Cell, Vol. 33, 103-114. May 1983, CopyrIght 1983 by MIT 0092.8674/83/050103-12$02.00/O

The Control of Protein Synthesis during Heat Shock in Drosophila Cells Involves Altered Polypeptide Elongation Rates

Dennis G. Ballinger and Mary Lou Pardue Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139

Summary

When Drosophila tissue culture cells are shifted from 25 to 36OC (heat shocked) the pre-existing mRNAs (25OC mRNAs) remain in the cytoplasm but their translation products are underrepresented relative to the induced heat shock proteins. Many of these undertranslated 25OC mRNAs are found in associa- tion with polysomes of similar size in heat-shocked and control cells. Furthermore, the messages en- coding a-tubulin, p-tubulin, and actin are found as- sociated with one-third to one-half as many total ribosomes in heat-shocked cells as in cells incu- bated at 25X. Increased temperature should lead to increased output of protein per ribosome. How- ever, the 25OC proteins are actually synthesized at less than 10% of 25OC levels in heat-shocked ceils. Thus, the rates of both elongation and initiation of translation are significantly (15- to 30-fold) slower on 25OC mRNAs than they are on heat shock mRNAs in heat-shocked cells.

Introduction

All organisms which have been examined, from bacteria to metazoans and higher plants, respond to elevated temperatures with an alteration in the spectrum of proteins they synthesize. Although this has been termed the heat shock response, similar alterations can be induced by a large variety of stressful conditions, suggesting that the response may be homeostatic in nature (see Ashburner and Bonner, 1979, and Schlesinger et al., 1982, for review). The heat shock response has been best characterized in Drosophila, where it was first shown that heat and a variety of respiratory inhibitors induce altered puffing of larval salivary gland polytene chromosomes (Ritossa, 1962, 1964). The changes in puffing reflect alterations in tran- scriptional activity of cells incubated at elevated tempera- tures Much of the transcription and processing is stopped, and new transcription is induced at a few loci (Spradling et al., 1975).

There are also alterations in the spectrum of proteins synthesized by heat-shocked Drosophila cells (Tissieres et al., 1974; Lewis et al., 1975). The synthesis of at least seven proteins (the so-called heat shock proteins, hsps) is induced by raising the temperature of growth from the normal 25 to 36 or 37°C while the synthesis of most proteins made at 25°C (25” proteins) is sharply curtailed. McKenzie et al. (1975) further demonstrated that this translational repression at 37°C is accompanied by a rapid disappearance of polysomes (within 15 min). Later poly-

somes are reformed and show a bimodal size distribution which is quite different from the unimodal polysome profile seen at 25°C.

It was not surprising to find that the hsps are encoded by heat-induced transcripts (36” mRNAs) (McKenzie and Meselson, 1977; Mirault et al., 1978). However, McKenzie (1976) found that heat-shocked cells also contain mRNAs encoding 25” proteins that are not synthesized at elevated temperatures. Messenger RNA extracted from cells incu- bated at 36-37°C and translated in vitro encodes both the hsps and 25” proteins (Mirault et al., 1978; Storti et al., 1980). Thus, the pattern of proteins synthesized during heat shock represents a specific recognition and selective translation of the 36” mRNAs from a pool of mRNAs which includes the pre-existing cytoplasmic messages (25” mRNAs). Once induced, this translational control mecha- nism is stable to alterations in temperature, as shown by the slow recovery over a 4-5-hr period of the synthesis of 25” proteins when heat-shocked cells are returned to 25°C (Lindquist, 1980a; Peterson and Mitchell, 1981) and by the stability of the control mechanism in cell-free lysates of heat-shocked cells (Storti et al., 1980; Kruger and Benecke, 1981; Scott and Pardue, 1981). At intermediate temperatures, the 36” mRNAs are produced efficiently, but the cells apparently do not discriminate between mes- sages and synthesize both the hsps and 25” proteins (see Lindquist, 1980a, and the results below).

There have been only a few other well characterized examples of translational control involving the preferential translation of specific mRNAs. Examples include discrimi- nation between different RNAs after fertilization of oocytes of the surf clam Spisula (Rosenthal et al., 1980) during the meiotic maturation of starfish oocytes (Rosenthal et al., 1982) during the development of the slime mold Dictyo- stelium (Alton and Lodish, 1977) during some virus-host interactions (Lodish, 1976; Lodish and Porter, 1980) and in the coordination of rRNA and ribosomal protein synthesis in E. coli and yeast (Dean et al., 1981a, 1981 b; Pearson et al., 1982). There have also been reports of altered translational efficiency during early embryogenesis of the mouse (Cascio and Wassarman, 1982) of Drosophila (Mermod et al., 1980) and of sea urchins (Infante and Heilman, 1981; Wells et al., 1981). The detailed mecha- nisms appear to differ among these systems. However, in all of these studies, the synthesis of a given protein is directly proportional to the amount of its RNA found asso- ciated with polysomes (Rosenthal et al., 1980, 1982; Lod- ish and Porter, 1980).

In this paper, we further characterize the mechanism of translational discrimination in heat-shocked Drosophila cells by examining the distribution between RNPs and polysomes of 25’ and 36” mRNAs in heat-shocked and control cells. We find that in heat-shocked cells many of the abundant 25” mRNA species are found in polysomes as well as in RNPs. These RNAs have the same average number of ribosomes per message in control and heat- shocked cells. There are a total of one-third to one-half as

Cell 104

many ribosomes associated with the mRNAs encoding (Y- tubulin, P-tubulin, and cytoplasmic actin in cells incubated at 36°C as there are in cells incubated at 25°C. Cells incubated at 36°C express these and many other 25” proteins at less than 10% of the level of cells incubated at 25°C (Lindquist, 1980b). Moreover, the output of protein per ribosome would be expected to increase more than 6- fold with an increase in temperature from 25 to 36°C (Hunt et al., 1969). These results indicate that the rates of both elongation and initiation on 25” mRNAs in heat-shocked cells are significantly (15. to 30-fold) reduced relative to the rates on the efficiently translated heat shock mRNAs.

Results

The Effects of Temperature on Protein Synthesis in Vivo As the temperature of Drosophila tissue culture cells is increased from the standard growth temperature of 25°C there is little change in the synthesis of 25” proteins at temperatures up to 33°C (Figure 1 B; Lindquist, 1980b), but above 34°C the synthesis of 25” proteins drops precipitously (Figure 1). The synthesis of the heat shock proteins begins to increase before there is any detectable reduction in the synthesis of 25” proteins (Tissieres et al., 1974; Lewis et al., 1975; Lindquist, 1980a). Under the conditions we use (see Experimental Procedures), cells produce the hsps maximally at 34-35°C while the synthe- sis of 25” proteins declines to almost undetectable levels at about 36°C (Figure 1). Lindquist (1980b) quantitated the production of 30 different 25” proteins in cells labeled with [3H]leucine, and estimated that 25” proteins are made at 2-10% of 25°C levels in cells incubated at 37°C for 1 hr. In our cells labeled with [35S]methionine at 36°C there is a similar decrease in the production of 25” proteins (data not shown).

Detergent-solubilized cytoplasmic extracts of cells incu- bated at 25, 33, and 36°C were analyzed on 0.5-1.5 M sucrose gradients in a buffer containing 0.5 M KCI to minimize nonspecific associations (Figure 2). As shown by McKenzie et al. (1975), there is a dramatic alteration in the distribution of ribosomes in polysomes which accompanies the alterations in protein synthesis induced by elevated temperatures. In extracts of cells incubated at 25°C there is a unimodal distribution of ribosomes in polysomes which peaks at about 12-13 ribosomes/message (b in Figure 2). Cells incubated at 33°C for 1 hr have a similar but some- what broader polysome profile. In cells incubated for 1 hr at 36°C there is a bimodal distribution of ribosomes in polysomes with one peak at 9 ribosomes/message (a in Figure 2) and a second peak at greater than 20 ribo- somes/message (c in Figure 2). The decrease in ribo- somes associated with the polysomal region of cells incu- bated at 33 and 36°C relative to cells incubated at 25°C is compensated for by an increase in the amount of free ribosomal subunits (fractions 1-3, Figure 2). This indicates that ribosomes are recovered in the postmitochondrial

A 25 3435 36 37 25

70 68

23 22

Figure 1, Polypeptides Synthesized by Drosophila Cells at Different Tem- peratures

A: A culture of Drosophilacells was maintained as described in Experimental Procedures, Split Into equal aliquots, and labeled with [Slmethio- nine in methionine-free medium for IO min beginning 55 min after shift to the indicated temperatures. Equal numbers of cells from each temperature were analyzed by electrophoresrs of proteins on a 15% polyacrylamrde gel. The ffgure shows an autoradfogram of the dried gel. 6: An autoradiogram of proteins labeled in cells whose polysomal extracts are utilized for the analysis presented rn Frgures 3-5 and in Table 1. The proterns were labeled wrth [%S]methionine in growth medrum for 20 min beginning 55 min after being shifted to the indicated temperatures, and analyzed on a 15% polyacrylamide gel. Equal numbers of cells were analyzed on each gel lane. A = actin.

supernatant with the same efficiency from cells incubated at the three temperatures.

In extracts of cells incubated at 25°C an average of 62% of the ribosomes sediment with two or more ribo- somes per message. The corresponding numbers for cells incubated at 33 and 36°C are 41 and 31%, respectively. These numbers vary somewhat between experiments, but there is always very close to a 50% decrease in the proportion of ribosomes in polysomes in cells incubated at 36°C relative to cells incubated at 25°C.

The Distribution of 25” mRNAs between RNP and Polysomes To identify the step of protein synthesis which is altered to account for the under-translation of 25” mRNAs in heat- shocked cells, we have studied the subcellular localization of mRNAs in cells incubated at 25,33, and 36°C. We have taken two experimental approaches, both of which involve the extraction of total RNA from fractions of sucrose gradients similar to those shown in Fig. 2 (see Experimental Procedures). In the first approach, the RNA in each fraction of the gradient was analyzed by in vitro translation. In the second approach, RNA from gradient fractions was iden- tified by hybridization with specific DNA sequences.

I: In Vitro Translation of Polysomal RNAs To characterize the functional RNAs in gradient fractions, we utilized the known capacity of the rabbit reticulocyte

Altered Peptide Elongation Rates 105

2.0 250

I I I I

I 5 IO 15 FRACTION

36O

Figure 2. The Optical Density Profile of Sucrose Gradient-Analyzed Polysomal Extracts of Cells Grown at 25, 33, and 36°C for 1 Hr

The graphs show the absorbance at 254 nm of extracts of cells analyzed on 0.5-I .5 M sucrose gradients. There is a 4-fold decrease in full scale absorbance at the break In the curve (indicated by the right marginal scale). The marks are at the same posrtion relative to the beginnrng of the gradient in all three panels (a and c indicate that most prominent peaks at 36°C and b is most prominent at 25°C). The horizontal axis indicates the posrtrons of fractions from which RNA was extracted for the analyses shown rn Frgures 3-5. The top of all gradients is to the left

lysate to indiscriminately translate 25” and 36” mRNAs (Mirault et al., 1978; Start et al., 1980; Peterson and Mitchell, 1981; Pardue et al., 1981). Using this method, we have visualized three classes of abundant mRNAs based on their sedimentation in sucrose gradients of extracts of cells incubated at 25, 33, and 36°C (Figure 3). Class I consists of the heat shock mRNAs and is found only in cells incubated at elevated temperatures, with the excep- tion of the mRNA for hsp 83, which is efficiently translated at all temperatures in vivo (Figure 1). This class is localized both in the polysomal fractions (4-15, Figures 2 and 3) and in the RNP fractions (l-4, Figures 2 and 3) of the sucrose gradients from cells incubated at 33 and 36°C (see the hsps in Figure 3). Class II appears to contain most of the abundant 25” mRNAs (characterized by, but not limited to, the RNAs encoding those proteins migrating between actin and the large hsps in Figure IB). The class

‘II mRNAs are found in both RNP and polysomal fractions in extracts of cells incubated at all three temperatures. Furthermore, specific mRNAs appear by sedimentation properties to be associated with approximately the same average number of ribosomes per message at each of the three temperatures (compare Q and R in Figure 3; also see below). It should be emphasized that, although these mRNAs are found in the polysomal region of the gradients from cells incubated at 36°C they are undertranslated at this temperature in vivo (Figure 1B). The third class of mRNAs is found in fractions containing RNPs and poly- somes in cells incubated at 25 and 33°C but only in fractions containing RNPs in cells incubated at 36°C (see Z in Figure 3; also below). Class Ill appears to be a minor subset of the 25” mRNAs, mostly encoding small polypep- tides.

II: Hybridization Analysis of Polysomal RNAs The presence of specific RNAs in fractions from the su- crose gradients was also assayed by hybridization. The phenol-extracted RNAs from each fraction were denatured with glyoxal, separated on agarose gels, and transferred to nitrocellulose filters. The filters were then probed with radioactive cloned DNA coding for known RNAs. This approach allows for the detection of a particular RNA with increased sensitivity over the translation assay, and makes quantitation easier. By comparison to the translation assay for functional mRNA, this approach would also have al- lowed for the detection of sequences which had been irreversibly converted to a nontranslatable form at the elevated temperatures. However, no irreversibly inactivated RNAs of this type were detected. Sequences complemen- tary to 12 cloned DNA probes encoding known mRNAs, as well as rRNA, have been localized in fractions from polysome gradients by this assay. The results of these studies confirm the existence of the three classes of mRNAs discussed above.

Class I mRNAs include those encoding hsp 83, hsp 70, hsp 27, hsp 26, hsp 23, and hsp 22 (see Figures 4A and 5A for hsp 70; all others are unpublished data). These RNAs are found only in cells incubated at 33 and 36°C and their sedimentation indicates that they are associated with the expected number of ribosomes for mRNAs of their size (based on globin as a standard: 7-9 ribosomes/ small 36” mRNA, and 20-25 ribosomes/hsp 70 mRNA).

Class II mRNAs include the RNAs encoding cy- and /?- tubulins and cytoplasmic actin, as well as RNAs tran- scribed from the intermediate repeat families copia and 297, and a transcript encoding a protein related to tropo- myosin found in non-muscle cells (transcript 3 of Bautch

Cell 106

36” c . . \ . * , /i ** ,_ ) , ._ * . I‘,” “ . ” .

E I 5 IO

25” . -. -* . . ,. >I *. II *. ._ ,. I’ ’

:

E I 5 IO

33 15 E

Figure 3. Autoradiograms of the in Vitro Translation Products Encoded by RNA Extracted from Fractions of Polysomal Sucrose Gradients

Phenol- and chloroform-extracted total RNA from fractions of sucrose gradients like those shown in Figure 2 was translated in the rabbit reticulo- cyte lysate. All the translations were done with RNA concentrations at which [35S]methionine counts incorporated into protein were proportional to the amount of RNA added. The positions of the major hsps are indicated In approximate M,; E, endogenous synthesis in the reticulocyte lysate without added mRNA; Q and R are also indicated with small arrows before lanes 8 and 5, respectively; Z migrates just above hsps 26 and 27 on these gels. The top of all gradients is to the left.

et al., 1982). The RNAs encoding oi-tubulin, P-tubulin, and actin are not translated efficiently in cells incubated at 36°C (Figure 1). We have not identified the proteins encoded by the last three RNAs among the in viva-labeled proteins, though these abundant mRNAs are found in cells at all three temperatures, and their behavior is similar to the

I 5 IO 15

Figure 4. Autoradiograms of Polysomal Sucrose Gradient Fractron RNA Analyzed by Filter Hybridization

Denatured total RNA from the gradient fractrons was separated on 1 .I % agarose gels, transferred to nitrocellulose, and hybridized to nick-translated probes encoding known RNAs. The probes used contain: A, hsp 70 DNA; 8, a-tubulrn DNA. Only the portion of the autoradiograms showing hybridi- zation to the probe is shown (for hsp 70, only the region of RNA from cells grown at 25°C which corresponds in size to the hybridization to RNA from cells at 36°C is shown). All hybridizations are within the linear range of the film as determined by serial dilution of the gradient fraction RNAs. These gradients are further analyzed in Figures 1 B and 3. The top of all gradients is to the left

other mRNAs in class II, Each of the Class II RNAs is found in polysomal fractions in extracts of cells incubated at all three temperatures (for a-, and P-tubulin, actin, and 297, see Figures 48 and 5, B-E). Messages in this class are found in similar amounts in cells incubated at 25 and 36°C but appear to be slightly decreased in cells incubated at 33°C. The proportion of these mRNAs found in the RNP fractions (l-4) of the gradients is different at the three temperatures. At 36°C a higher proportion of these se- quences are in RNP fractions than at 25°C while at 33’C very little RNA at all is found in RNP fractions (Figures 3 and 5). In contrast, those class II mRNA molecules in the polysome region of the gradients are associated with approximately the same average number of ribosomes at each of the three temperatures (Table 1). The constant

Altered Peptide Elongation Rates 107

A: hsp70

B: b’ -tubulln

c: 4 -?ubulln

D: actin

E; 297

F: ribosomal protein rp49

IO- C

25’

25”

2.5

L 5 IO 15

ND

36”

2.5

7.5 36“

5

2.5

L 5 IO I5

IO

7.5 36’

5

L

2.5

5 IO 15

Figure 5. The Amount of Specific RNA In Fractrons from Sucrose Gradients

Autoradiograrns like those shown in Figure 4 were traced with a Joyce-Loebl mrcrodensrtometer and the area under hybridizatron peaks in each fraction was determined with a Numonics area integrator to determrne hybridization intensity. Each data point represents the average of intensrty measurements made on two serial dilutrons of the gradient fraction RNAs, and represents hybridization that IS wtthin the linear response of the film. The graphs show the intensity in arbitrary units (ordinate) as a function of fraction number (abscrssa). The top of all gradients is to the left. Tracings A and C further analyze the autoradiograms shown rn Figure 4. 8, D, and E also analyze the RNA extracted from the gradrents shown in Figure 2, while part F is from another set of gradient RNAs which resembles the gradient fraction RNAs described in Figures 2-5 in all respects.

size of the polysomes of a given mRNA indicate that, for the class II mRNAs, those molecules that are being trans- lated have a ratio of elongation rate to initiation rate that is similar at the three temperatures.

In order to estimate the potential synthesis of protein directed by Class II mRNAs at the three temperatures, one requires an indication of the total number of ribosomes associated with these mRNAs. To estimate this value, we multiplied the intensity (in arbitrary units) corresponding to the hybridization of a given DNA probe to each gradient fraction RNA by the average number of ribosomes per mRNA in that fraction, and summed these values over the entire gradient. Table 1 lists the total number of ribosomes associated with each of the 25” mRNAs studied at each temperature, relative to the values at 25°C. We find that one-third to one-half as many ribosomes are associated with each of the class II 25” mRNAs at 36°C as there are at 25°C. Since the in vivo production of 25” proteins is about 2-10% as great at 36°C as it is at 25°C (Lindquist, 1980b), the ribosomes associated with these 25” mRNAs in cells incubated at 36°C do not synthesize protein at the same rate that they do at 25°C. The simplest explanation of these data is that both elongation and initiation are specifically blocked on class II mRNAs in heat-shocked cells.

Only one of the transcripts of Class III has been identi- fied. In cells incubated at 25°C the RNA-encoding ribo- somal protein rp49 (700 nucleotides in length; P. O’Con- nell, personal communication) is associated with as many as 13-I 5 ribosomes/message, which represents ribo-

Table 1. Summary of the Number of Ribosomes Associated with Specific mRNAs in Heat-Shocked and Control Ceils

Relative Total Number of Relative Average Ribosomes Number of Associated with the Ribosomes/ RNA” Polysomal RNA”

Probe 25” 33” 36” 25” 33” 36”

hsp 70” 0.21 1.0 0.96 1.0

a-TubulinC 1.0 0.42 0.48 1.0 1.04 0.94

@-Tubuli# 1.0 0.71 0.54 1.0 0.98 0.94

Cytoplasmic actir? 1.0 0.32 0.30 1.0 1.10 0.80

2976 1.0 0.15 0.53 1.0 0.64 0.62

rp49’ 1.0 ND‘ 0.06 1.0 ND 0.15

a Estimated by multiplying the intensity of hybridization in each fraction by the average number of ribosomes in the fraction, and summing over the entrre gradient. b Estimated by dividing the total number of ribosomes associated with an RNA by the amount of that RNA in the polysomal fractions (4-15). ‘This set of gradrent fraction RNAs is the one analyzed in Figure 3. The in vivo-labeled proteins made at the same time as these extracts are shown rn Figure IB. d This set of gradient fraction RNAs is from the gradients shown in Figure 2, and analyzed in Figures 48 and 5, B, D, and E. e Summary of Figure 5F, using a third set of gradient fractron RNAs. ‘ND: not done.

somes packed as closely as possible along the mRNA molecule. In extracts of 25” cells, there is sometimes a bimodal distribution of this RNA in the polysome region (Figure 5F); however, in some experiments, only the smaller of these two peaks is seen (data not shown). We do not know what influences the presence or absence of the large polysome peak in particular cultures of cells, though the feeding history of the culture may play a role in this variability. In cells incubated at 36°C however, the RNA encoding this ribosomal protein is always found predominantly in RNP fractions (Figure 5F). This distin- guishes rp49 mRNA from Class II mRNAs, and indicates that a specific block of initiation is responsible for the under-translation of rp49 mRNA in heat-shocked cells.

“Polysome-Associated” 25’ mRNAs Have the Density Expected of Polysomal RNA In experiments described thus far, polysomal mRNAs have been separated from nonpolysomal RNAs solely on the basis of their velocity sedimentation in sucrose gradients. It is possible that large RNP or aggregate structures might sediment with polysomes in these gradients; however, a number of observations indicate that the RNAs we have studied are, in fact, associated with ribosomes.

Artifactual sticking of RNA to polysomes has been minimized by the use of detergent and 0.5 M KCI during polysome preparation. Also, the absence of class Ill mRNAs from polysomes of heat-shocked cells argues that any RNA sticking must show some specificity. To directly test for artifactual sticking, we have preincubated phenol- extracted RNA (metabolically labeled for 1 hr with [3H]- uridine in cells incubated at 25°C) with extracts of cells incubated at 25 and 36°C before separating these extracts on polysome gradients. All of the labeled RNA sedimented in the first three fractions of the gradients, regardless of whether the RNA was run alone or after preincubation with extracts of cells incubated at 25 or 36°C. This indicates that little nonspecific association of free RNA with polyso- mal material occurs during sedimentation. In another set of experiments, 25 mM EDTA released all of the translat- able RNA from the polysomal region into the RNP region of sucrose gradients used to analyze extracts of cells incubated at 25 and 36°C. This EDTA treatment is known to release mRNA from polysomes, though it may also dissociate RNPs or aggregates.

We have also directly demonstrated that RNA comple- mentary to Class II mRNAs has the density characteristic of polysomes in cells incubated at all three temperatures. Equilibrium density centrifugation in metrizamide gradients was used to separate native RNPs and polysomes (Buck- ingham and Gros, 1975). Figure 6 shows the hybridization of the cloned cytoplasmic actin gene to RNA extracted from fractions of metrizamide gradients loaded with ex- tracts of 25, 33, and 36°C cells (a- and @-tubulin se- quences show a similar distribution; unpublished results). Material sedimenting in fractions 2 and 3 of the gradients have an equilibrium density characteristic of polysomes (p = 1.25-l .40; Figure 6). Actin sequences are found in

$;red Peptide Elongation Rates

Ii IO 9 8 7 6 5 4 3 2 I

36’

1.08 I.1 8 1.22 1.23 1.28 1.47

Ffgure 6. Hybridization Analysis of RNA from Metrizamide Gradients

Polysomal extracts of cells grown at 25, 33, and 36°C for 1 hr were sedimented to equflibrium densfty in metrizamide gradients. RNA extracted from fractions of the gradients was denatured, separated by electrophore- sis, and transferred to nitrocellulose. The figure shows an autoradiogram of the filter hybridized to nick-translated cloned DNA encoding cytoplasmic actin. The top of all gradfents IS to the left. The approximate density at 4°C of every other fraction is shown at the bottom of the figure. The two high molecular weight bands in fraction 7 of the 36’C gradient are probably due to contaminating DNA.

these fractions from extracts of cells incubated at all three temperatures (Figure 6). Some actin sequences are also seen in RNP fractions in extracts of cells incubated at 36°C (fractions 4-9; p = 1.18-1.25; Figure 6). Approxi- mately half of the actin sequences are polysomal and half are associated with RNPs in extracts of cells incubated at 36°C when analyzed on either metrizamide or sucrose gradients.

The gradient fraction containing material of polysomal density in extracts of cells incubated at 25 and 33°C directs the synthesis of much the same pattern of proteins in vitro as the cells make in vivo (Figure 7). However, at 36°C there is a sharp discrepancy between those mRNAs of polysomal density and those which are efficiently trans- lated in vivo (Figure 7). These data again provide strong evidence that the mRNAs found in polysomes at 36°C do not all produce protein at the same rate.

25 33 36 End

End

Ffgure 7. Comparfson of in Viva-Labeled Proteins wfth the In Vitro Transla- tfon Products Dfrected by Polysomal Density RNA

Total RNA was extracted from the polysomal fraction (p = 1.25-l .40) of extracts of cells incubated for 1 hr at 25, 33, and 36°C and analyzed on metrfzamfde gradfents. Equal aliquots of this RNA were then translated In the rabbft retfculocyte lysate. The [?3]methionfne-labeled products of these translatrons were analyzed on a 15% polyacrylamrde gel side by side with the proteins labeled in viva with [%S]methionine in growth medium for 20 min- beginning 50 mfn after shifting to the Indicated temperatures. Equal numbers of cells and equal sfzed aliquots of the in vitro translations were analyzed from each temperature. End: endogenous synthesis by reticulo- cyte lysate without added mRNA. A: In vfvo-labeled proteins. 5: In vitro translation products of polysomal density RNA.

Cell 110

Discussion

The data presented above indicate that cells incubated at 36°C have two populations of mRNAs which direct the synthesis of proteins at different rates, The first population contains the efficiently translated heat shock mRNAs. The second population contains many of the 25” mRNAs whose translational products are underrepresented among the proteins labeled in vivo at 36°C. These class II mRNAs are found on polyribosomes in cells incubated at 36°C. We have shown that this class includes the mRNAs for (Y- tubulin, fi-tubulin, and cytoplasmic actin. Approximately one-third to one-half as many ribosomes are associated with these particular class II mRNAs in cells incubated at 36°C as in cells incubated at 25°C.

The output of protein from a ribosome should increase with increasing temperature. Hunt et al. (1969) have meas- ured the effects of temperature on the synthesis of A+ and ,&globulin in rabbit reticulocytes. Using their values, we have calculated a 6-fold increase in the output of protein per ribosome for a temperature increase from 25 to 36°C. Lindquist (1980a) has estimated the rates of initiation and elongation for ribosomes translating hsp 70 and hsp 83 at 37°C and found these values to be within the range of similar measurements made in other systems at 37°C. In other words, the kinetic properties of Drosophila ribosomes are not significantly different than other eukaryotic and prokaryotic ribosomes. Lindquist (1980a) also calculated an overall 3-fold increase in translational efficiency when cells were shifted from 25 to 37°C by assuming that all of the ribosomes in the polysomes of heat-shocked cells produce hsps. This is a lower estimate of the actual increase in translational efficiency since our results show that a number of 25” mRNAs are also associated with polysomes in heat-shocked cells. Thus, ribosomes syn- thesizing at least the large hsps at 36-37°C appear to make protein at nearly the theoretical 6-fold increase over the output of a ribosome at 25°C. If those ribosomes associated with Class II 25” mRNAs at 36°C also produced protein at this rate, one would expect the combined effects of temperature and reduced number of ribosomes to yield a 1.5- to 3-fold increase in protein output relative to 25°C; instead, there is more than a IO-fold decrease in the production of these proteins (Lindquist, 1980b). These data indicate that in cells incubated at 36°C a ribosome translating a 25” mRNA produces I.530.fold less protein than a ribosome translating a 36” mRNA. The simplest explanation for these results is that elongation is specifically blocked or slowed on Class II 25” mRNAs in heat-shocked cells, though we cannot rule out the unlikely possibility that newly synthesized 25” proteins are specifically degraded in heat-shocked cells.

The number of ribosomes associated with a message depends on the relative rates of initiation and elongation on that message. Thus, the observation that each specific translated 25” mRNA molecule is associated with about the same numbers of ribosomes at 25 as at 36°C indicates

that if elongation is not completely blocked on Class II 25” mRNAs in heat-shocked cells, then there must be a com- pensating decrease in the rate of initiation on these mRNAs. There is evidence that some elongation must proceed on 25” mRNAs in heat-shocked cells. For in- stance, McKenzie has shown that within IO min after cells are shifted to 37°C fewer than 10% of the ribosomes are associated with polysomes. Ribosomes then begin to slowly accumulate back into polysomes at about 30-40 min, and reach a plateau at about l-3 hr after heat shock (McKenzie et al., 1975; McKenzie, 1980a). This buildup of polysomes may involve the slow accumulation of ribo- somes on 25” mRNAs, as well as the rapid accumulation of ribosomes onto newly transcribed 36” mRNAs.

At first glance, it appears that there may be a discrep- ancy in the data in that we observe a 50% reduction in the proportion of ribosomes associated with polysomes, and about a 50% reduction in total ribosomes associated with several different 25” mRNAs in cells incubated at 36°C relative to cells incubated at 25°C. We must somehow account for the ribosomes which translate the 36” mRNAs if this generalization holds for other Class II mRNAs, as we think it will based on our translation assays. One possibility is that ribosomes are made available by a specific inhibition block on Class Ill mRNAs. Class III mRNAs are not asso- ciated with polysomes in cells incubated at 36°C. The single member of this class identified so far encodes a ribosomal protein, and the sizes of the other class Ill mRNA translation products suggests that they may also be ribo- somal proteins. It is interesting that ribosomal proteins in E. coli are translationally autoregulated. Under conditions of overproduction of some specific ribosomal proteins in vivo, the translation of parts of the polycistronic message encoding these proteins is specifically reduced (Dean et al., 1981a, 1981 b). This is most likely due to the excess free protein binding to the mRNA, rendering it untranslat- able (Dean et al., 1981 b). A similar phenomenon appears to act in yeast to regulate the production of a ribosomal protein whose gene is carried on a multicopy plasmid (Pearson et al., 1982). If such ribosomal protein autoregu- lation also exists in Drosophila cells, then the interruption of rRNA processing during heat shock (Lengyel and Par- due, 1975) would account for the presumed block of initiation on the ribosomal protein mRNA we have studied (and, by extrapolation, other Class Ill mRNAs). A significant number of ribosomes could be made available for the translation of 36” mRNAs by such a specific block of initiation on the abundant Class Ill mRNAs. In addition, we do not know the fate of the high complexity-low abundance class of mRNAs in heat-shocked cells. If these mRNAs were to belong to Class Ill, then the ribosomes translating them would also be made available for the translation of 36” mRNAs.

Lysates made from heat-shocked cells are able to rec- ognize and specifically translate 36” mRNAs from a mixed population of mRNAs (Storti et al., 1980; Kruger and Benecke, 1981). Studies on the mechanism of translational

Altered Peptrde Elongation Rates 111

selection in these lysates have demonstrated that some component of a crude ribosomal pellet of control lysates is able to rescue the synthesis of 25” mRNAs in heat- shocked lysates (Scott and Pardue, 1981). Moreover, ex- periments involving the mixing of lysates from heat- shocked and control cells indicate that there are no ex- changeable inhibitors or activators in these lysates (Storti et al., 1980). These data are consistent with the hypothesis that lysates of heat-shocked cells lack a factor required for the translation of 25” mRNAs but not for the translation of 36” mRNAs, and that some ribosomal alteration may be involved in the under-translation of 25” mRNAs in heat- shocked cells. We do not yet know if the mechanisms of translational control identified in the lysates and in the experiments of this paper are the same, but it is not difficult to envision a mechanism which regulates elongation at the level of the ribosome, perhaps by the reversible dephos- phorylation of an SG-like ribosomal protein (Glover, 1982).

There is one obvious structural difference between 2.5” mRNAs in heat-shocked and control cells. In Drosophila, most mRNA species contain at least some molecules with poly(A) tails sufficiently long to cause their retention on oligo(dT)-cellulose. However, after a I-hr incubation at 36°C most of the 25” mRNAs no longer bind oligo(dT)- cellulose (Storti et al., 1980). Unfortunately, this loss of oligo(dT) binding is not fully correlated with the translational control of these 2.5” mRNAs. After a 30.min incubation at 36°C 25” mRNAs are not efficiently translated, yet they are retained on oligo(dT)-cellulose (Storti et al., 1980). Thus, it appears that the loss of poly(A) is a result and not a cause of the translational control in Drosophila cells. It is interesting that short poly(A) tails are found on inefficiently translated mRNAs during early Spisula development, and that these tails lengthen when the mRNAs are utilized more efficiently (Rosenthal et al., 1983).

Several laboratories have previously studied the mRNAs associated with polysomes in heat-shocked cells. Mc- Kenzie and Meselson (1977) failed to observe a significant proportion of 2.5” mRNAs in their preparations of poly(A)- containing polysomal mRNA. However, the fact that most of the 25” mRNAs are unable to bind to oligo(dT)-cellulose after 60 min of heat shock (Storti et al., 1980) would preclude their detection in such an assay. These mRNAs which do not bind oligo(dT)-cellulose contain either short poly(A) tails, or internal A-rich regions that can hybridize [3H]oligo(U) (Storti et al., 1980). These data may account for the results of Biessman et al. (1978) who found that 50% of the oligo(dT)-primed cDNA copied from heat- shocked polysomal RNA was complementary to 25” mRNA. Finally, pre-existing mRNA has been found by in vitro translation to be on polysomes of heat-shocked Dro- sophila cells (Sondermeijer and Lubsen, 1978; Kruger and Benecke, 1981; Pardue et al., 1981) as well as HeLa cells treated with amino acid analogues (Thomas and Mathews, 1982).

There is little precedent for a specific control of transla- tion at the level of elongation. Recent work on the secretory

protein prolactin, in a system where elongation was syn- chronized by an artificial block of initiation, demonstrates that elongation of this polypeptide chain can be blocked at a single site on the mRNA in vitro by association with a specific factor (Walter et al., 1981). However, this factor is associated with all sizes of polysomes in a system where initiation is not blocked (Walter and Blobel, 1981) and it is probable that the natural function of this inhibitory factor is to slow elongation until the nascent chain can associate with membranes. There is also anecdotal evidence that the ratios of different isoaccepting tRNAs can alter the message selection of a system (see Jackson and Hunt, 1982). This type of control is probably due to differences in elongation rates because of differences in the codon usage of different mRNAs. There appear to be several differences between the tRNA populations of heat- shocked and control cells (C. French, personal communi- cation), but there is not enough information on codon usage in heat shock and control mRNAs to allow specu- lation about the potential role of this type of control system in heat-shocked cells.

One of the striking features of the heat shock response is that untranslated 25” mRNAs are not degraded in heat- shocked cells. Association of mRNAs with ribosomes has long been considered important for mRNA stability in prokaryotes (see the discussion of polarity in Dean et al., 1981a). In yeast, nonsense mutations affect message stability in a mannr consistent with the hypothesis that mRNAs are stabilized by association with polysomes (Los- son and Lacroute, 1979). The heat shock response may be another system in which association with polysomes is correlated with message stability. This may be particularly evident in cells at 33°C where little mRNA is found in the RNP fractions (Figures 3-5) and there appears to be less of each particular 25” mRNA studied than there is at 25 or 36°C. We are currently investigating the validity and extent of this decrease in message stability in cells incubated at 33°C. In any case, particular mRNAs are distributed differ- ently between RNP and polysomal fractions at the three temperatures. Moreover, particular RNA molecules in the polysomal compartment are always associated with similar numbers of ribosomes at the three temperatures. This could indicate a cooperativity of ribosome association with mRNAs, or it may reflect a finely tuned relationship be- tween initiation rate and elongation rate.

Experimental Procedures

Buffers The buffers used Included the following: Int K buffer: 250 mM KCI, 2.5 mM MgCI,, 20 mM HEPES, pH 7.2, 10 mM EGTA; equilibration buffer: 750 mM KU, 7.5 mM Mg&, 20 mM HEPES, pH 7.2; gradient buffer: 500 mM KCI, 5 mM MgCI*, 20 mM HEPES, pH 7.2, + 10 mM EGTA; NET buffer: 100 mM NaCI, 2.5 mM EDTA, 20 mM Tris. pH 7.4; SSC: 150 mM NaCI, 15 mM Na citrate: SET: 150 mM NaCI, 30 mM Trrs-HCI, 2 mM EDTA, pH 8.0.

Cell Growth, Labeling, and Polysome Extraction Schneider line 2-L cells (Storti et al., 1980) were grown at 25°C in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 0.5%

Cell 112

lactalbumin hydrolysate, MEM nonessential amino acids, 50 U/ml penicillin, and 50 pg/ml streptomycin (all from Gibco). Cells were maintained at 3-15 x IO6 ceils/ml by dilution every 2-3 days. During an experiment, growth conditions were standardized by maintaining the ceils at 3-8 X 106/ml by daily feeding at least 3 days prior to the experiment. For polysome extraction, cells were fed with 0.1 volume of fresh medium at 4 and 1 hr before the culture was split into aliquots and incubated at the indicated temperatures, Cells were harvested by centrifugation, washed twice with phosphate-buffered saline, lysed in 0.01 volume (relative to the original culture volume) of Int K buffer containing 5% rat liver supernatant (Start et al., 1980) and 1% Triton X-l 00. The lysis was carried out for 15 min on ice with occasional vortexing. The extract was spun at 12,000 X g for 20 min, and the resulting postmitochondrial supematant was frozen immediately in liquid NP, and stored at -70°C for further analysis.

For in vivo labeling, cells were either concentrated 3-4 times in medium lacking methionine (Storti et al., 1980) and allowed to recover at 25°C for 20 min before labeling, or labeled in normal growth medium. In either case, cells were labeled after incubation at the various temperatures for the indicated times with i-2/200 of the original culture volume of [%S]methio- nine (about 12 mCi/ml and 1200 Ci/mmol; Amersham). The presence or absence of methronine in the medium made no detectable alterations in the pattern of proteins labeled under all conditions tested. The cells were harvested by centrifugation and resuspended in 0.1-0.2 of the original culturevolume of SDS sample buffer, and polypeptides were separated on polyacrylamide gels containing 15% (w/v) acrylamide, 0.087% (w/v) bisa- crylamide, in the discontinuous buffer system of Laemmli (1970). A l-ml aliquot of the cells from which polysome extracts were made was labeled for 20 min beginning at 55 min after Incubation at the elevated temperatures to monitor the rn vivo protein syntheses in the cells to be used for the polysome experiments.

Sucrose Gradient Analysis of Polysome Extracts and RNA Extraction Aliquots of the polysomal extracts were thawed, adjusted to gradrent salts by the addition of 1 volume of equilibration buffer, and analyzed on 0.5- 1.5 M linear sucrose gradients in gradient buffer. A polysomal extract (0.4 ml) representing about l-3 X 10’ cells was analyzed on each 36.ml SW 27 gradient, and about ‘/4 as much on each 5.ml SW 50.1 gradient. The gradients were analyzed through an ISCO UA5 absorbance monitor and an ISCO gradient fractronator (Instrument Specialties Co., Lincoln, NE; kindly made available by A. Rich). Fractions were collected into 2 volumes of ethanol and precipitated overnrght at -20°C. The precipitate was har- vested and resuspended in 0.25-I volume of NET buffer containing 1% SDS, and immediately mixed with 1 volume of phenol (previously equili- brated with NET buffer). The aqueous phase was further extracted with phenol, twice with phenol:chloroform:isoamyl alcohol (25:24:1), twice with chloroform:isoamyl alcohol (24:1), and precipitated twrce in 2 volumes of ethanol at -20°C. The RNA was then resuspended in pure water and lyophilized. Equal volumes of water were added to equal fractions from the gradient (except fractions I, 2, and 3, which received twice the volume of water per fraction). RNA was stored in water at -70°C.

Metrizamide Gradient Analysis of Polysome Extracts Polysomal extracts (0.1 ml, representing about 5 x IO7 cells; plus 1 volume of equilibration buffer containing 0.2% 0.mercaptoethanol), were layered over metrrzamide step gradients containing 0.1 ml 60%, 4.6 ml 40%, and 0.2 ml 35% metrizamide (Nyegaard and Co. A/S, Oslo; Accurate Chemical and Supply Corp., Hicksville, NY) in gradient buffer (without EGTA, plus 0.1% P-mercaptoethanol) (see Buckingham and Gros, 1975). The gradients were spun 60 hr at 4°C 35,000 rpm in an SW 50.1 rotor, and 0.5 ml fractions were collected into 2 volumes of ethanol containrng 10 CLg of carrier calf liver tRNA (Boehringer Mannheim). RNA was extracted as described above.

In Vitro Translation The RNA from gradient fractions was translated in the rabbit reticulocyte lysate prepared as descrrbed In Pelham and Jackson (1976). and modrfied by Jackson and Hunt (1982). The translation products were analyzed after the addition of 5 volumes of SDS sample buffer on 15% polyacrylamrde gels as described above. All translations were wrthrn the range of propor-

tionality between RNA concentratron and [?S]methionine counts incorpo- rated into protein, as assayed by serial dilution of the RNA.

Recombinant DNA and Hybridization Analysis All recombinant DNA manipulations were carried out as described in Mischke and Pardue (1982). The gradient fraction RNAs were denatured using glyoxal, separated on 1 .I % agarose gels, and transferred to nitro- cellulose according to the procedure of Thomas (1980). The baked filters were prehybrrdized for >2 hr at 60°C in 4x SET (containing 0.2% each polyvinylpyrrolidone, Ficoll, and bovine serum albumin; and 0.1% SDS). Hybridrzation was overnight at 60°C in this buffer also contarnrng the nick- translated probe (lo’-10’ cpm 32P/pg DNA) and 200 pg unlabeled E. coli DNA/ml. Filters were washed in 1 x SSC, 0.1% SDS at 60°C.

These studies would not have been possible without the generous gifts of recombinant DNA phages and plasmids from many people: D. Mischke, LY- and fi-tubulin (Mischke and Pardue, 1982); V. L. Bautch, R. V. Storti, D. Mischke, and M. L. Pardue (1982) tropomyosin; S. L. Tobin, cytoplasmic actrn; P. O’Connell, ribosomal protein rp49 (Vaslet et al., 1980); E. A. Craig, the small hsps (Crarg and McCarthy, 1980); B. Young, the large hsps; G. M. Rubin, copia and 297 (Dunsmuir et al., 1980); and P. Rae and B. Young, rDNA (Dawrd et al., 1978; and unpublished results).

All manipulations involving cells containing recombinant molecules were done in accordance with the National Institutes of Health guidelines for research rnvolving recombrnant DNA molecules.

Acknowledgments

We thank Dietmar Mischke, Tim Hunt, Andrew Murray, and Harvey Lodish for helpful ideas and fruitful discussions. We also acknowledge V. L. Bautch, E. A. Craig, D. Mischke, P. O’Connell, P. Rae, G. M. Rubin, R. V. Storti, S. L. Tobin, and B. Young for their generous gifts of recombinant phage and plasmids. We are indebted to K. S. Kabnick, J. Toffenetti, and H. R. Horvitz for help with this manuscript. This work was supported by grants from the National lnstrtutes of Health. D. G. B. is a graduate fellow of the Whitaker Health Sciences Fund.

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 indrcate this fact.

Received January 6, 1983; revised February 11, 1983

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