Jan 5, 2018 - Printed in U. S.A.. Activation of Hemin-regulated Initiation Factor-2 Kinase in. Heat-shocked HeLa Cells*. (Received for publication, July 8, 1985).
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc
Vol. 261, No. 1, Issue of January 5, pp. 338-342,1986 Printed in U.S.A.
Activation of Hemin-regulated Initiation Factor-2 Kinase in Heat-shocked HeLa Cells* (Received for publication, July 8, 1985)
Arrigo De Benedetti and Corrado Baglioni From the Departmentof Biological Sciences, State University of New York a t Albany, Albany, New York 12222
Protein synthesis was drastically inhibited in HeLa cells incubated for 5 min at 42.5 “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-tRNAr to 4 0 S ribosomal subunits was greatly reduced and a polypeptide identified by immunoprecipitation with the a subunit of eukaryotic initiation factor-2 (eIF-2) was phosphorylated. Extracts prepared from control and hsat-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 M , 90,000 polypeptide and eIF-2a werephosphorylated 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-2a phosphorylation. Furthermore, the initial inhibition of protein synthesis and eIF-2a phosphorylation after heat shock were reduced byadding hemin to intact HeLa cells. These cells synthesized heat-shock proteins with some delay relative to cells without added hemin. The binding of MettRNAf to 40 S ribosomal subunits was inhibited by about 50% inextracts preparedfrom cells heatshocked for 40 min, and eIF-2a phosphorylation was increased in these cells. These results suggest that heme-controlled repressor is activated in heat-shocked cells and that eIF-2a phosphorylation limits mRNA translation even after partial recovery of protein synthesis.
gregate (8,9). After prolonged incubation at this temperature, thesynthesis of “normal”proteinsispartiallyrestored, whereas that of heat shock proteins proceeds at a high rate (8).The inhibition of protein synthesis during the early stages of the heat shockresponsewasrecently attributed to an 40 S ribosomal subunits (10) impaired Met-tRNAf binding to and toincreased phosphorylationof the a subunit of initiation factor eIF-2 (11). In the present study, we investigatedin HeLa cells the mechanism responsible for the inhibitionof protein synthesis after heat shock. Phosphorylation of eIF-2a was maximal when protein synthesiswas drastically inhibited, butdeclined when protein synthesis recovered. A polypeptide of M , 90,000 was phosphorylated when eIF-201 was phosphorylated. This polypeptide was tentatively identified with an eIF-2a kinase which is activated inreticulocyte lysate not supplemented with hemin (12) or incubated at high temperature (13, 14). The new finding in this work is that this kinase, which is designated HCR,’ is activated in HeLa cells during heatshock. MATERIALS ANDMETHODS
Cell Culture and Treatment-HeLa cells were grown in spinner cultures at 5 X 105/ml, as described (15). For labeling experiments, the cells were collected by centrifugation, and the supernatant was saved (conditioned medium)to resuspend thecells a t 3 X 106/ml. The cells were heat-shocked by resuspensioninconditioned medium heated a t 42.5 “C. Five volumes of ice-cold spinner salts solution were added to stop this treatment, and thecells were collected by centrifugation. Analysis of 40 S . Met-tRNA, Complexes-Cells were incubated for 5 min before heat shock with 0.1 mCi/ml of [35S]methionine. Cell extracts were prepared and centrifuged through sucrose gradients, as described(16). Gradientfractions were precipitated according to Panniers and Henshaw (10).To measure binding of Met-tRNAf to 40 S ribosomal subunits in cell extracts, binding assays were carried All cells respond to hyperthermia (heat shock) with rapid out in 50-pl reactions containing the componentsdescribed below for changes in the patternof gene expression (1). These changes cell-free protein synthesis and0.1 mM emetine. After 2 min a t 30 “C, occur at both the transcriptional and translational levels, and 25,000 cpm of [36S]Met-tRNAfwere added and the incubation continfor 2 min before fractionation on sucrose gradients (16). Purified lead to the synthesis of a set of proteins designated heat shock ued rabbit liver Met-tRNAf was aminoacylated as previously described proteins (2). Specific mechanisms regulate translationof heat (17). shock proteins in different organisms. In yeast, heat shock Phosphoprotein Analysis-HeLa cells were incubated for 2 h in medium minus phosphate and then for 1 h with 0.1 mCi/ml of 32P mRNAs are induced and translated, whereas other mRNAs are degraded (3, 4). In Drosophila cells, the mRNAs induced before heat shock. The cells were collected by centrifugation and by heat shock associate with polyribosomes, whereas the other resuspended in an equal volume of lysis buffer containing 20 mM mM Mg(OAc)2,1mM dithiothreitol, and20 mM Hepes buffer, mRNAs are stored in a non-polyribosomal pool (5, 6). In KC1,1.5 pH 7.4. The cells were broken by homogenization and a supernatant chicken reticulocytes, the level of heat-shock mRNAs is only fraction was obtained by centrifugation for 10 min at 30,000 X g. 5 pl slightly increased, in contrast toa greatly elevated synthesis of this fraction were directly analyzed by gel electrophoresis. Alterof heat shock proteins andrepression of globin synthesis (7). natively, 20 p1 of cell extract were immunoprecipitated with 2 p1 of ProteinsynthesisisdrasticallyinhibitedinHeLa cells an antiserum againstpurified rabbit reticulocyte eIF-2 (a kindgift of Dr. William C. Merrick, Case Western Reserve University Medical incubated at 42 “C and concomitantly polyribosomes disagSchool) ina reaction containing 10 mM NaF to inhibit phosphatases
* This work was supported by Grant CA 29895 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The abbreviations used are: HCR, heme-controlled receptor; eIF, eukaryotic initiation factor; Hepes,4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; eIF-Z(aP), eukaryotic initiation factor-2 phosphorylated in the a subunit.
338
eIF-2 Heat Kinase and (18, 19). After 20 min on ice, 25 pl of SAC buffer (1% Triton X-100, 0.5% sodium dodecyl sulfate,and 2.5 mM EDTAinphosphatebuffered saline) were added together with 15 pl of I@-Sorb (Enzyme Center, Inc.). After 30 min on ice, 0.2 ml of SAC buffer were added and the samples were filtered through CVWP filters in a Multiwell Millititer filtration plate (Millipore). Each well was washed with 2 mlof SAC buffer and the filters were then cut out and eluted by boiling in sample buffer. All the samples were analyzed by electrophoresis in 10% polyacrylamide gels. T o phosphorylate proteins in cell extracts, pelleted cells were resuspended in 2 volumes of lysis buffer, and a supernatant fraction was prepared as described above. 5 pl of this fraction were incubated for 4 min a t 30 "C,in 10-pl reactions containing 0.5 pCi of [yJ2P]ATP,20 ng of purified rabbit reticulocyte eIF-2 (a gift of W. C. Merrick), where indicated, and the other components previously described (17). To inhibit HCR, 2 pl of an antiserum raised against rabbit reticulocyte HCR (a gift of Dm. Gisela Kramer and Boyd Hardesty, University of Texas at Austin) were added for 10 min a t 0 "C before the incubation. The reactions were stopped by the addition of 2 X sample buffer and analyzed by gel electrophoresis. CdI-frec Protein Synthesis-Cell extracts with or without 50 p M hemin were prepared as described (20) and used immediately in 50pl reactions containing 35 pl of cell extract, 5 pCi of ['Hllysine, and the other componentspreviously reported (20). RESULTS
Inhibition of ProteinSynthesisin Heat-shocked HeLa Cells-Protein synthesis was drastically inhibited5 min after transferring HeLa cells from 37 to 42.5 PC, but resumed after 20 min at a rate approximately 50% thatof control cells kept a t 37 "C (Fig. 1). This reduced rate of translation was maintained for at least 3 h (data not shown). Subsequent experiments were designed to establish whether this inhibition of proteinsynthesis was caused by inactivation of initiation factor eIF-2, which promotes the binding of initiator MettRNAf to 40 S ribosomal subunits. When the a subunit of eIF-2 is phosphorylated, the level of Met-tRNAr.40 S complexes decreases (12, 21). These complexes were measured in intact HeLacells labeledwith ["Slmethionine by centrifuging cell extracts through sucrose gradients, described as (16). The ["S]Met-tRNAf in the 40 S peak was reduced by about 65% after 10 min of heat shock (6850 cpm a t 37 "Cuersus 2350 a t
Shock
339
42 "C). eIF-Sa Phosphorylation in Heat-shocked Cells-To establish whether eIF-2a phosphorylation increased during heatshock, HeLa cells were incubated 2 h with "?P a t 37 "C and then at 42.5 "C for 10 min. The level of eIF-2a phosphorylation was examined by sampling cells transferred to 42.5 "C over 30 min. Unfractionated cell extracts were analyzed by gel electrophoresis. Maximal phosphorylation of a M, 37,000 polypeptide was detected after 10 min of heat shock (Fig. 2). This polypeptide was identified with eIF-B(aP) by immunoprecipitationwithanantiserumpreparedagainst purified rabbit reticulocyte eIF-2. The analysis of the immunoprecipitated samples showed that eIF-2a was maximally phosphorylated after 10 min a t 42.5 "C and was gradually dephosphorylated afterwards. The dephosphorylation of eIF-2a after20 min was correlated with the resumptionof protein synthesis shown in Fig. 1. It should be pointed out that the phosphorylation of a number of polypeptides beside eIF-2a increased in response to heat shock (Fig. 3A). Mechanism of e l F - 2 ~Phosphorylation ~ in Heat-shocked Cells-These results suggested that phosphorylation of eIF2a was involved in the inhibitionof initiation in heat-shocked cells. However, it was difficult to investigate the mechanism 0
20
IO
X)
minutes 0 IO
20
" "
eIF-2 ~
-P L -a
1
I
A
B
FIG. 2. Analysis of proteins labeled with 32Pfrom control cells ( 0 )or from cells heat-shocked at 42.5"Cfor the indicated min ( A , 10-30) and of the corresponding immunoprecipitates obtained with anti-eIF-2antiserum ( B ) .5 pl of cell extract were directly analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresisand 20 pl were immunoprecipitated with antiserum against rabbit reticulocyte eIF-2. Asample of purified eIF-2 phosphorylated as described (17) was run in parallel and the position of its a and p subunits is indicated.
B
FIG. 1. Inhibition of protein synthesis in heat-shocked HeLa cells. The cells were incubated a t 3'7 ( 0 )or 42.5 "C (A)with 25 pCi/ ml of' ["Hllysine. 0.1-ml aliquots were removed at the times indicated, filtered through glass fiber filters, washed with saline and 5Tij trichloroacetic acid, dried, and counted.
-50
-hemin
FIG. 3. Time course ofprotein synthesis of cell-free systems containing extract from control cells (0)or cells heat-shocked for 10 min a t 42.5 "C (A). The cell extracts were prepared in homogenization buffer containing 50 p~ hemin (left) or no hemin (right). The reactions were incuhated at 30 "C, as described under "Materials and Methods."
340
eIF-2 Kinase and Heat Shock
of eIF-2a phosphorylation in intact cells. For this analysis, extracts from control and heat-shocked cells were prepared following a procedure developed to obtain an active cell-free system from HeLa cells (20). These extracts were assayed for protein synthesis at 30 "C, but no significant difference was detected between control and heat-shocked cells (Fig. 3A). Both cell extracts synthesized protein linearly for at least 35 min and were therefore quite active in initiation. This result contrasted with a recent report that a cell-free system prepared from heat-shocked Ehrlich ascites cells has little initiation activity (10). One difference between these cell-free systems was the addition of 50 p~ hemin to the buffer used to lyse HeLa cells (20). Preparation of cell extracts in the presence of hemin is most effective in obtaining a cell-free system active in initiation, although hemin can also be added to HeLa cell extracts before freezing them, with satisfactory results (20). To establish whether the addition of hemin was responsible for the discrepancy between our results and those with Ehrlich ascites cells, we prepared extracts from HeLa cells without this addition. These extracts were immediately assayed for protein synthesis (Fig. 3B). Extracts from control cells showed an initial translation rate almost identical to that of control extracts supplemented with hemin, but protein synthesis declined drastically after 10 min (Fig. 3B). Extracts from heatshocked cells showeda reduced rate of protein synthesis even at early time points. Much of the radioactivity incorporated by the latter extractscould beaccounted for by the elongation of nascent polypeptides, as reported by Panniers and Henshaw (10) and confirmed (datanot shown) by inhibiting initiation with aurintricarboxilic acid (20). Further evidence for a loss of initiation activity in extracts of heat-shocked cells and for a recovery after prolonged incubation at 42.5 "C was obtained by measuring the binding of [35S]Met-tRNArto 40 S ribosomal subunits. Cell extracts prepared without addition of hemin were incubated for 2 min with rabbit liver [35S]Met-tRNAfand fractionated by gradient centrifugation. After 10 min of heat shock, the binding of initiator tRNA to 40 S initiation complexes was reduced to 1990 cpm or 20% that of control cells (9960 cpm). However, after 40 min at 42.5 "C this binding increased to 5200 cpm (50% that of control cells). The effect of hemin on eIF-2a phosphorylation in extracts of heat-shocked cells was next investigated. Extracts with or without added hemin were prepared from cells incubated for 10 min at 42.5 "C. These extracts were labeled with [r-"P] ATP in incubations with or without added eIF-2. Extracts from control cells did not phosphorylate eIF-Za, whereas extracts from heat-shocked cells phosphorylated eIF-2a in the absence of added hemin but not when supplemented with hemin (Fig. 4). Moreover, a M , 90,000 polypeptide was phosphorylated in extracts of heat-shocked cells without added hemin (Fig. 4, lanes C and D).The M , of this band corresponded to thatof a polypeptide phosphorylated by HCR (13, 21), suggesting that this kinase was activated in cells heatshocked for 10 min. The addition of hemin specifically prevented the phosphorylation of this polypeptide in extracts of heat-shocked cells and correspondingly inhibited eIF-2a phosphorylation, suggesting that the activation of HCR was reversed by hemin. It should be pointed out that another polypeptide of M, 95,000 was poorly phosphorylated in extracts of heat-shocked cells not supplemented with hemin, whereas it was phosphorylated in extractsof control cells and of heat-shocked cells supplemented with hemin. A similar observation has been reported by Ernst et al. (22) for rabbit reticulocyte lysate incubated at 42 "C, but this polypeptide
- hemln
+hemin
"
A
B
C
D
H
l
J
K
oac
eIF-2
-
+,
37O
-
+11
-
+,
,-
37
42O
+
42O
FIG. 4. Analysis of proteins phosphorylated in incubations of cell extracts prepared without added hemin or with 50 PM hemin. Cell extracts were prepared from control cells (37 ") or from cells heat-shocked for 10 min a t 42.5 "C (42 "). 5 pl of cell extract were incubated for 4 min in 10-pl reactions containing 0.5 pCi of [ y 32P]ATP, as described under "Materials and Methods." 20 ng of purified eIF-2 were added to the reactions indicated with a + at the bottom. A sample of eIF-Z(aP) is shown in the first lune and the position of a M,90,000 polypeptide is indicated.
I eIF-2 ( a P b
--
eIF-2 anti-HCR
-
+
-
-k i-
FIG. 5. Effect of anti-HCR on the phosphorylation of eIF2a in extracts of heat-shocked cells. The incubations were carried out as described in the legend to Fig. 4, with 2 pl of antiserum against HCR added where indicated.
could not be identified. The experiment shown in Fig. 5 was repeated with extract fromcells incubated for 40 min at 42.5 "C, but the M , 90,000 polypeptide and added eIF-2 were less phosphorylated than in incubations with extract prepared after 10 min of heat shock (data not shown). Evidence for activation of HCR in heat-shocked cells was obtained by adding an antiserum prepared against rabbit HCR (21) to reactions containing extract of heat-shocked cells with no added hemin (Fig. 5). The phosphorylation of both eIF-2a and the M , 90,000 polypeptide was inhibited by anti-HCR, but in addition other bands showed decreased phosphorylation in the presence of anti-HCR. It should be pointed out that the antiserum used was raised against the M , 90,000 polypeptide of HCR, which contains severalpolypeptides ranging from M , 85,000 to 100,000 (21). Several of these polypeptides are phosphorylated and dephosphorylated during the activation of HCR (21). Although the polyclonal antiHCR specifically inhibits the eIF-2a kinase activity of HCR, it precipitates several phosphoproteins, some of which may be components of the HCR protein kinase complex? Control B. Hardesty and G . Kramer, personal communication.
Kinase eIF-2
and Heat Shock
experiments with nonimmune sera were carried out,but failed to show inhibition of eIF-2a phosphorylation (datanot shown). A role of hemin in the inhibition of protein synthesis in heat-shocked cells was shown by adding different concentrations of this compound to HeLa cells before a 20-min incubation at 42.5 "C. During this incubation, protein synthesis was measured relative to control cells kept at 37 "C (Fig. 6). Low hemin concentrations (0.3 p ~ reversed ) to a small extent the inhibition of protein synthesis in heat-shocked cells, but hemin concentrations 100-fold greater allowed protein synthesis to proceed at about 50% the rate of control cells. The addition of 30 p~ hemin had a very slight stimulatory effect on protein synthesis in control cells (Fig. 6, legend). Since hemin has been shown to be taken upby cells in culture (23), these results suggested that hemin prevented at least in part the activation of HCR and eIF-2a phosphorylation in heatshocked cells. This was demonstrated by an experiment de-
341 minutes
5(
%
2!
"
- hemin
+hemin
FIG. 8. Analysis of proteins synthesized by control cells and by cells heat-shocked without or with 30 p~ hemin added to the culture medium. At the top are indicated the time of labeling after transferring the cells to 42.5 "C. The cells were labeled with [35S]methionineas described in the legend to Fig.6.150,000 cpm were applied in each track. The positions of two prominent heat shock proteins (HSP 70 and HSP 80) are indicated.
signed to measure the phosphorylation of eIF-2a incells heatshocked after addition of 30 p~ hemin (Fig. 7). Hemin greatly FIG. 6. Effect of hemin on theinhibition of protein synthesis reduced eIF-2a phosphorylation, suggesting that it prevented in heat-shocked cells. HeLa cells were preincubated for 20 min a t HCRactivation in heat-shocked cells. Moreover, extracts 37 "C with the hemin concentrations indicated on the abscissa and then transferred to 42.5 "C. After 5 min, 15 pCi of [35S]methionine from these cells did not significantly phosphorylate eIF-2a in were added for 15 min to 1-ml aliquots of cell cultures. Acid-precipi- kinase assays (data not shown). To establish whether hemin addition alteredthe heatshock table radioactivity was measured as described in the legend to Fig. 1 and is expressed as a per cent of that incorporated by cells incubated response, the proteins synthesized by control cells and by 15 min at 37 "C without added hemin (126,000 cpm). These cells cells incubated with 30 p~ hemin were analyzed by gel elecincorporated 131,000 cpm when supplemented with 30 p~ hemin. trophoresis. The cells were labeled in 20-min pulses with [35S] methionine (Fig. 8). The rate of synthesis of heat shock A B C proteins of M, 70,000 and 80,000 was significantly increased in control cells after 50 min of heat shock, whereas a comparable increase was observed in hemin-treated cells 70 min after heat shock. Therefore, the addition of hemin delayed somewhat either thesynthesis of heat shock mRNAs or their translation. DISCUSSION
Incubation of HeLa cells at 42.5 "C results in a drastic inhibition of initiation, but after 30 min protein synthesis resumes at about 50%the normal rate (8,9). Initiation factor eIF-2 plays a central role in the regulation of initiation in animal cells (24). When eIF-Pa is phosphorylated, there is a loss of native 40 S ribosomal subunits containing Met-tRNAr (12). In heat-shocked HeLa cells, 40 S Met-tRNAf complexes were drastically reduced. When phosphoproteins were labeled in intactcells and examined by gelelectrophoresis, eIF-B(aP) was found to be significantly phosphorylated 10 min after heat shock. This finding confirms the previous report that eIF-2a is phosphorylated in HeLa cells incubated at 44"C
-
FIG. 7. Effect of hemin on eIF-2a phosphorylation in heatshocked cells. Phosphoproteins were labeled at 37 ( A )or 42.5 "C ( B and C ) and analyzed after immunoprecipitation as described in the legend to Fig. 2. 30 p~ hemin was added in B 20 min before heat shock.
342
eIF-2 Kinase and HeatShock
(11).However, in cells incubated at 42.5 "C eIF-2a was dephosphorylated after 20 min, when protein synthesisresumed at 50% the rate of control cells. Correspondingly, eIF-2(aP) did not quite return to control levels in cells continuously incubated at 42.5 "C and Met-tRNAfbinding to 40 S subunits was reduced by about 50%. When reticulocyte lysate is incubated without added hemin, protein synthesisis inhibited by the eIF-2akinase designated HCR and a M , 90,000 polypeptide is phosphorylated (12, 21, 24). Moreover, HCR can be activated by incubation of reticulocyte lysate at high temperature and a correlation was established between temperature of incubation and concentration of added hemin required to maintain protein synthesis (13, 14). When the proteins of extracts from heat-shocked HeLa cells without added hemin were analyzed, the phosphorylation of a M , 90,000 polypeptide was detected. Moreover, these extracts had little initiationactivity. Addition of hemin restored initiation activity and inhibited the phosphorylation of the M , 90,000 polypeptide. Similarly, addition of an antiserum against HCR prevented the phosphorylation of this polypeptide and of eIF-2a in extracts of heat-shocked cells not supplemented with hemin. Activation of HCR and phosphorylation of eIF-2a is a likely explanation for the initial drastic inhibition of protein synthesis in HeLa cells heat-shocked at 42.5 "C. It is not known whether a lack of hemin is responsible for the activation of HCR and inhibition of protein synthesis in these cells, although this is suggested by the reversal of inhibition obtained by adding hemin to the culture medium prior to heat shock (Fig. 6). Maximal effect is obtained with 30 KM hemin, which maintains protein synthesis at about the rate observed after 20-40 min of continuous incubation at 42.5 "C. At this time, the heat-shocked cells recover 50% of their protein synthetic activity. It seems possible that a persistent inhibition of MettRNAf binding to 40 S ribosomal subunits may be responsible for this incomplete recovery, since this binding is inhibited about 50% in extracts prepared after 40 min of heat shock. It should be pointed out that the experiments described were carried out only in cells heat-shocked at 42.5 "C. This condition was chosen because it appeared to be reasonably close tothe hyperthermia to which human cells maybe exposed during fever. It is quite possible that higher temperatures may inhibit protein synthesisby different mechanisms. Duncan and Hershey (11)have reported that in HeLa cells incubated at 44 "C initiationfactors eIF-2, eIF-4B, eIF-3, and eIF-4F may be inhibited. Panniers et al. (25) recently reported that eIF-4F activity is reduced in extracts prepared from Ehrlich ascites cells heat-shocked at 44 "C. Addition of purified eIF-4F stimulates protein synthesisin these lysates (25). However, it cannotbe excluded that theoverall rate of protein synthesis in HeLa cells heat-shocked at 42.5 "C may in part be limited by the ability of the cells to efficiently translate only heat shock mRNAs. Addition of hemin to intact cells cannot overcome this effect, but itprevents the initial drastic inhibition of protein synthesis and delays the synthesis of heat shock proteins. It seems possible, therefore, that an inhibition of protein synthesis initiation immediately after heat shock may somehow facilitate translation of heat shock mRNAs when protein synthesis resumes. We are currently investigating whether heat-shock mRNAs are preferentially translated when the initiation activity is reduced and the
temperature of incubation is increased. It is not known how hemin may regulate protein synthesis in HeLa cells. The intracellular concentration of "free" versus protein-bound hemin andits subcellular distribution vary with different physiological conditions (23), but itis not clear whether this may be relevant for the regulation of protein synthesis. Possibly, the concentration of free hemin may initially decrease in heat-shocked cells and be restored to its normal level after the cells become adapted to high temperature. Further studies on hemin levels in intact cells may help to elucidate its role in the inhibition of protein synthesis during heat shock. Acknowledgments-We are grateful to Dr. William C. Merrick for the gift of purified eIF-2 and anti-eIF-2antiserum and to Dr. Gisela Kramer and Dr. Boyd Hardesty for the gift of anti-HCR antiserum. REFERENCES 1. Schlesinger, M. J., Ashburner, M., and Tissieres, A. (1982) Heat Shock: From Bacteria toMan, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 2. Di Domenico, B. J., Bugaisky, G . E., and Lindquist, S. (1982) Cell 31,593-603 3. Kurtz, S., and Lindquist, S. (1984) Proc. Natl. Acad. Sci. U. S. A . 81, 7323-7327 4. Lindquist, S. (1981) Nature 293, 311-314 5. Mc Kenzie, S. L., and Meselson, M. S. (1977) J . Mol. Biol. 177, 279-283 6. Storti, R. V., Scott, M. P., Rich, A., and Pardue, M. L. (1980) Cell 22,825-834 7. Banerjee, S. S., Theodorakis, N. G., and Morimoto, R. I. (1984) Mol. Cell. Biol. 4, 2437-2448 8. Hickey, E. D., and Weber, L. A. (1982) Biochemistry 21, 15131521 9. McCormick, W., and Penman, S. (1969) J. Mol. Biol. 3 9 , 315333 10. Panniers, R., and Henshaw, E. C. (1984) Eur. J. Biochem. 140, 209-214 11. Duncan, R., and Hershey, J. W. B. (1984) J. Biol. Chem. 259, 11882-11889 12. Farrel, P. J.,Balkow, K., Hunt, T., and Jackson, R. J. (1977) Cell 11,187-200 13. Bonanu-Tzedaki, S. A., Sohi, M., and Arnstein, H. R. V. (1981) Eur. J. Biochem. 1 1 4 , 69-77 14. Mizuno, S. (1975) Biochim. Biophys. Acta 414,273-282 15. Minks, M. A., Benvin, S., Maroney, P. A., and Baglioni, C. (1979) J. Biol. Chem. 254,5058-5064 16. De Benedetti, A., and Baglioni, C. (1983) J . Biol. Chem. 258, 4556-14562 17. De Benedetti, A,, and Baglioni, C. (1985) J. Biol. Chem. 260, 3135-3139 18. Grankowski, N., Lehmusvirta, D., Kramer, G., and Hardesty, B. (1980) J. Biol. Chem. 255, 310-317 19. Wong, S. T., Mastropaolo, W., and Henshaw, E. C. (1982) J . Biol. Chem. 257,5231-5238 20. Weber. L. A.. Feman. E. R.. and Baelioni. C. (1975) Biochemistrv 14,5315-5321 21. Wallis. M. H.. Kramer., G.., and Hardestv. " , B. (1980) Biochemistrv 19,798-804 22. Ernst, V., Zukofsky Baum, E., and Reddy P. (1982) in Heat Shock: From Bacteria to Man, (Schlesinger, M. J., Ashburner, M., and Tissieres, A., eds) pp. 215-225, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 23. Granick, S., Sinclair, P., Sassa, S., and Grieninger, G. (1975) J. Biol. Chem. 250,9215-9225 24. Jagus, R., Anderson, W. F., and Safer, B. (1981) Prog. Nucleic Acid Res. Mol. Biol. 25, 127-185 25. Panniers, R., Steward, E. B., Merrick, W. C., and Henshaw, E. C. (1985) J . Biol. Chem. 260, 9648-9653 -
I
