may be universal. In four heat shock proteins (hsp 22, 23, 26, and 27) have been found in nuclei (A. Arrigo, S. Fakan, and A. Tissieres, 1980, Develop. Biol. 78, 86-103). Eight heat shock-induced proteins of Dictyostelium discoideum were found to be preferentially localized in nuclei. They ranged in size from 26,000 to 32,000 daltons and could be recognized among the chromatin-associated proteins. Partial degradation of the chromatin released the low-molecular-weight heat shock proteins to the same extent as the histones. The heat shock response has been shown to result in protection of cells to the lethal effects of high temperature in a variety of organisms including Dictyostelium We found that this response is extremely rapid in Dictyostelium being maximal by 30 min. The low-molecular-weight heat shock proteins enter the nuclei rapidly and so could play a role there in thermal protection. A mutant strain was isolated which is impaired in the protection afforded by a heat shock. This strain synthesizes most proteins normally but specifically fails to synthesize the low-molecular-weight heat shock proteins under conditions which result in their induction in wild-type cells. Drosophila
weight heat shock proteins could be observed in Dietyostelium following heat shock of either growing or developing cells (Loomis and Wheeler, 1980). However, they are not as prevalent in whole cell extracts as hsp70 and are difficult to distinguish against the background of residual synthesis of proteins made during growth. In this study we have isolated nuclei from heat shocked Dictyostelium and found that heat shock proteins make up a considerable portion of the newly synthesized nuclear proteins.
INTRODUCTION
Exposure of cells to sublethal temperatures outside of the range permissive for growth results in a heat shock response in a variety of organisms including Drosophila, yeast, Tetrahymena, Naegleria, Dictyostelium, plants, and vertebrates (Ashburner and Bonner, 1979, McAlister and Finkelstein, 1980, Guttman et al., 1980, Walsh, 1980; Loomis and Wheeler, 1980; Barnett et ah, 1980; Kelley and Schlesinger, 1978). The response is characterized by a transient high rate of synthesis of a small number of proteins and a reduction in the synthesis of most other proteins. The major heat shock protein, hsp70, has been highly conserved since antiserum prepared against chick hsp70 cross-reacts with hsp70 of human, mouse, nematode, and Dictyostelium (Kelley and Schlesinger, personal communication). In yeast, Dictyostelium, and Drosophila the synthesis of heat shock proteins has been shown to afford protection from the lethal effects of higher temperatures (McAlister and Finkelstein 1980, Loomis and Wheeler 1980, Mitchell et ak, 1979). Thus, it would appear that the heat shock response has been highly conserved during evolution to function in thermal protection. In Drosophila there are four low-molecular-weight heat shock proteins (22,000 to 27,000 daltons) which are preferentially localized in the nucleus associated with chromatin (Arrigo et al, 1980, Velasquez et al., 1980). They appear to be coordinately expressed from closely linked genes (Craig and McCarthy 1980; Corces et al, 1980). An increase in the rate of synthesis of low-molecular0012-1606/82/040412-07$02.00/O Copyright All rights
Q 1982 by Academic Press, Inc. of reproduction in any form reserved.
MATERIALS
AND METHODS
Strains. Dictyostelium discoideum strain AX3 was grown in HL-5 medium (Loomis 1971). To screen for mutant strains defective in heat shock responses, a population of strain AX3 was mutagenized with N-methylN’-nitro-N-nitrosoguanidine to lop3 survival and innoculated into multitest wells at an average of one viable cell per well (Brenner et aL, 1976). After 2 weeks the multitest trays were replicated to two sets of trays containing HL-5 medium and grown for 24 hr at 22°C. The replicas were shifted to 30°C to induce the heat shock response. After 3 hr one set was shifted to 34°C to test for thermal protection. After another 4 hr both sets were returned to 22°C. Clones which grew in the set kept at 3O”C, but not in the one shifted to 34”C, were picked to test tubes containing 2 ml of HL-5 medium. After 2 or more days of growth at 22°C the cells were again incubated at 30°C for 4 hr and then 34°C for 4 hr. Samples were taken before and after the 34°C challenge, diluted, and plated on nutrient agar plates 412
LOOMIS
AND WHEELER
Chrmtin-Associated
Heat Shock Proteins
FIG. 1. Subcellular localization. Cells of strain AX3 growing at 22°C were transferred to 30°C. After 1 hr, [%I methionine was added the newly made proteins were labeled for 2 hr (B and D). Control cells were labeled for 2 hr at 22°C (A and C). The cells were broken the nuclei (A and B) and supernatant fractions (C and D) were separated. Proteins containing lo5 cpm from each fraction were analyzed two-dimensional gel electrophoresis. The acid end (pH 3.5) is on the left.
spread with Klebsiella (aerogenes. After incubation of the plates at 22°C for 4 dlays, viable cells were estimated from the number of plaques. of nuclei. Nuclei were isolated as dePreparation scribed by Pong and Loomis (1973). Cells were washed twice with cold 7% sucrose and suspended at 5 X lo7 cells/ml in 0.04 M Tris-HCl, pH 7.8, containing 15% sucrose, 0.1 mM EDTA, 6.0 mM MgCls, 0.04 M KCl, 5 mM mercaptoethanol, and 0.4% NP-40. After shaking vigorously by hand for 1 min, unbroken cells were removed by centrifuging ‘2 min at 5009. The supernatant was centrifuged for 10 min at 4000g to pellet nuclei. Nuclei were washed once with the above buffer containing no detergent and resuspended in 20 mM Tris, 2 mM CaCI,, pH 8.8. of chromatin. Chromatin was prepared Preparation by the method of Bakke and Bonner (1979). Nuclei were washed twice with a 0.05 M NaCl solution containing 5 mM EDTA, 5 mM EG-TA, and 1 mM PMSF at pH 8.0. The chromatin was pelleted by centrifugation at 12,000g for 3 min. The pellet was then washed twice with 10
413
and and by
mM Tris, 0.1 mM EGTA, and 1 mM PMSF at pH 8.0, once with 10 mM Tris and 1 mM PMSF at pH 8.0, and finally with nuclease shearing buffer (5 mM Tris/acetate, pH 7.8, 20 mM ammonium acetate, 0.4 mM CaCl,, 0.2 mM EDTA, 1 mM PMSF). The chromatin was suspended in nuclease shearing buffer at 35 OD alkaline AZ60 units/ml prior to digestion with staphylococcal nuclease (Worthington). N&ease treatments. Chromatin was suspended at 35 AZ60units/ml in nuclease shearing buffer. Staphylococcal nuclease (Worthington) was added to 100 units/ml and samples were incubated for 5 min at 25°C. Undigested chromatin was pelleted at 8000g for 5 min and the supernatant withdrawn. The chromatin was resuspended in nuclease shearing buffer and digested again with nuclease for 5 min. After spinning 8000g for 5 min the second supernatant was withdrawn and combined with the first. The undigested chromatin pellet was suspended in a volume equal to that of the combined supernatants prior to gel electrophoresis. Protein analysis. Cells at 107/ml were labeled with
414
DEVELOPMENTAL
BIOLOGY
100 to 200 &i of [35S]methionine or [14C]tryptophan in 200 ~1 HL-5 medium. Extracts were prepared and analyzed as previously described (Loomis and Wheeler, 1980). Two-dimensional electrophoresis was carried out as described by Garrels and Gibson (1976) using ampholines of pH 3.5 to 10. The amount of incorporation into each spot was determined by cutting out the spots and counting them in a liquid scintillation counter. Duplicate determinations agreed within 10%.
VOLUME
Kd
90, 1982
A
-hsp _-
LMW
3
histon
Nuclear Localization Nuclei prepared from heat-shocked cells of D. discoideum were found to contain all of the heat shock proteins but to be enriched in eight species with apparent molecular weights of 26,000 to 32,000 (Fig. 1). The major heat shock protein, hsp70, was about equally distributed between the fractions while hsp82 was predominantly found in the soluble fraction. Mixing of labeled and unlabeled nuclear and soluble fractions followed by separation did not result in redistribution of the labeled species. The low-molecular-weight heat shock proteins focused in the range from pH 4 to pH 8 and could not be observed among the proteins synthesized in un-heatshocked cells even upon much longer exposures of the gels (Fig. 1, Table 1). Synthesis of the low-molecularweight heat shock proteins was completely inhibited when actinomycin D (125 pg/ml) was added 10 min before the cells were placed at 30°C but not when added 30 min after the heat shock, suggesting that their synthesis requires de no~o transcription following heat shock induction (data not shown).
Strain
TABLE IN MUTANT
1 AND WILD-TYPE
Percentage
of total
AX3
countsb Strain
Proteina
22°C
30°C
3O”C/22”C
22°C
30°C
hsp’70 LMW actin
0.8 <0.08 7.42
6.5 1.86 1.0
8.1
0.8 10.05 7.2
1.5 KO.05 2.5
HL122
12
0.13
34
12
es
34
FIG. 2. Chromatin-associated proteins. (A) Coomassie blue stain of chromatin proteins from heat shocked cells separated by SDS-gel electrophoresis (5-15s gradient acrylamide). (B) Autoradiogram of the same gel. (C) Coomassie blue stain of chromatin proteins from control cells. (D) Autoradiogram of the control gel. Cells were heat shocked at 30°C for 30 min and then labeled with [%]methionine for 3 hr at 30°C (A and B) or labeled 3 hr at 22’C (C and D). Half of the heat shocked sample was treated with staphylococcal nuclease (lanes 1 and 2) and the other half was left untreated (lanes 3 and 4) before repelleting the chromatin (lanes 1 and 3) and collecting the solubilized material (lanes 2 and 4).
Chromatin
Association
The low-molecular-weight heat shock proteins sedimented along with chromatin prepared from heat shocked cells (Fig. 2). Treatment of the chromatin with micrococcal nuclease released some of the histones and a similar proportion of the low-molecular-weight heat shock proteins. hsp82 could not be observed in the purified chromatin and most likely was found in trace amounts in the nuclear fraction as a cytoplasmic contaminant. hsp70 and actin were found among the chromatin proteins but were loosely associated since they appeared in the wash in the absence of nuclease treatment (Fig. 2). Unlike the histones, the low-molecularweight proteins were not soluble in a 10% perchloric acid extraction of chromatin (data not shown).
3O”C/22”C
Kinetics >23
actin
1
RESULTS
INDUCTION
70
1.9 0.35
‘hsp70 refers to the four major spots at the position of 70,000 daltons while LMW refers to the eight low-molecular-weight heat shock proteins (Fig. 1). *Spots were cut from the gels and counted. The percentage of total [%]methionine incorporated into TCA-precipitable protein during a 2-hr period was calculated for each spot. The label was added to the cells 1 hr after shifting the population to 30°C. Total TCA-precipitated material from 3 X lo5 cells of strain AX3 was 1.3 X lo5 cpm at 22°C and 1.1 X lo5 cpm at 30°C, total TCA-precipitated material from 3 X lo5 cells of strain HL122 was 1.5 X lo5 cpm at 22°C and 1.0 X lo5 cpm at 30°C. Repeat experiments confirmed these results.
of Protection
Heat shock treatment of Dictyostelium and other organisms protects the cells from high lethal temperatures (Loomis and Wheeler, 1980; McAlister and Finkelstein, 1980; Mitchell et a& 1979). The protection of Dictyostelium to 34°C was found to occur very rapidly, being significant at 10 min and maximum at 30 min after placing the cells at 30°C (Fig. 3). The protection from thermal killing persisted for several hours after shifting the cells back to 22°C but was insignificant by 6 hr (Fig. 4). If the nuclear localization of the low-molecularweight proteins plays a role in heat resistance, they
LOOMIS
AND WHEELER
Chrmatin-Associated
415
Heat Shock Proteins
I
I
2
4
TIME
AFTER
SHIFT (HOURS)
-DOWN
FIG. 4. Loss of protection. Cells which had been heat shocked at 30°C for 1 hr were shifted back to 22°C for various periods of time before being placed at 34°C. The viable titer was determined every hour for 4 hr at 34°C. The half-life was estimated assuming firstorder killing kinetics from two or more determinations at each time point following shift down (0). The half-life of un-heat-shocked cells at 34°C was also determined (0).
were kept at 30°C and half were shifted down to 22°C. Samples were taken 3 and 6 hr later and nuclei prepared to see if the low-molecular-weight preferentially exited (Fig. 6). By 3 hr most of the labeled low-molecularweight proteins had disappeared from the nuclei of cells chased at 22°C while they persisted in the nuclei kept at 30°C during the chase. Thus the kinetics of loss of the chromatin-associated proteins is similar to those for the loss of thermal protection (Fig. 4). Heat Shock Mutant As an initial approach to genetically defining the mechanism by which the heat shock response protects I
I
I
I
I
0
1
2
3
4
TIME
(hours)
FIG. 3. Protection from thermal killing. Cells of strain AX3 growing at 22°C were heat shocked at 30°C for 0 min (O), 10 min (0), 30 min (O), or 60 min (A) before being shifted to 34°C. Samples were taken at various times after the cells were shifted to 34”C, diluted and plated with K. aerogenes. After incubation of the plates at 22°C for 3 days, plaques were counted to give the viable titer.
must enter the nuclease rapidly. We analyzed the newly made proteins present in nuclei 10 and 30 min after heat shock and found that all of the low-molecularweight species were present in nuclei after 30 min (Fig. 5) and were faint but detectable in nuclei after 10 min (data not shown). We also labeled cells heat shocked at 30°C for 3 hr and then blocked further incorporation by removing the r5S]methionine and adding cycloheximide (500 pg/ml) to inhibit subsequent protein synthesis. Half the cells
FIG. 5. Entry of heat shock protein into nuclei. Cells of strain AX3 growing at 22°C were shifted to 30°C and labeled with [%]methionine for 30 min. Nuclei were prepared and their proteins were analyzed by two-dimensional gel electrophoresis.
416
DEVELOPMENTAL
BIOLOGY
the cells, we screened for mutants defective in the response. Five thousand clones from a mutagenized population were replicated in multitest wells, grown at 22”C, and then heat shocked at 30°C for 3 hr. One set was challenged by 4 hr at 34°C and the other was kept at 30°C. Clones which grew in the unchallenged set but not in the challenged set were rescreened by quantitating the number of survivors in challenged and unchallenged populations. From 155 clones rescreened, one strain, HL122, proved to be defective in the thermal protection afforded by treatment at 30°C (Fig. 7). The strain grows well at 22°C and has a half-life at 30°C which is comparable to that of wild type. While the amount of [35S] methionine incorporated at either 22 or 30°C did not differ significantly between the mutant and the wild type, the pattern of newly made proteins in cells of strain HL122 after heat shock is markedly different from that in wild-type cells (Fig. 8, Table 1). The low-molecular-weight heat shock proteins have never been observed to be synthesized in strain HL122 while they make up about 1.9% of the proteins
t
hsp70
VOLUME
90, 1982
1 2 3 TIME (hours)
4
1 2 3 4 TIME AT 3CP (hours)
FIG. ‘7. Thermal protection in a heat shock mutant. (A) Cells strain HL122 growing at 22°C were heat shocked at 30°C for 1 before being transferred to 34°C (A); or were directly transferred 34°C (0) or 30°C (0). (B) The half-life of cells of strain Ax3 (0) strain HL122 (0) at 34°C was determined after various periods heat shock at 30°C as described in the legend to Fig. 3.
of hr to or of
synthesized in heat shocked Ax3 cells. The family of hsp70 proteins is synthesized as slightly less than 1% of total protein in wild-type and mutant cells at 22”C, but while this proportion increases &fold in strain AX3 following heat shock it increases less than 2-fold in strain HL122 at 30°C. Likewise the proportion of newly synthesized proteins made up by actin decreases 7.4fold following heat shock in strain Ax3 but only 2.9fold in strain HL122 (Table 1). hsp82 has not been observed to be rapidly synthesized in heat shocked cells of strain HL122. Moreover, the heat shock proteins were not induced in strain HL122 when incubated at either 2'7 or 32°C.
LMW DISCUSSION
1
2
3
4
5
FIG. 6. Chase from the nucleus. Cells were heat shocked at 30°C for 3 hr and labeled with [%G]methionine between 1 and 3 hr. They were then washed and resuspended in unlabeled medium containing cycloheximide (500 pg/ml) and chased at either 22 or 30°C. Nuclei were prepared from 2 X 105/cells at various times following the chase. Lane 1: 0 hr; lane 2: 3-hr chase at 22°C; Lane 3: 3-hr chase at 30°C; lane 4: 6-hr chase at 22°C; lane 5: 6-hr chase at 30°C. The nuclear proteins were separated electrophoretically on a 10% aerylamide gel which was prepared for autoradiography.
A brief treatment at nonlethal temperatures just above those permissive for growth protects cells of several organisms from thermal killing at higher temperatures (Loomis and Wheeler, 1980; McAlister and Finkelstein, 1980; Mitchell et al, 1979). The mechanism by which high temperatures result in irreversible damage to cells is unknown but occurs at temperatures which would not be expected to result in denaturation of most proteins (34°C in Dictyostelium; 42°C in Drosophila). At least in Dictyostelium death does not come from breaching the integrity of the cellular membrane since cells which have been rendered nonviable at 34°C continue to exclude trypan blue for several hours (Loomis, unpublished). There are many other ways in which high temperatures may be lethal including nuclear damage. It is therefore intriguing that among the proteins in-
LOOMIS AND WHEELER
Chromatin-Associated
Heat
Shock
417
Proteins
strain defective in the heat shock response leading to thermal protection is not induced to synthesize the chromatin-associated proteins by heat shock supports the idea that they may be at least partially responsible for the protection. The mutant strain is not impaired in growth or incorporation of labeled amino acids at either 22 or 30°C and so does not appear to carry a generally debilitating mutation. The most abundant protein synthesized following heat shock is hsp70. In Dictyostelium there are at least two distinct but closely related proteins, hsp7Oa and hsp70b (Loomis and Wheeler, 1980). During growth at 22°C they account for almost 1% of the total protein synthesized and accumulate to the expected degree. Since they are abundant even before heat shock, it is not apparent how continued synthesis for only 30 min can be essential for thermal protection. Nuclear localization of the low-molecular-weight proteins occurs with kinetics comparable to the kinetics of acquisition of thermal protection. If the synthesis of the heat shock proteins is blocked by the addition of cycloheximide (Loomis and Wheeler, 1980) or as a consequence of the mutation in strain HL122, then heat shock does not confer heat resistance. Further mutational analysis of the heat shock response may better define the processes involved. This work was supported by a grant from the NIH. FIG. 8. Protein synthesized by mutant strain HL122 at 30°C. Nuclei (A) and supernatant (B) fractions were prepared from lo6 cells which had been labeled with [%]methionine for 3 hr at 30°C after 1 hr of preincubation at 30°C. Proteins containing 3 X lo5 cpm were analyzed by two-dimensional electrophoresis.
duced by a heat shock are a class which rapidly becomes tightly associated with the chromatin. In Dictyostelium there are at least eight chromatin-associated heat shock proteins which differ in isoelectric point but have similar mobility in SDS electrophoresis. In Drosophila there are four proteins of similar molecular weight (22,000 to 27,000 daltons) which are found preferentially in the nucleus (Arrigo et ab, 1980). One of them contains little or no tryptophan (Arrigo et ah, 1980). Likewise we found that none of the low-molecular-weight heat shock proteins of Dictyostelium were labeled by [14C]tryptophan while hsp70 was strongly labeled (data not shown). This is another case of similarity of the heat shock proteins between highly diverse organisms. In Dictyostelium heat shock results in the rapid coordinate induction of the eight low-molecular-weight chromatin-associated proteins by a factor of more than 23. This is the most dramatic environmental response known in this organism. The finding that a mutant
REFERENCES ARRIGO, A., FAKAN, S., and TISSIERES, A. (1980). Localization of the melanogaster tissue culheat shock-induced proteins in Drosophila ture cells. Develop. BioL 78, 86-103. ASHBURNER, M., and BONNER, J. J. (1979). The induction of gene activity in Drosophila by heat shock. Cell 17, 241-254. BAKKE, A., and BONNER, J. (1979). Purification of the histones of Dietyostelium discoideum chromatin. Biochemistq/ 18, 4556. BARNETT, T., ATTSCHULER, M., MCDANIEL, C., and MASARENHAS, J. (1980). Heat shock induced proteins in plant cells. Develop. Genet 1, 331-336. BRENNER, M., DIMOND, R., and LOOMIS, W. F. (1976). Rapid biochemical screening of large numbers of animal cells clones. Methods Cell BioL
14,187-194.
CORCES,V., HOLMGREN, R., FREUD, R., MORIMOTO, R., and MESELSON, meknwgaster M. (1980). Four heat shock proteins of Drosophila coded within a 12-kilobase region in chromosome subdivision 6’7B. Proc.
Nat.
Acad
Sci. USA
77, 5390-5393.
CRAIG, E., and MCCARTHY, B. (1980). Four Drosophila heat shock genes at 67B: Characterization of recombinant plasmids. Nucl Acids Res. 8, 4441-445’7. GUTTMAN, S., GLOVER, C., ALLIS, C., and GOROVSKY, M. (1980). Heat shock, deciliation and release from anoxia induce the synthesis of the same set of polypeptides in starved T. pyrifbrmti. Cell 22,299309.
GARRELS, J., and GIBSON, W. (1976). Identification tion of multiple forms of actin. Cell 9, 793-805.
and characteriza-
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DEVELOPMENTAL
BIOLOGY
KELLEY, P., and SCHLESINGER, M. (1978). The effect of amino acid analogues and heat shock on gene expression in chick embryo fibroblasts. CeU 15, 12’77-1286. LOOMIS, W. F. (1971). Sensitivity of Dictyostelium diswideum to nucleic acid analogues. Exp. Cell Res. 64, 484-486. LOOMIS, W. F., and WHEELER, S. (1980). Heat shock response of Dietyostelium Develop. Bid 79, 399-408. MCALISTER, L., and FINKELSTEIN, D. (1980). Heat shock proteins and thermal resistance in yeast. Biochem Biophys. Rec. Commun 93, 819-824. MITCHELL, H., MOLLER, G., PETERSEN, N., and LIPPS-SARMIENTO, L.
VOLUME 90, 1982 (1979). Specific protection from phenocopy induction by heat shock. Develop Genet. 1, 181-192. PONG, S., and LOOMIS, W. F. (1973). Multiple nuclear RNA polymerases during development of Dictyostelium disc&dewn. J. Biol Chem. 248, 3933-3939. VELASQUEZ, J., DIDOMENICO, B., and LINDQUIST, S. (1980). Intracellular localization of heat shock proteins in Drosophila. Cell 20,679689. WALSH, J. (1980). Appearance of heat shock proteins during the induction of multiple flagella in Naegleria grub& J. Biol. Chem. 255, 2629-2631.
BIOLOGY
90,
412-418 (1982)
Chromatin-Associated WILLIAM Department
of Biology, Received
August
Heat Shock
Proteins
F. LOOMIS AND STEVEN University 20, 1981;
of California accepted
at
of Dic~yostelium
A. WHEELER
San Diego, Lo Jolla, CalZfornia 92099
in revised
fcvw
November
SO, 1981
A heat shock response has been observed in a wide variety of eukaryotic organisms and may be universal. In four heat shock proteins (hsp 22, 23, 26, and 27) have been found in nuclei (A. Arrigo, S. Fakan, and A. Tissieres, 1980, Develop. Biol. 78, 86-103). Eight heat shock-induced proteins of Dictyostelium discoideum were found to be preferentially localized in nuclei. They ranged in size from 26,000 to 32,000 daltons and could be recognized among the chromatin-associated proteins. Partial degradation of the chromatin released the low-molecular-weight heat shock proteins to the same extent as the histones. The heat shock response has been shown to result in protection of cells to the lethal effects of high temperature in a variety of organisms including Dictyostelium We found that this response is extremely rapid in Dictyostelium being maximal by 30 min. The low-molecular-weight heat shock proteins enter the nuclei rapidly and so could play a role there in thermal protection. A mutant strain was isolated which is impaired in the protection afforded by a heat shock. This strain synthesizes most proteins normally but specifically fails to synthesize the low-molecular-weight heat shock proteins under conditions which result in their induction in wild-type cells. Drosophila
weight heat shock proteins could be observed in Dietyostelium following heat shock of either growing or developing cells (Loomis and Wheeler, 1980). However, they are not as prevalent in whole cell extracts as hsp70 and are difficult to distinguish against the background of residual synthesis of proteins made during growth. In this study we have isolated nuclei from heat shocked Dictyostelium and found that heat shock proteins make up a considerable portion of the newly synthesized nuclear proteins.
INTRODUCTION
Exposure of cells to sublethal temperatures outside of the range permissive for growth results in a heat shock response in a variety of organisms including Drosophila, yeast, Tetrahymena, Naegleria, Dictyostelium, plants, and vertebrates (Ashburner and Bonner, 1979, McAlister and Finkelstein, 1980, Guttman et al., 1980, Walsh, 1980; Loomis and Wheeler, 1980; Barnett et ah, 1980; Kelley and Schlesinger, 1978). The response is characterized by a transient high rate of synthesis of a small number of proteins and a reduction in the synthesis of most other proteins. The major heat shock protein, hsp70, has been highly conserved since antiserum prepared against chick hsp70 cross-reacts with hsp70 of human, mouse, nematode, and Dictyostelium (Kelley and Schlesinger, personal communication). In yeast, Dictyostelium, and Drosophila the synthesis of heat shock proteins has been shown to afford protection from the lethal effects of higher temperatures (McAlister and Finkelstein 1980, Loomis and Wheeler 1980, Mitchell et ak, 1979). Thus, it would appear that the heat shock response has been highly conserved during evolution to function in thermal protection. In Drosophila there are four low-molecular-weight heat shock proteins (22,000 to 27,000 daltons) which are preferentially localized in the nucleus associated with chromatin (Arrigo et al, 1980, Velasquez et al., 1980). They appear to be coordinately expressed from closely linked genes (Craig and McCarthy 1980; Corces et al, 1980). An increase in the rate of synthesis of low-molecular0012-1606/82/040412-07$02.00/O Copyright All rights
Q 1982 by Academic Press, Inc. of reproduction in any form reserved.
MATERIALS
AND METHODS
Strains. Dictyostelium discoideum strain AX3 was grown in HL-5 medium (Loomis 1971). To screen for mutant strains defective in heat shock responses, a population of strain AX3 was mutagenized with N-methylN’-nitro-N-nitrosoguanidine to lop3 survival and innoculated into multitest wells at an average of one viable cell per well (Brenner et aL, 1976). After 2 weeks the multitest trays were replicated to two sets of trays containing HL-5 medium and grown for 24 hr at 22°C. The replicas were shifted to 30°C to induce the heat shock response. After 3 hr one set was shifted to 34°C to test for thermal protection. After another 4 hr both sets were returned to 22°C. Clones which grew in the set kept at 3O”C, but not in the one shifted to 34”C, were picked to test tubes containing 2 ml of HL-5 medium. After 2 or more days of growth at 22°C the cells were again incubated at 30°C for 4 hr and then 34°C for 4 hr. Samples were taken before and after the 34°C challenge, diluted, and plated on nutrient agar plates 412
LOOMIS
AND WHEELER
Chrmtin-Associated
Heat Shock Proteins
FIG. 1. Subcellular localization. Cells of strain AX3 growing at 22°C were transferred to 30°C. After 1 hr, [%I methionine was added the newly made proteins were labeled for 2 hr (B and D). Control cells were labeled for 2 hr at 22°C (A and C). The cells were broken the nuclei (A and B) and supernatant fractions (C and D) were separated. Proteins containing lo5 cpm from each fraction were analyzed two-dimensional gel electrophoresis. The acid end (pH 3.5) is on the left.
spread with Klebsiella (aerogenes. After incubation of the plates at 22°C for 4 dlays, viable cells were estimated from the number of plaques. of nuclei. Nuclei were isolated as dePreparation scribed by Pong and Loomis (1973). Cells were washed twice with cold 7% sucrose and suspended at 5 X lo7 cells/ml in 0.04 M Tris-HCl, pH 7.8, containing 15% sucrose, 0.1 mM EDTA, 6.0 mM MgCls, 0.04 M KCl, 5 mM mercaptoethanol, and 0.4% NP-40. After shaking vigorously by hand for 1 min, unbroken cells were removed by centrifuging ‘2 min at 5009. The supernatant was centrifuged for 10 min at 4000g to pellet nuclei. Nuclei were washed once with the above buffer containing no detergent and resuspended in 20 mM Tris, 2 mM CaCI,, pH 8.8. of chromatin. Chromatin was prepared Preparation by the method of Bakke and Bonner (1979). Nuclei were washed twice with a 0.05 M NaCl solution containing 5 mM EDTA, 5 mM EG-TA, and 1 mM PMSF at pH 8.0. The chromatin was pelleted by centrifugation at 12,000g for 3 min. The pellet was then washed twice with 10
413
and and by
mM Tris, 0.1 mM EGTA, and 1 mM PMSF at pH 8.0, once with 10 mM Tris and 1 mM PMSF at pH 8.0, and finally with nuclease shearing buffer (5 mM Tris/acetate, pH 7.8, 20 mM ammonium acetate, 0.4 mM CaCl,, 0.2 mM EDTA, 1 mM PMSF). The chromatin was suspended in nuclease shearing buffer at 35 OD alkaline AZ60 units/ml prior to digestion with staphylococcal nuclease (Worthington). N&ease treatments. Chromatin was suspended at 35 AZ60units/ml in nuclease shearing buffer. Staphylococcal nuclease (Worthington) was added to 100 units/ml and samples were incubated for 5 min at 25°C. Undigested chromatin was pelleted at 8000g for 5 min and the supernatant withdrawn. The chromatin was resuspended in nuclease shearing buffer and digested again with nuclease for 5 min. After spinning 8000g for 5 min the second supernatant was withdrawn and combined with the first. The undigested chromatin pellet was suspended in a volume equal to that of the combined supernatants prior to gel electrophoresis. Protein analysis. Cells at 107/ml were labeled with
414
DEVELOPMENTAL
BIOLOGY
100 to 200 &i of [35S]methionine or [14C]tryptophan in 200 ~1 HL-5 medium. Extracts were prepared and analyzed as previously described (Loomis and Wheeler, 1980). Two-dimensional electrophoresis was carried out as described by Garrels and Gibson (1976) using ampholines of pH 3.5 to 10. The amount of incorporation into each spot was determined by cutting out the spots and counting them in a liquid scintillation counter. Duplicate determinations agreed within 10%.
VOLUME
Kd
90, 1982
A
-hsp _-
LMW
3
histon
Nuclear Localization Nuclei prepared from heat-shocked cells of D. discoideum were found to contain all of the heat shock proteins but to be enriched in eight species with apparent molecular weights of 26,000 to 32,000 (Fig. 1). The major heat shock protein, hsp70, was about equally distributed between the fractions while hsp82 was predominantly found in the soluble fraction. Mixing of labeled and unlabeled nuclear and soluble fractions followed by separation did not result in redistribution of the labeled species. The low-molecular-weight heat shock proteins focused in the range from pH 4 to pH 8 and could not be observed among the proteins synthesized in un-heatshocked cells even upon much longer exposures of the gels (Fig. 1, Table 1). Synthesis of the low-molecularweight heat shock proteins was completely inhibited when actinomycin D (125 pg/ml) was added 10 min before the cells were placed at 30°C but not when added 30 min after the heat shock, suggesting that their synthesis requires de no~o transcription following heat shock induction (data not shown).
Strain
TABLE IN MUTANT
1 AND WILD-TYPE
Percentage
of total
AX3
countsb Strain
Proteina
22°C
30°C
3O”C/22”C
22°C
30°C
hsp’70 LMW actin
0.8 <0.08 7.42
6.5 1.86 1.0
8.1
0.8 10.05 7.2
1.5 KO.05 2.5
HL122
12
0.13
34
12
es
34
FIG. 2. Chromatin-associated proteins. (A) Coomassie blue stain of chromatin proteins from heat shocked cells separated by SDS-gel electrophoresis (5-15s gradient acrylamide). (B) Autoradiogram of the same gel. (C) Coomassie blue stain of chromatin proteins from control cells. (D) Autoradiogram of the control gel. Cells were heat shocked at 30°C for 30 min and then labeled with [%]methionine for 3 hr at 30°C (A and B) or labeled 3 hr at 22’C (C and D). Half of the heat shocked sample was treated with staphylococcal nuclease (lanes 1 and 2) and the other half was left untreated (lanes 3 and 4) before repelleting the chromatin (lanes 1 and 3) and collecting the solubilized material (lanes 2 and 4).
Chromatin
Association
The low-molecular-weight heat shock proteins sedimented along with chromatin prepared from heat shocked cells (Fig. 2). Treatment of the chromatin with micrococcal nuclease released some of the histones and a similar proportion of the low-molecular-weight heat shock proteins. hsp82 could not be observed in the purified chromatin and most likely was found in trace amounts in the nuclear fraction as a cytoplasmic contaminant. hsp70 and actin were found among the chromatin proteins but were loosely associated since they appeared in the wash in the absence of nuclease treatment (Fig. 2). Unlike the histones, the low-molecularweight proteins were not soluble in a 10% perchloric acid extraction of chromatin (data not shown).
3O”C/22”C
Kinetics >23
actin
1
RESULTS
INDUCTION
70
1.9 0.35
‘hsp70 refers to the four major spots at the position of 70,000 daltons while LMW refers to the eight low-molecular-weight heat shock proteins (Fig. 1). *Spots were cut from the gels and counted. The percentage of total [%]methionine incorporated into TCA-precipitable protein during a 2-hr period was calculated for each spot. The label was added to the cells 1 hr after shifting the population to 30°C. Total TCA-precipitated material from 3 X lo5 cells of strain AX3 was 1.3 X lo5 cpm at 22°C and 1.1 X lo5 cpm at 30°C, total TCA-precipitated material from 3 X lo5 cells of strain HL122 was 1.5 X lo5 cpm at 22°C and 1.0 X lo5 cpm at 30°C. Repeat experiments confirmed these results.
of Protection
Heat shock treatment of Dictyostelium and other organisms protects the cells from high lethal temperatures (Loomis and Wheeler, 1980; McAlister and Finkelstein, 1980; Mitchell et a& 1979). The protection of Dictyostelium to 34°C was found to occur very rapidly, being significant at 10 min and maximum at 30 min after placing the cells at 30°C (Fig. 3). The protection from thermal killing persisted for several hours after shifting the cells back to 22°C but was insignificant by 6 hr (Fig. 4). If the nuclear localization of the low-molecularweight proteins plays a role in heat resistance, they
LOOMIS
AND WHEELER
Chrmatin-Associated
415
Heat Shock Proteins
I
I
2
4
TIME
AFTER
SHIFT (HOURS)
-DOWN
FIG. 4. Loss of protection. Cells which had been heat shocked at 30°C for 1 hr were shifted back to 22°C for various periods of time before being placed at 34°C. The viable titer was determined every hour for 4 hr at 34°C. The half-life was estimated assuming firstorder killing kinetics from two or more determinations at each time point following shift down (0). The half-life of un-heat-shocked cells at 34°C was also determined (0).
were kept at 30°C and half were shifted down to 22°C. Samples were taken 3 and 6 hr later and nuclei prepared to see if the low-molecular-weight preferentially exited (Fig. 6). By 3 hr most of the labeled low-molecularweight proteins had disappeared from the nuclei of cells chased at 22°C while they persisted in the nuclei kept at 30°C during the chase. Thus the kinetics of loss of the chromatin-associated proteins is similar to those for the loss of thermal protection (Fig. 4). Heat Shock Mutant As an initial approach to genetically defining the mechanism by which the heat shock response protects I
I
I
I
I
0
1
2
3
4
TIME
(hours)
FIG. 3. Protection from thermal killing. Cells of strain AX3 growing at 22°C were heat shocked at 30°C for 0 min (O), 10 min (0), 30 min (O), or 60 min (A) before being shifted to 34°C. Samples were taken at various times after the cells were shifted to 34”C, diluted and plated with K. aerogenes. After incubation of the plates at 22°C for 3 days, plaques were counted to give the viable titer.
must enter the nuclease rapidly. We analyzed the newly made proteins present in nuclei 10 and 30 min after heat shock and found that all of the low-molecularweight species were present in nuclei after 30 min (Fig. 5) and were faint but detectable in nuclei after 10 min (data not shown). We also labeled cells heat shocked at 30°C for 3 hr and then blocked further incorporation by removing the r5S]methionine and adding cycloheximide (500 pg/ml) to inhibit subsequent protein synthesis. Half the cells
FIG. 5. Entry of heat shock protein into nuclei. Cells of strain AX3 growing at 22°C were shifted to 30°C and labeled with [%]methionine for 30 min. Nuclei were prepared and their proteins were analyzed by two-dimensional gel electrophoresis.
416
DEVELOPMENTAL
BIOLOGY
the cells, we screened for mutants defective in the response. Five thousand clones from a mutagenized population were replicated in multitest wells, grown at 22”C, and then heat shocked at 30°C for 3 hr. One set was challenged by 4 hr at 34°C and the other was kept at 30°C. Clones which grew in the unchallenged set but not in the challenged set were rescreened by quantitating the number of survivors in challenged and unchallenged populations. From 155 clones rescreened, one strain, HL122, proved to be defective in the thermal protection afforded by treatment at 30°C (Fig. 7). The strain grows well at 22°C and has a half-life at 30°C which is comparable to that of wild type. While the amount of [35S] methionine incorporated at either 22 or 30°C did not differ significantly between the mutant and the wild type, the pattern of newly made proteins in cells of strain HL122 after heat shock is markedly different from that in wild-type cells (Fig. 8, Table 1). The low-molecular-weight heat shock proteins have never been observed to be synthesized in strain HL122 while they make up about 1.9% of the proteins
t
hsp70
VOLUME
90, 1982
1 2 3 TIME (hours)
4
1 2 3 4 TIME AT 3CP (hours)
FIG. ‘7. Thermal protection in a heat shock mutant. (A) Cells strain HL122 growing at 22°C were heat shocked at 30°C for 1 before being transferred to 34°C (A); or were directly transferred 34°C (0) or 30°C (0). (B) The half-life of cells of strain Ax3 (0) strain HL122 (0) at 34°C was determined after various periods heat shock at 30°C as described in the legend to Fig. 3.
of hr to or of
synthesized in heat shocked Ax3 cells. The family of hsp70 proteins is synthesized as slightly less than 1% of total protein in wild-type and mutant cells at 22”C, but while this proportion increases &fold in strain AX3 following heat shock it increases less than 2-fold in strain HL122 at 30°C. Likewise the proportion of newly synthesized proteins made up by actin decreases 7.4fold following heat shock in strain Ax3 but only 2.9fold in strain HL122 (Table 1). hsp82 has not been observed to be rapidly synthesized in heat shocked cells of strain HL122. Moreover, the heat shock proteins were not induced in strain HL122 when incubated at either 2'7 or 32°C.
LMW DISCUSSION
1
2
3
4
5
FIG. 6. Chase from the nucleus. Cells were heat shocked at 30°C for 3 hr and labeled with [%G]methionine between 1 and 3 hr. They were then washed and resuspended in unlabeled medium containing cycloheximide (500 pg/ml) and chased at either 22 or 30°C. Nuclei were prepared from 2 X 105/cells at various times following the chase. Lane 1: 0 hr; lane 2: 3-hr chase at 22°C; Lane 3: 3-hr chase at 30°C; lane 4: 6-hr chase at 22°C; lane 5: 6-hr chase at 30°C. The nuclear proteins were separated electrophoretically on a 10% aerylamide gel which was prepared for autoradiography.
A brief treatment at nonlethal temperatures just above those permissive for growth protects cells of several organisms from thermal killing at higher temperatures (Loomis and Wheeler, 1980; McAlister and Finkelstein, 1980; Mitchell et al, 1979). The mechanism by which high temperatures result in irreversible damage to cells is unknown but occurs at temperatures which would not be expected to result in denaturation of most proteins (34°C in Dictyostelium; 42°C in Drosophila). At least in Dictyostelium death does not come from breaching the integrity of the cellular membrane since cells which have been rendered nonviable at 34°C continue to exclude trypan blue for several hours (Loomis, unpublished). There are many other ways in which high temperatures may be lethal including nuclear damage. It is therefore intriguing that among the proteins in-
LOOMIS AND WHEELER
Chromatin-Associated
Heat
Shock
417
Proteins
strain defective in the heat shock response leading to thermal protection is not induced to synthesize the chromatin-associated proteins by heat shock supports the idea that they may be at least partially responsible for the protection. The mutant strain is not impaired in growth or incorporation of labeled amino acids at either 22 or 30°C and so does not appear to carry a generally debilitating mutation. The most abundant protein synthesized following heat shock is hsp70. In Dictyostelium there are at least two distinct but closely related proteins, hsp7Oa and hsp70b (Loomis and Wheeler, 1980). During growth at 22°C they account for almost 1% of the total protein synthesized and accumulate to the expected degree. Since they are abundant even before heat shock, it is not apparent how continued synthesis for only 30 min can be essential for thermal protection. Nuclear localization of the low-molecular-weight proteins occurs with kinetics comparable to the kinetics of acquisition of thermal protection. If the synthesis of the heat shock proteins is blocked by the addition of cycloheximide (Loomis and Wheeler, 1980) or as a consequence of the mutation in strain HL122, then heat shock does not confer heat resistance. Further mutational analysis of the heat shock response may better define the processes involved. This work was supported by a grant from the NIH. FIG. 8. Protein synthesized by mutant strain HL122 at 30°C. Nuclei (A) and supernatant (B) fractions were prepared from lo6 cells which had been labeled with [%]methionine for 3 hr at 30°C after 1 hr of preincubation at 30°C. Proteins containing 3 X lo5 cpm were analyzed by two-dimensional electrophoresis.
duced by a heat shock are a class which rapidly becomes tightly associated with the chromatin. In Dictyostelium there are at least eight chromatin-associated heat shock proteins which differ in isoelectric point but have similar mobility in SDS electrophoresis. In Drosophila there are four proteins of similar molecular weight (22,000 to 27,000 daltons) which are found preferentially in the nucleus (Arrigo et ab, 1980). One of them contains little or no tryptophan (Arrigo et ah, 1980). Likewise we found that none of the low-molecular-weight heat shock proteins of Dictyostelium were labeled by [14C]tryptophan while hsp70 was strongly labeled (data not shown). This is another case of similarity of the heat shock proteins between highly diverse organisms. In Dictyostelium heat shock results in the rapid coordinate induction of the eight low-molecular-weight chromatin-associated proteins by a factor of more than 23. This is the most dramatic environmental response known in this organism. The finding that a mutant
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CORCES,V., HOLMGREN, R., FREUD, R., MORIMOTO, R., and MESELSON, meknwgaster M. (1980). Four heat shock proteins of Drosophila coded within a 12-kilobase region in chromosome subdivision 6’7B. Proc.
Nat.
Acad
Sci. USA
77, 5390-5393.
CRAIG, E., and MCCARTHY, B. (1980). Four Drosophila heat shock genes at 67B: Characterization of recombinant plasmids. Nucl Acids Res. 8, 4441-445’7. GUTTMAN, S., GLOVER, C., ALLIS, C., and GOROVSKY, M. (1980). Heat shock, deciliation and release from anoxia induce the synthesis of the same set of polypeptides in starved T. pyrifbrmti. Cell 22,299309.
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BIOLOGY
KELLEY, P., and SCHLESINGER, M. (1978). The effect of amino acid analogues and heat shock on gene expression in chick embryo fibroblasts. CeU 15, 12’77-1286. LOOMIS, W. F. (1971). Sensitivity of Dictyostelium diswideum to nucleic acid analogues. Exp. Cell Res. 64, 484-486. LOOMIS, W. F., and WHEELER, S. (1980). Heat shock response of Dietyostelium Develop. Bid 79, 399-408. MCALISTER, L., and FINKELSTEIN, D. (1980). Heat shock proteins and thermal resistance in yeast. Biochem Biophys. Rec. Commun 93, 819-824. MITCHELL, H., MOLLER, G., PETERSEN, N., and LIPPS-SARMIENTO, L.
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