Oct 25, 2017 - Lysates from normally growing (25 âC) or heat- shocked (37 âC, 45 min) Drosophila melanogaster em- bryos were obtained and the effect of ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc
Vol. 263, No. 30,Issue of October 25, pp. 15720-15725,1988 Printed in U.S.A.
Translational Control in Heat-shocked Drosophila Embryos EVIDENCE FOR THE INACTIVATION OF INITIATIONFACTOR(S) mRNA CAP STRUCTURE*
INVOLVED IN THE RECOGNITION OF
(Received for publication, March 28, 1988)
Federico G. MarotoS and Jose M. Sierra4 From the Centro deBiologia Molecular, Consejo Superior de Znvestigaciones Cientificas and UniuersidadAutomma de Madrid, Cantoblanco, 28049 Madrid, Spain
Lysates from normally growing (25 “C) or heatshocked (37 “C, 45 min) Drosophila melanogaster embryos were obtained and the effect of analogues of the mRNA 5”terminal cap, m7G(5’)ppp(5’)N structure and ofpotassium ions on their endogenous protein synthesis activity was studied. At optimal concentration of KCHsCOO (75-80 mM), protein synthesis in normal lysates is strongly inhibited by cap analogues (m7GpppG,m7GDP, and m’GMP).At the same ionic conditions, heat shock lysates translate preferentially the heat shock messengers, and this translation is almost unaffected by the cap analogues. In contrast, residual synthesis ofnormal proteins in heat shock lysates was reduced by these compounds. By lowering the concentration of potassium ions it was possible to gradually reverse the inhibitory effect of the cap analogues in normal lysates and also to increase specifically the translation of normal mRNAs in heat shock lysates. Translation of normal mRNAs is also partial but specifically rescued by supplementing heat shock lysates with polypeptide chain initiation factors partially purified from rabbit reticulocytes. These data are consistent with the notion that the failure of normal mRNAs to be translated under heat shock conditions might be due, at least to some extent, to the inactivation of polypeptide chain initiation factor(s) involved in the recognition of the mRNA 5”terminal cap structure. ~~~
~
simple model (11) of competition between these and heat shock messengers (4, 7, 9, 10, 12-14) underlies this mechanism. It is known that a structural feature present in the leader sequence of the heat shock messengers is required for their efficient translation during heat shock (15-17). In addition, it appears that the mechanism by which Drosophila heat shock and normal mRNAs are discriminated also involves a change in some component(s) of the translational machinery, although its characterization hasremained elusive until now (9, 10, 18, 19). It is even unsettled whether this component(s) is present in the postribosomal supernatant (10) or associated to ribosomes (9). On the other hand, although the mechanisms for translational regulation underlying the heatshock response in mammalian and Drosophila cells might be different, at least to some extent (1, 2, 20), several polypeptide chain initiation factors appear to be modified in response to heat shock in HeLa (21-23) and Ehrlich (24, 25) cells. However, a correlation of the rapid inhibition of protein synthesis with the phosphorylation of eIF-2a and the dephosphorylation of the 28-kDa subunit of eIF-4F observed in mammalian cells under heat shock remains to be clearly established (21-26). We report here that endogenous translation of heat shock mRNAs in lysates from heat-shocked Drosophila melanogaster embryos is very resistant to inhibition by analogues of the mRNA cap structure. We also show the partial but specific rescue of normal mRNA translation in these lysates by lowering the concentration of K+ or the addition of partially purified polypeptide chain initiation factors of rabbit reticulocytes. These results are consistentwith the notion that cap recognition is involved in translational control during heat shock in Drosophila.
A brief exposure of cells to higher than physiological temperatures resultsin profound changes of their gene expression. This heat shock response is being studied in several organisms, particularly in Drosophila where it appears to be regulated not only at the level of gene transcription but also at EXPERIMENTALPROCEDURES the level of mRNA translation (for recent reviews see Refs. 1 Embryo Collection and Heat Shock-Wild type D. melanogaster and 2). Within minutes after heat shock, a mechanism of (Oregon R strain) were grown in mass culture, and embryos 6-12-htranslational control is established that leads to the prefer- old were collected as described previously (27).The embryos were ential translation of messenger RNAs coding for the heat thoroughly washed with saline solution (0.7% NaCl), dechorionated shock proteins (hsps)’ over the preexisting normal messages. for 2 min in Clorox/ethanol ( H ) ,and finally washed five times with Neither the degradation of normal mRNAs (3-10) nor a ice-cold buffered saline (1.5 mM KH~POI,6.5 mM Na2HPOI, 2.7 mM
KCl, 137 mM NaCl). Embryos were subjected to the heat shock treatment (45min, 37 “C)before dechorionation. Preparation of Embryo Lysates-The procedure outlined by SandAsesora de Investigacibn Cientifica y Tbcnica and the Fondo de Investigaciones Sanitarias (Spain). The costs of publication of this ers et al. (10) for the preparation of lysates from Drosophila cells was article were defrayed in part by the payment of page charges. This followed with slight modifications. All operations were performed a t article must therefore be hereby marked “advertisement” in accord- 0-4 ‘C. Washed embryos were suspended in 1 volume of buffer A (10 mM Hepes, pH 7.6,5 mM dithiothreitol) and homogenized in a tightance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of a fellowship from the Fondo de Investigaciones fitting Dounce homogenizer (15 strokes), and one-ninth volume of buffer B (100 mM Hepes, pH 7.6, 1 M KCH&OO, 10 mM Sanitarias. Mg(CH3C00)2,50 mM dithiothreitol) was added. The homogenate To whom correspondence should be addressed. The abbreviations used are: hsp, heat shock protein; eIF, eukar- was centrifuged for 30 min at 40,000 X g, and thetop 75% of the clear yotic initiation factor; Hepes, 4-(2-hydroxyethyl)-l-piperazineeth- supernatant was removed and made 0.2 mg/ml in creatine phosphoanesulfonic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide kinase and 10% in glycerol. This fraction, which will be referred to as embryo lysate, was kept frozen a t -70 ‘C until use. gel electrophoresis.
* This investigation was supported by grants from the Comisi6n
15720
Translational Control in Heat-shocked Drosophila Embryos
15721
Cell-free Translation Assay-Unless otherwise noted, samples con- of that obtained in lysates from normal embryos (Fig.lA). A tained, in a final volume of 20 pl, 30 mM Hepes adjusted to pH 7.6 significant increase in the translation rate is observedin with KOH, 74 mM KCH3CO0, 0.5 mM Mg(CH&00)2, 0.1 mM lysates from embryos which wereallowed to recover (3 h, spermidine, 4 mM dithiothreitol, 0.15 mM EDTA, 5% glycerol, 25 p M of each of 19 unlabeled amino acids, 28-56 X lo6 cpm of [35S]Met 25 "C) from heat shock (Fig. lA).Fig. 1B shows the patterns (Amersham Corp., 1020-1475 Ci/mmol), 15 mM creatine phosphate, of translation products in lysates from normal (truck 1 ) or 2 pg (0.8-1.0 units) of creatine phosphokinase, 10 units of RNasin heat-shocked (truck 2) embryos. In heat shock lysates, the (Promega-Biotec),and 10 pl of embryo lysate. Whenpresent, synthesis of heat shock proteins (hsp 83, hsp 70, hsp 27-26, m7GpppG, m7GDP, and GDP were added together with and hsp 23-22) isclearly detected, while the synthesis of Mg(CH&00)2 at equimolar concentration. Reaction mixtures were normal proteins is strongly reduced. This patternof polypepincubated for 45 mina t 28 "C. T o determine the radioactivity incorporated into hot acid-insoluble materials, 5-pl samples were with- tide synthesis is basically identical to thatdescribed in Drodrawn and applied to WhatmanNo. 3MM filter squares,which were sophila cells and tissues grown in culture. In addition, two processed as described (28). non-heat-shock polypeptides (MI= 16,000and 17,000), which Gel Electrophoresis-After incubation,reactionmixtures were probably correspond to histones, are actively synthesized in made 62.5 mM in Tris-HCI, pH 6.8,2% in SDS, 10% in glycerol, and heat shock lysates from Drosophila embryos. Synthesis of 5% in 2-mercaptoethanol, and the [35S]Met-labeled polypeptides were analyzed by SDS-PAGE (12.5% acrylamide, 0.34% bisacrylamide; or histones and their messages has been reported to bevery 0.21% bisacrylamide in the experimentof Fig. 6) accordingto Laem- resistant to heat shock inhibition (3). As expected, a prefermli (29), followed by fluorography using Amplify (Amersham Corp.). ential increase in the synthesis of normal proteins relative to Gel calibration was made using following the molecular mass markers: heat shock proteins isobserved in lysates fromrecovered phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbuminembryos (not shown). (43 kDa), carbonic anhydrase (30 kDa), and trypsin inhibitor (20.1 Resistance of Heut Shock mRNA Translation to Inhibition kDa). Preparation of Initiation Factors from Rabbit Reticulocytes-A ri- by Cup Amlogues-At an optimal concentration of KCH3COO to the method (-75mM), protein synthesis in normal lysates is strongly bosomal high-salt wash fraction was obtained according of Schreier and Staehelin(30). To isolate highly purified eIF-4E and inhibited by cap analogues (m'GpppG, m'GDP, and m7GMP) eIF-4F, the ribosomal high-salt washwas furtherfractionated by (Fig. 2A). This inhibition requires not only the presence of precipitation with ammonium sulfate, sucrose gradient sedimentation the methyl-7 group but also one phosphate group at least, in a buffer containing 0.5 M KC1 and affinity chromatography on which is consistent with a specific effectof these compounds. m'GTP-Sepharose essentially as described (31-33). RESULTS
Lysates fromnormallygrowing (25 "C) or heat-shocked (37 "C, 45 min) D. melarwguster embryos were obtained as indicated under "Experimental Procedures." As shown in Fig. I A , protein synthesis is linear for 260 min in both types of lysates.However, therate of protein synthesis in lysates prepared from heat-shocked embryos is usually about 10-25% 1
2
15
- hh ss pp 87 30
Ii
In heat shock lysates, however, a 40-50% maximuminhibition of protein synthesis activity is usually obtained at the same ionic conditions, even in the presence of relatively high concentrations of cap analogues (Fig.2B). This reduction appears to be mainly due to the specific inhibition of the residual synthesis of normal proteins observed in these lysates (Fig. 3). When the pattern of translation products in heat shock lysates supplemented with m7GpppG is analyzed, it is found that the synthesis of the heat shock proteins (except for hsp 83) is almost unaffected, whereas the residual synthesis of normal proteins is inhibited (Fig. 3, trucks 1-5). Synthesis of p16 and p17 appears to be more resistant to cap analogue inhibition than the synthesis of other normal proteins (this is more clearly observed when an equal number of counts/ min are applied to thegel to analyze the translationproducts
"P 4 3
- hsp 26,27 - hsp 22,23
. )
I
30 60 Time ( min)
90
FIG. 1. A, time course of protein synthesis in lysates prepared from normally growing (25 "C) (0)or heat-shocked (37 "C, 45 min) (0)D. melanogaster embryos, or from embryos which were allowed to recover (25 "C, 180 min) after heat shock (0).Values represent [:%]Met incorporated in 5 pl of reaction mixture. B , electrophoretic analysis of the translation products in lysatesfrom normal (track I ) or heat-shocked (track 2) embryos. 2 pl(68,OOO cpm) or 10 pl(69,OOO cpm) of each reaction mixturewere applied in tracks I and 2, respectively, and subjected to SDS-PAGE andfluorography. The positions of the heat shock (hsp 83, hsp 70, hsp 27, hsp 26, hsp 23, and hsp 22) and some of the normal (p43, p17, and p16) proteins are indicated. Translation assays in A and B were carried out as described under "Experimental Procedures."
I
I
I
0.5
1.0
I
[Capanalogue]
I
I
0.5
1.0
(mM1
FIG. 2. Effect of cap analogues on protein synthesis in lysates from normal (A) or heat-shocked ( B )D. melanogacrter embryos. Reaction mixtures containing m'GpppG (O), m7GDP (a), m'GMP (A), m7G (A),GDP (O), or GMP (M) at the concentrations indicated were incubated for 45 min a t 28 "C in the standard conditions. 5-pl samples were taken to assay for protein synthesis, which is plottedas a percentage of the control incubated without nucleotide. 100% values were (cpm X lo-'): 170.0 (0).157.9 (01,166.0 (A), 144.6 (A),157.9 (O), and 144.6 (m) in A, and 34.7 (O), 45.2 (a),and 39.7 (A) in B.
Translational Control in Heat-shocked Drosophila Embryos
15722 1
hsp83 hsp70
-
p43
-
2
3
4
5
6 7 8 9 1 0
TABLE I Influence of potassium ions on the inhibition of protein synthesis by m7GDPin normal lysates from D. melamgaster embryos Lysates were prepared from normal embryos as described under "Experimental Procedures" (experiment 2) or without the addition of KCH3COO (experiment 1).Translation assays were carried out in the standard conditions for 45 min a t 28 "C in the absence or the presence (0.5 and 0.37 mM in experiments 1 and 2, respectively) of m7GDP as indicated. Values represent ["S]Met incorporated in 5 pl of reaction mixture. ~
-""-
hsp 26,27
-
hop 22.23
-
p17
-
p16
0
FIG. 3. Translation products in heat shock (tracks 2 - 5 ) or normal (tracks 6-20) lysates supplemented with m'GpppG. Standard reaction mixtures (31 p l ) without further additions (tracks 1 and 6) or supplemented with 0.07 mM (tracks 2 and 7), 0.15 mM (tracks 3 and 8 ) , 0.29 mM (tracks 4 and 9),or 0.59 mM (tracks 5 and 1 0 ) of m'GpppG were incubated for 45 min a t 28 "C. 10 pl (tracks 15) or 2 pl (tracks 6-10) of each reaction mixture were analyzed by SDS-PAGE and fluorography. The radioactivity applied was (cpm x 69.4,54.4,45.6,41.1,34.8,68.1,49.3,35.4,23.1, and 15.0 in tracks 1-10, respectively.
of samples containing or lacking cap analogue). The synthesis of normal proteins appears to be more sensitive to cap analogue inhibition in normalthan in heat shock lysates, possibly because in the latter the synthesis of normal proteins is already inhibited. When these kindof experiments are carried out using lysates prepared from recovered embryos, a selective inhibition of normal mRNA translation is also obtained (not shown). A preferential reduction in the synthesis of normal proteins withrespect to thatof the heatshock proteins is also obtained with m7GDP and m7GMP (data notshown). On the other hand, we have ruled out thepossibility that thesynthesis of the heat shock proteins observed in heat shock lysates supplemented with cap analogues was mainly due to elongation and terminationof the heat shock polypeptides already initiated because this synthesis is abolished by low concentrations of pactamycin (not shown). Together, the above data are consistent with the notion that in Drosophila the bulk of normal mRNA translation takes place, at optimal concentration of potassium ions, by a cap recognition mechanism. However, the requirement of this mechanism for the translationof the heat shock messengers, at the same ionic conditions, appears to be much less stringent. Rescue of Normal mRNA Translation under Heat Shock by Lowering the Ionic Strength-It has been reported (34-36) that the effectiveness of the cap analogues as inhibitors of protein synthesis in both rabbit reticulocyte and wheat germ cell-free translation systems is clearly diminished at lower than optimalconcentrations of potassium ions, due to a reduced importance of the 5'-terminal cap in mRNA recognition a t these ionic conditions (36). We have found that this is also true for the cell-free system prepared from normal embryos of Drosophila. As shown in Table I, it is possible to reverse gradually the inhibitory effect of m7GDP by lowering the concentration of KCH&OO at which protein synthesis assay is carried out in lysates from normal embryos. In thelight of the previous result, we have studied the effect of potassium ions (KCH&OO) on the translational activity
~~
[%]Met incorporated KCH3COO
+m'GDP -m'GDP epm x
mM
Experiment 1 10.9 85 33 22 11 0 Experiment 2 104 94 74 64 54
96
47.5 35.2 25.4 21.8 12.3
19.0 22.8 20.0 13.8
71 46 10 8 0
72.5 73.2 77.7 61.9 61.3
27.6 28.5 35.4 35.0 38.0 36.9
62 61 54 43 38 31
52.2
44
A 1
2
3 5
4 6
Inhibition
7 1
B 8 2
3
4
5
6
7
FIG. 4. Translation products in heat shock (A, tracks 2-4; B, tracks 2-7) and normal (A, tracks 5-8) lysates at various KCHsCOO concentrations. Preparation of lysates was as described under "Experimental Procedures" ( A ) or without the addition of KCHICOO ( B ) .Standard conditions for translation were used, except for the final concentration of KCH&OO, which was as follows: 44 mM (A, tracks 1 and 5), 64 mM ( A , tracks 2 and 6), 84 mM (A, tracks 3 and 7), 104 mM (A, tracks 4 and 8),and 0, 11,33,43,67,109, and 144 mM in tracks 1-7 of B,respectively. 8 pl ( A , tracks 1-4), 3 PI (A, tracks 5-8), or the volume needed to apply an equal amount of radioactivity per sample ( B )of each reaction mixture were analyzed by SDS-PAGE and fluorography. The radioactivity applied was (cpm X lov3)68,64,45,29,69,84,72, and 54 in tracks 1-8 of A , respectively; and 12 in each track of B.
of heat shock lysates and found that, while the optimum concentration in normal lysates is around 75 mM, heat shock lysates show a clear shift to lower values with an optimum around 45 mM (Table I and data not shown). Interestingly, this shift is due to a partial but specific rescue of normal mRNA translation (Fig. 4 A , tracks 1 3 ; Fig. 4B, tracks 3-5). It should be noted that thisrescue is obtained in the range of KCH3COO concentration that reverses the inhibition of protein synthesis by cap analogues in normal lysates (Table I). Even a t very low concentrations of KCH3CO0, at which an
Translational Control
Embryos
in Heat-shocked Drosophila
overall inhibition of protein synthesis activity occurs, a preferential synthesis of normal over heat shock proteins is observed (Fig. 4B, tracks 1 and 2). As for the results obtained on the effect of cap analogues in heat shock lysates (Fig. 3), translation of the mRNA coding for hsp 83 looks more like the translationof normal mRNAs than of heat shock messengers (Fig. 4). Conversely, by increasing the concentration of K+, above the optimum value obtained in normal lysates but in a range probably physiological (75-110 mM) (37), overall protein synthesis in normal lysates is almost unaffected (Fig. 4A, tracks 6-8),whereas it is significantly reduced in heat shock lysates as a result of the specific inhibition of normal mRNA translation (Fig. 4A, tracks 2-4; Fig. 4B, tracks 5-7). Inother words, the regulatory mechanism establishedin Drosophila embryos under heat shock conditions, which leads tothe preferential translation of heat shock messengers, becomes more apparent at close to physiological concentrations of K+ at which recognition of the 5”terminal cap for mRNA translation appears to be more stringent. Rescue of Normal mRNA Translationunder Heat Shock by Polypeptide Chain Initiation Factors of Rabbit ReticulocytesTogether, the above data are consistent with the notion that the failure of normal mRNAs to be translated under heat shock conditions might be due, a t least to some extent, to the inactivation of polypeptide chain initiation factor(s) involved in the recognition of the 5”terminal cap in mRNA. This hypothesis also implies that translationof heat shock messengers is not affected, just because it takes place largely by a mechanism less dependent on the function of the putative inactive factor(s). As a first attempt tolook for this factorb), we have studied the effect of polypeptide chain initiation factors partially purified from rabbit reticulocytes on mRNA translation in lysates from heat-shocked Drosophila embryos. Fig. 5B shows that protein synthesis in these lysates, but not in lysates from normal embryos (Fig. 5A), is significantly increased in thepresence of either a ribosomal high-salt wash fraction of rabbit reticulocytes or an ammonium sulfate fraction obtained from this high-salt wash. In both cases, this increase is due to the specific rescue of the translation of normal mRNAs (Fig. 6). In light of these results, the ribosomal high-salt wash was further fractionated, as described under “Experimental Procedures,” to obtain highly purified eIF-4E
15723
A 1
2
3
I3 4
5
1
2
3
4
5
6
” ”-hhsp70
pv
St o 0
”43
-hsp 27 - hsp 26 ”hsp23 -hSp 22
FIG.6. Translation products in lysates from heat-shocked Drosophila embryos supplemented with polypeptide chain initiation factors from rabbit reticulocytes. 8 pl of each reaction mixture shown in Fig. 5B (0 in A; in B; samples containing the highest amount of the high-salt wash and the ammonium sulfate fractions correspond totracks SA and 6B,respectively) were analyzed by SDS-PAGEand fluorography. The radioactivity applied was (cpm X 31.0, 31.8, 41.7, and 51.6, 55.6 in tracks 1-5 of A , respectively, and 21.6,22.2,31.4,36.9,40.5, and 39.2 in tracks 1-6of B, respectively.
and eIF-4F. Although both initiationfactors were able to reverse cap analogue inhibition of translation in the rabbit reticulocyte system (38), we have consistently found that neither of them have an effect on protein synthesis of Drosophila heat shock lysates (data not shown). As will be discussed, these results do not rule out the hypothesis that, in Drosophila, heat treatment produces the inactivation of an initiation factor(s) involved in cap recognition. DISCUSSION
We have reported here the preparation of cell-free translation systems from Drosophila embryos grown at normal temperatures or subjected previously to heat shock. As expected from the available data onlysates from cultured cells (3, 7,9, lo), lysates from heat-shocked embryos retain the ability to preferentially translate heatshock over normal mRNAs. The availability of embryo lysates could be advantageous for several reasons, in particular for the analysis of mutants defective in the mechanism of translational regulation of the heatshock 0.05 0.15 0.1 0.05 0.1 0.15 response. It is also shown in this report that compounds structurally Protein (A280 units) related to the 5”terminal cap structure of mRNA are good FIG. 5. Effect of polypeptide chain initiation factors from rabbit reticulocytes on protein synthesis in lysates from nor- inhibitors of the translation of the non-heat-shock mRNAs, mal ( A )or heat-shocked ( B ) Dmelanogmter . embryos. Trans- irrespective of the type of Drosophila lysate used and provided lationassayssupplementedintheamountsshown with either a thatthe reaction is carried out at close to physiological ribosomal high-salt wash fraction (O), obtainedaccording to the concentrations (37) of potassium ions. However, translation (30).or a 0-4096 ammonium sulfate of the majority of the heat shock mRNAs observed in lysates method of Schreier and Staehelin cut of this high-salt wash (B) were incubated for 45 min at 28 ‘C in from heat-shocked embryos appears to be very resistant to the standard conditions. 5-plsamples were taken to determine [%I Met incorporated into hotacid-insoluble material which is plotted as the inhibitory effect of these compounds. Inhibition of protein synthesis by cap analogues were reported to occur in several a percentage of the control incubated without the rabbit reticulocyte fractions. 100% values: 90,343 cpm (0)and 76,829 cpm (B) in A ; systems under appropriateionic conditions and appears to be 19,362 cpm (0)and 13,521 cpm (B)in B. due to their interference with the mechanism of recognition
15724
Translational Control
in Heat-shocked Drosophila
of the 5”terminal cap structure, which is required for binding of the mRNA tothe smaller ribosomal subunit (39, 40). Therefore, the evidence provided in this report supports the notion that, while a mechanism highly dependent on cap recognition is required for the translation of the bulk of normal mRNAs in Drosophila, this recognition appears to be less stringent for the translation of the heat shock messengers. As an interesting inference from the above conclusion, we wouldlike to suggest that the mechanism of translational regulation of the heat shock response in Drosophila might involve the inactivation of polypeptide chain initiation factor(s) needed for the recognition of the 5”terminal cap structure. Only translation of the normal messengers, which is dependent on cap recognition, but not that of the heat shock mRNAs, will be affected as a result of this inactivation. In support of this hypothesis, this study shows a partial but specific rescue of normal mRNA translation when heat shock lysates were incubated under ionic conditions at which overall protein synthesis activity is less dependent on 5”terminal cap recognition (34-36). An additional support for our hypothesis is the observation that it is also possible to attain the specific rescue of normal mRNA translation by supplementing Drosophila heat shock lysates with partially purified preparations of polypeptide chain initiation factors from rabbit reticulocytes. Several initiation factors, including eIF-2, eIF-4B, eIF-4E, and eIF-4F, were shown to bemodified under heat shock conditions in mammalian cells (21-25). In addition, Panniers et al. (24) have shown that the inhibition of translation in heat-shocked Ehrlich cells involves reduction of eIF-4F activity. At present, we have no direct experimental evidence for assigning the “rescue” activity, detected in our crude protein fractions of rabbit reticulocytes, to one of the initiation factors already characterized from these cells. Nevertheless, it should be noted that the inability of rabbit reticulocyte eIF-4F and eIF-4E to rescue normal mRNA translation in heat shock lysates is still compatible with the inactivation of an initiation factor(s) involved in cap recognition. It is possible that, in Drosophila, heat treatmentproduces the inactivation of a cap binding protein which cannot be replaced by rabbit reticulocyte eIF-4F (or eIF-4E) justbecause the latterfactor is unable to interact properly with other initiationfactors of Drosophila embryos needed for the initial steps of mRNA translation. This explains the lack of effect of the highly purified reticulocyte factors. However, the active crude fractions still contain several of the initiation factors involved in cap recognition and unwinding of the 5’-proximal secondary structure of mRNA (40-44). It is, therefore, reasonable that these factors, including eIF-4F (or eIF-4E), can function together to provide the defect of the heat shock lysates. In agreement with our hypothesis, Abramson et al. (45) have recently found that, in a complete translation system from wheat germ, mammalian factors required for mRNA binding to ribosomes (eIF-4A, eIF-4B, and eIF-4F) partially substitute for wheat germ factors, whereas the wheat germ factors are ineffective in the mammalian system. Interestingly, a good interchangeability was onlyobtained in partialreactions in which the interaction between heterologous proteins was not required. In order to directly test our hypothesis, we have begun with the isolation and characterization of cap binding proteins from Drosophila embryos. The reason why the translation of heat shock messengers is less dependent on cap recognition than the translation of normal mRNAs is unclear. It is clearly established, however, that a signal required for the preferential translation of, at least, mRNAs for hsp 70 (16) and hsp 22 (17) under heat
Embryos
shock conditions is found very close to the 5’ end of their leader sequences. Although the natureof this regulatory signal is unknown, it appears to be related to a conserved sequence found within the first 30 nucleotides of the leader sequences of the heat shock mRNAs, with the exception of the mRNA coding for hsp 83 (17). Interestingly, we have shown in this report that this messenger behaves more like normal than heat shock messengers at the translational level. It has been suggested that leader sequences of heat shock messengers contain less secondary structure than othermessenger leaders (16),but it is stillunsettled whether that is due to the existence of the conserved sequence. On the other hand, we know that protein factors involved in the recognition and binding of mRNA to theribosome may play a role in unwinding the secondary structure of the mRNA (40, 41, 43, 44). From these observations and the datashown in this paper, it is tempting to speculate that heat shock messengers of Drosophila are preferentially translated under heat shock conditions just because they have a reduced secondary structure within their leader sequences. This will make their translation much less dependent on polypeptide chain initiation factors involved in cap recognition and subsequent unwinding of the messenger. Therefore, according to our hypothesis, if inactivation of some of these factors occurs in response to heat shock, it will preferentially prevent translation of normal mRNAs. Acknowledgment-The expert technical assistance of M. A. Andres is acknowledged. REFERENCES 1. Lindquist, S. (1986) Annu. Reu. Bwchem. 55, 1151-1191 2. Burdon, R. H. (1986) Biochem. J. 240,313-324 3. Storti, R. V., Scott, M. P., Rich, A., and Pardue, M. L. (1980) Cell 22,825-834 4. Lindquist, S. (1981) Nature 2 9 3 , 311-314 5. DiDomenico, B.J., Bugaisky, G. E., and Lindquist, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 7 9 , 6181-6185 6. Mirault, M. E., Goldschmidt-Clermont,M., Moran, L., Arrigo, A. P., and Tissieres, A. (1978) Cold Spring Harbor Symp. Quant. Biol. 42,819-827 7. Kriiger, Ch., and Benecke, B.-J. (1981) Cell 2 3 , 595-603 8. Petersen, N. S., and Mitchell, H. K. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,1708-1711 9. Scott, M. P., and Pardue, M.L. (1981) Proc. Natl.Acad. Sci. U. S. A. 78,3353-3357 10. Sanders, M. M., Triemer, D. F., and Olsen, A. S. (1986) J. BioL Chem. 261,2189-2196 11. Lodish, H. F. (1976) Annu. Reu. Biochem. 45,39-72 12. McKenzie, S. L., Henikoff, S., and Meselson, M.(1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1117-1121 13. Lindquist, S. (1980) J. Mol. Biol. 1 3 7 , 151-158 14. Scott, M. P., Fostel, J. M., and Pardue, M. L. (1980) Cell 22, 929-941 15. Klemenz, R., Hultmark, D., and Gehring, W. J. (1985) EMBO J. 4,2053-2060 16. McGarry, T . J., and Lindquist, S. (1985) Cell 42,903-911 17. Hultmark, D., Klemenz, R., and Gehring, W. J. (1986) Cell 44, 429-438 18. Glover, C. V. C. (1982) Proc. Natl. Acad. Sci. U. S. A . 79, 17811785 19. Olsen, A. S., Triemer, D. F., and Sanders, M. M . (1983) Mol. Cell. Biol. 3,2017-2027 20. Hickey, E. D., and Weber, L. A. (1982) Biochemistry 21, 15131521 21. Duncan, R., and Hershey, J. W. B. (1984) J. Biol. Chem. 2 5 9 , 11882-11889 22. De Benedetti, A., and Baglioni, C. (1986) J. Biol. Chem. 2 6 1 , 338-342 23. Duncan, R., Milburn, S. C., and Hershey, J . W. B. (1987) J. Biol. Chem. 262,380-388 24. Panniers, R., Stewart, E. B., Merrrick, W. C., and Henshaw, E. C. (1985) J . Biol. Chem. 260,9648-9653
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