THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 263, No. 28, Issue of October 5, pp. 14579-14585,1988 Printed in U.S.A.

HSP70 mRNA Translation in Chicken Reticulocytes Is Regulated at the Level of Elongation*

(Received for publication, March 21,1988)

Nicholas G . Theodorakis, Sunandita S. BanerjiS, and Richard I. Morimotog From the Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern Uniuersity, Euanston, Illinois 60208

During heat shock of chicken reticulocytes the syn- thesis of a single heat shock protein, HSP70, increases greater than 10-fold, while the level of HSP70 mRNA increases less than 2-fold during the same period. Com- parison of the in vivo levels of HSP70 and B-globin synthesis with their mRNA abundance reveals that the translation of HSP7O mRNA is repressed in normal reticulocytes and is activated upon heat shock. In its translationally repressed state HSP70 mRNA is func- tionally associated with polysomes based on sedimen- tation analysis of polysomes from untreated or puro- mycin-treated cells and by analysis of in vitro %un- off” translation products using isolated polysomes. Treatment of control and heat shocked cells with the initiation inhibitor pactamycin reveals that elongation of the HSP70 nascent peptide is not completely ar- rested, but is slower in control cells. Furthermore, the inefficient translation of HSP7O mRNA in vivo is not due to the lack of an essential translation factor; HSP70 mRNA is efficiently translated in chicken re- ticulocyte translation extracts as well as in heterolo- gous rabbit reticulocyte extracts. Our results reveal that a major control point for HSP70 synthesis in reticulocytes is the elongation rate of the HSP70 nas- cent peptide.

The heat shock response is one of the most dramatic examples of rapid changes in the pattern of gene expression. All organisms examined to date respond to elevated temper- ature or other environmental insults by the induced synthesis of a small set of evolutionarily conserved proteins, the heat shock proteins (reviewed in Schlessinger et al., 1982; Craig, 1985; Lindquist, 1986). In many cases, one response to heat shock is the preferential translation of heat shock mRNAs over normal cellular mRNAs (Storti et al., 1980; Lindquist, 1980, 1981; Hickey and Weber, 1982). Thus, heat shock pro- vides a convenient method to alter translational specificity.

Translational control of protein synthesis represents an effective method by which a cell can alter its pattern of gene expression. For example, changes in gene expression during

* This work was supported by the National Institutes of Health, Leukemia Foundation, Inc., the March of Dimes Foundation, a Fac- ulty Research Award FRA313 (to R. I. M.) from the American Cancer Society, and a Graduate Fellowship from the Nicolson Foundation (to N. G. T.). 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.

$ Present address: Memorial Sloan-Kettering Cancer Center, Schwartz Bldg., Rm. 801, 1275 York Ave., New York, NY 10021,

5 To whom correspondence should be addressed Dept. of Biochem- istry, Molecular Biology, and Cell Biology, Northwestern University, 2153 Sheridan Rd., Evanston, IL 60208.

development in Spisula (Rosenthal et al., 1980) and Voluor (Kirk and Kirk, 1985) are controlled at the translational level. Although much progress has been made in understanding the mechanisms by which global changes in protein synthesis can occur (Mathews, 1986), the mechanisms that allow for selec- tive translation among a population of cytoplasmic mRNAs are obscure.

One system that we have used to study the regulatory mechanisms of protein synthesis utilizes heat shock to effect rapid changes in the pattern of gene expression together with the reticulocyte as the experimental system. We previously observed that chicken reticulocytes respond to incubation at elevated temperature by the synthesis of one heat shock protein, HSP70, and the repression of non-heat shock protein (principally globin) synthesis (Morimoto and Fodor, 1984; Banerji et al., 1984). The heat shock-induced changes in the expression of globin and HSP70 appear to be mediated at the translational level; neither HSP70 nor globin mRNA levels change appreciably (Banerji et al., 1984).

In this study we examine the translational control of hp70 expression. We find that HSP70 mRNA is translationally repressed in non-heat shocked reticulocytes. Nevertheless, the majority of HSP70 mRNA is found on polysomes in both control and heat shocked cells. HSP70 mRNA is slowly re- leased from polysomes in the presence of the initiation inhib- itor pactamycin in control cells. We conclude that the syn- thesis of HSP7O in chicken reticulocytes is regulated at the level of elongation.

MATERIALS AND METHODS

General Methods-Isolation of reticulocytes from anemic hens, metabolic labeling with [36S]methionine, purification of RNA, in uitro translation in rabbit reticulocyte lysates, and gel electrophoresis were performed as described (Banerji et al., 1984; Theodorakis and Mori- moto, 1987). The level of radioactivity in gel slices was measured by excising the appropriate region of the gel, rehydrating in water, solubilizing in Protosol (DuPont-New England Nuclear) at 65 “C, and scintillation counting. Chicken reticulocyte translation extracts were prepared and nuclease-treated as described for those made from rabbit reticulocytes (Pelham and Jackson, 1976).

Sedimentation Analysis-Chicken reticulocytes were suspended at a concentration of lo9 cells/ml in Dulbecco’s modified Eagle’s medium and incubated at 37 “C (control) or 43 “C (heat shock) for 30 min. Where appropriate, translation inhibitors (puromycin or pactamycin) were added, and the cells were incubated at 37 ‘C for the indicated times. The cells were then quick-chilled by agitating for 5-10 s in a dry ice/methanol bath and then immersed in an ice water bath. The cells were then centrifuged in a cold (4 ‘C) microcentrifuge for 10 s; the supernatant was removed, and the cells were frozen in a dry ice/ methanol bath. Frozen cell pellets (0.5-1.0 X lo9 cells each) were lysed by addition of 0.6 ml of ice-cold Buffer A (0.3 M KC1, 5 mM MgCL, 10 mM HEPES,’ pH 7.4) containing 0.5% Nonidet P-40, 100

The abbreviations used are: HEPES, N-2-hydroxyethylpipera- zine-N”2-ethanesulfonic acid; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis.

14579

14580 Translation Elongation Control of HSP70 Synthesis pg/ml cycloheximide, and 10 mM vanadyl adenosine. The lysate was centrifuged at 12,000 X g for 5 min at 4 "C to pellet nuclei and cell debris. A portion was diluted in an equal volume of Buffer A or Buffer A containing 20 mM EDTA; 200 pl of extract (1-2 X loR cell eq) was layered on a 12-ml linear sucrose gradient in Buffer A and centrifuged for 90 min at 38,000 rpm at 4 "C in a Beckman SW41 rotor. The gradients were fractionated into 0.5-ml aliquots a t a rate of 1 ml/min with continuous monitoring at 260 nm.

Nascent peptides were released from ribosomes by incubating the cells at 37 "C for 10 min in the presence of 100 pg/ml puromycin, a concentration that inhibits greater than 99% of [35S]methionine incorporation into trichloroacetic acid-precipitable material. Puro- mycin was purchased from Calbiochem and stored at -20 "C in a 10 mg/ml stock in water. Pactamycin, a generous gift of Dr. Richard Keene (The Upjohn Co.), was stored as a 1 mM stock in absolute ethanol a t -20 "C. Pactamycin was added to 0.1 p~ and the cells were incubated at 37 'C for the appropriate times.

RNA was purified from post-mitochondrial supernatants or sucrose gradient fractions by Proteinase K digestion followed by phenol extraction. Samples were adjusted to 0.2 M LiCI, 10 mM Tris-HC1, pH 7.8, 10 mM EDTA, and 0.5% SDS. Proteinase K (Boehringer Mannheim) was added to 250 pg/ml; the samples were incubated at 45 "C for 30 min and then extracted with phenol/chloroform. The aqueous phase was recovered and the RNA precipitated by the addi- tion of 2.5 volumes of ethanol or 1.5 volumes of isopropyl alcohol.

Dot blot hybridization analysis was performed on formaldehyde- denatured samples (Krawczyk and Wu, 1987). Purified RNA or untreated gradient fractions were diluted with an equal volume of 2 x NaPF (1 x NaPF = 1 M NaCl, 40 mM sodium phosphate buffer, pH 7, 6% formaldehyde), heated to 65 "C for 5 min, cooled to room temperature, and applied to nitrocellulose using a Schleicher and Schuell Minifold apparatus. The filters were soaked in 20 X SSC (1 X SSC = 0.15 mM NaC1, 0.015 M sodium citrate, pH 7) before use. After the samples were applied, the Minifold wells were rinsed with 1 M sodium phosphate, pH 7. The filter was probed with nick- translated plasmids containing the chicken @-globin gene (pblBR15; Dolan et al., 1983) or the chicken h p 7 0 gene (pC1.8; Morimoto et al., 1986) as described (Banerji et al., 1984). In some experiments, DNA was labeled using the random primer method (Feinberg and Vogel- stein, 1983) according to manufacturer's specifications (Amersham

Pelleted Polysomes and in Vitro Elongation Assays-Frozen cell pellets were resuspended in 2 volumes of Buffer B (10 mM KCI, 1.5 mM MgC12, 10 mM HEPES, pH 7.4) and lysed by gentle mixing on ice. One volume of ice-cold Buffer C (85 mM KCI, 5 mM MgC12, 10 mM HEPES, pH 7.4) containing 0.5 M sucrose was added and the lysate was centrifuged at 10,000 X g for 10 min a t 4 "C. The post- mitochondrial supernatant was collected, underlaid with 10 ml of 1.4 M sucrose in Buffer C, and centrifuged for 4 h a t 50,000 rpm in a Beckman Type 60 Ti rotor. The polysome-containing pellet was resuspended a t a concentration of 1 A m unit/5 pl and stored at -80 "C. In vitro run-off translations were performed as described (Yamamoto et al., 1983) using 0.5-1.0 AZW units of polysomes in a typical 25-p1 translation reaction. Aurintricarboxylic acid (Sigma) was added to a final concentration of 0.1 mM to inhibit initiation (Lodish et al., 1971; Huang and Grollman, 1972).

Corp).

RESULTS

HSP70 mRNA Is Translationally Repressed in Control Re- ticulocytes-The induced synthesis of HSP70 without a pro- portional increase in HSP70 mRNA levels suggests that HSP70 mRNA in control cells is in a translationally repressed state (Baneji et at., 1984). To test this, we examined the levels of HSP70 and globin synthesis and their corresponding mRNAs. Chicken reticulocytes were incubated at 37 or 43 "C for 30 min; cells were labeled with [35S]methionine or proc- essed for mRNA purification to program mRNA-dependent rabbit reticulocyte in uitro translation extracts. Proteins la- beled in uiuo and in vitro were analyzed in parallel by gel electrophoresis and fluorography (Fig. 1). Heat shock causes a dramatic increase in the synthesis of HSP70, although mRNA levels encoding HSP70 increase only slightly (Figs. 1 and 2). Moreover, it is immediately apparent from Fig. 1 that HSP70 is an under-represented translation product in control

C HS E C HS

HSP70

GLOBIN

in v ivo invitro

A B FIG. 1. Comparison of protein synthesis and mRNA levels

in control and heat shocked chicken reticulocytes. A, reticulo- cytes were incubated at 37 "C (C) or 43 "C ( H S ) for 30 min and pulse- labeled with [D"S]methionine for 30 min. The labeled proteins were analyzed by SDS-PAGE and fluorography. The positions of HSP70 and globin are indicated. B, reticulocytes were incubated at control or heat shock temperatures; RNA was isolated and used to program a message-dependent rabbit reticulocyte lysate translation system. The translation products were analyzed by SDS-PAGE and fluorog- raphy. Lane E, endogenous synthesis of the lysate in the absence of added RNA.

cells in vivo relative to its mRNA abundance revealed by in uitro translation.

The relative synthesis rates and mRNA levels for HSP70 and @-globin were determined by analysis of the radioactivity associated with each protein in the fluorogram shown in Fig. 1. Bands corresponding to the positions of HSP70 and globin were excised from the gel, the proteins were solubilized, and the radioactivity was determined by liquid scintillation count- ing. Corrections were made for the number of methionine residues in each protein as derived from the published nucleo- tide sequence for the respective genes (Dolan et al., 1983; Dodgson and Engel, 1983; Morimoto et al., 1986) and for the fraction of globin synthesis specific for @-globin (Moss and Thompson, 1969).

The relative amounts of @-globin and HSP70 mRNA were also determined by hybridization analysis to gene-specific DNA probes. Serial dilutions of RNA isolated from control and heat shocked cells were immobilized on nitrocellulose filters and hybridized to 32P-labeled HSP70 and @-globin gene probes. The linear range of hybridization is indicated in Fig. 2. These results provide a measure of the relative levels of HSP70 and @-globin mRNAs after corrections are made for the specific activity of each probe and the fraction of each plasmid that is homologous to mRNA.

Quantification of HSP70 and @-globin synthesis and mRNA levels are given in Table I. Although HSP70 mRNA is present at approximately 0.5% of the level of @-globin mRNA, the synthesis of HSP7O in control cells is less than 0.1% of the level of @-globin synthesis. After heat shock, the level of HSP70 mRNA increases only 30%, whereas HSP70 synthesis increases greater than 10-fold. Identical results were obtained

Translation Elongation Control of HSP70 Synthesis 14581

28 -

24 -

20 -

N l e 16- X

U 5 1 2 -

8 -

4 -

P P / /

/

/ /

$. 0

~

0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 f i g R N A (HSP 70)

4 8 12 16 20 24 28 32 ng R N A ( P - g l o b i n )

FIG. 2. Quantification of the relative levels of HSP70 and j3-globin mRNA in control and heat shocked chicken reticu- locytes. Polyadlenylated RNA was isolated from reticulocytes incu- bated at 37 or 43 "C, denatured with formaldehyde, and applied to nitrocellulose filters using a dot blot apparatus. The filters were hybridized to 3ZP-labeled plasmids containing HSP70 (pC1.8) or @- globin (pplBR15) sequences. The levels of hybridization were deter- mined by scintillation counting. The ordinate indicates the level of radioactivity associated with each dot; the abscissa indicates the amount of RNA applied to each dot. Note that the levels of RNA for HSP70 and fl-gllobin use different scales. The slopes of the lines are as follows: HSF'70 control (open circles), 637 cpm/pg; HSP7O heat shock (open triangles), 844 cpm/pg; @-globin control and heat shock (filled symbols), 43,500 cpm/pg.

using cytoplasmic rather than total cellular mRNA (data not shown). We conclude that HSP70 mRNA is translationally repressed in control chicken reticulocytes. Heat shock results in a level of HSP70 synthesis that approximates HSP70 mRNA abundance.

Distribution. of HSP70 mRNA on Polysomes-The presence of HSP70 mRNA in a translationally repressed state in con- trol cells suggested the possibility that this mRNA might not be associated with the translational apparatus. Therefore it was of interest. to determine the distribution of HSP70 mRNA on polyriboso~mes, monosomes, and ribonucleoprotein parti- cles. Accordingly, a post-mitochondrial supernatant was pre- pared from co:ntrol and heat shocked cells and sedimented on sucrose gradients as described under "Materials and Meth- ods." Gradients were monitored at 260 nm and collected into 24 fractions.

The absorbance profiles of polysomes from control and heat shocked cells <are shown in Fig. 3, A and E. In control cells a typical po1yso:me profile is observed, with a distribution up to seven ribosomes per mRNA, characteristic of cells engaged in protein synthesis. Heat shock causes dissociation of poly- somes concomitant with an increase the amount of free ribo- somes, indicat.ive of an inhibition of translational initiation.

We assayed the distribution of HSP70 mRNA on polysomes by dot blot hybridization to radioactive gene probes. Gradient fractions were denatured with formaldehyde, applied to nitro- cellulose, and hybridized to the 32P-labeled plasmid pC1.8, which contains the chicken hsp70 gene. Surprisingly, we found that the majolrity of HSP70 mRNA in both control and heat shocked cells raedimented with large polysomes (Fig. 3, B and

TABLE I Quantitation of HSPM and &globin synthesis and mRNA levels

Protein synthesis in uiuo"

cpm' Corrected cpm' Relative level

Control @-Globin 46,651 18,600 1296 HSP70 187 14.4 1

@-Globin 37,473 14,969 117 HSP70 1.667 128 1

Heat shock

Protein svnthesis in vitrod

Control 0-Globin 51,009 20,404 293 HSP70 905 69.6 1

@-Globin 45,073 18,029 234 HSP70 1,002 77.1 1

Heat shock

mRNA levels'

cpm/pg RNA Relative level Corrected level'

Control @-Globin 43,500 68.3 181 HSP70 637 1 1

Heat shock &Globin 43,500 51.5 HSP70

137 844 1 1

"Chicken reticulocytes were incubated at control or heat shock temperatures for 30 min; the proteins were labeled with [35S]methio- nine and analyzed by SDS-PAGE.

*The amount of [36S]methionine incorporated into @-globin and HSP7O was determined by liquid scintillation counting of a region of the gel that contained the appropriate band.

'The radioactivity associated with globin was corrected for the amount of radioactive methionine specific for @-globin (40%), assum- ing that the ratio of the synthesis of @-globinla*-globin/aD-globin is 4:3:1 and that the number of methionine residues in each globin is 1, 1, and 3, respectively. The radioactivity associated with HSP7O was corrected for 13 methionine residues.

Polyadenylated RNA was isolated from control and heat shocked cells and used in a message-dependent rabbit reticulocyte translation extract. The proteins synthesized were analyzed by fluorography.

'Polyadenylated RNA from control and heat shocked cells was spotted onto nitrocellulose and hybridized to 32P-labeled plasmids containing chicken @-globin (pj31BR15) and the chicken hp70 (pC1.8) gene. The level of hybridization was a linear function of the amount of RNA applied to the filter.

'Corrections were made for the specific activities of the probes (4.5 X lo7 cpmlpg for p@lBR15 and 5.7 x 10' cpmlpg for pC1.8) and for the fraction of the plasmid homologous to mRNA (8.5% for pPlBR15 and 17.9% for pC1.8).

F ) . Treatment of the cell lysate with EDTA released HSP7O mRNA to the post-polysomal region of the gradient (Fig. 3, C and G ) , suggesting that HSP70 mRNA is associated with polysomes.

To establish further that HSP70 mRNA is polysome asso- ciated, we used puromycin to disrupt polysomes. Control and heat shocked cells were incubated in the presence of 100 pg/ ml puromycin for 10 min, and the polysome distribution of HSP70 mRNA was determined. Incubation of either control or heat shocked cells with puromycin released HSP70 mRNA to the post-polysomal region of the gradient (Fig. 3, D and H ) . As puromycin is a specific inhibitor of peptide chain formation (Yarmolinsky and de la Haba, 1959; Vazquez, 1979), release of HSP70 mRNA from polysomes strongly indicates that HSP70 mRNA, although in a translationally repressed state, is functionally associated with polysomes.

In control cells 77% of HSP70 mRNA detected in the gradient is in the polysome region of the gradient (fractions 1-15), whereas in heat shocked cells the fraction of HSP7O mRNA detected in the polysome region was 68%. We have

14582

0 . 2 0 (0 (Y a

0.1

b o t t o m 8 0 s 'OP

Translation Elongation Control of HSP70 Synthesis b o t t o m 8 0 s lop

w r A

15 I B ' O N

5 10 15 20 5 10 15 2 0 fraction no.

FIG. 3. Sedimentation analysis of polysomes from chicken reticulocytes. Cells were incubated at 37 or 43 "C for 30 min; in some samples puromycin was added to 100 pg/ml and the cells were further incubated for 10 min. A post-mitochondrial supernatant was prepared and sedimented on 15-45% sucrose gradients. Gradient fractions were denatured with formaldehyde, spotted onto nitrocel- lulose, and hybridized to the chicken h p 7 0 gene (pC1.8). HSP7O mRNA levels were determined by scanning densitometry. A, absorb- ance profiles of polysomes from control (solid line) and puromycin- treated (dotted line) samples. The direction of sedimentation is from right to left. The 80 S monosome peak is indicated by the arrow. There is a 4-fold increase of the absorbance scale at the break. B-D, HSP70 mRNA distribution in control, EDTA-treated, or puromycin- treated samples. E, absorbance profile of polysomes from heat shocked cells (solid line) and from cell lysates treated with EDTA (dashed line). F-H, HSP70 mRNA distribution in heat shocked, EDTA-treated, or puromycin-treated samples.

frequently observed that a greater proportion of HSP70 mRNA is in the post-polysomal fractions in heat shocked cells than in control cells. The variability in polysome distri- bution and mean polysome size of HSP70 mRNA observed among various experiments reflects the varying protein syn- thetic capabilities of different reticulocyte preparations. Nevertheless, a strikingly consistent feature was that the majority of HSP70 mRNA in control cells was found on polysomes despite its poor translatability.

We were concerned with the possibility that only a small fraction of the HSP70 mRNA in the reticulocyte could be detected in the sucrose gradients and that the polysome- associated material might not be truly representative of the total mRNA population. Therefore, we quantified the recov- ery of HSP70 mRNA from the gradients and compared the levels with HSP70 mRNA levels in whole cell lysates. A cell lysate was prepared from control or puromycin-treated cells

as described and divided into two equal portions. One set was sedimented on sucrose gradients as described; the remainder was treated with Proteinase K, extracted with phenol/chlo- roform, and the RNA precipitated with ethanol. Gradient fractions and serial dilutions of RNA purified from reticulo- cyte lysates were applied to nitrocellulose as described. The filter was probed for HSP7O mRNA, and the hybridization intensities were quantified by scanning densitometry of the exposed x-ray film. Serial dilutions of RNA purified from the lysate provided a standard to measure recovery of mRNA and allowed us to determine that the hybridization intensities in the gradient fractions did not exceed the linear response range of the film.

The fraction of HSP70 mRNA recovered from gradients is given in Table 11. In a representative experiment, we could recover from the gradients 56% of HSP70 mRNA from control cell lysates, 69% from EDTA-treated lysates, and 93% from puromycin-treated cell lysates. Although the recovery of HSP70 mRNA from sucrose gradients varied among several different experiments, the recovery of HSP70 mRNA from gradients using heat shocked cells was always similar to that of control cells (data not shown). The increase in recovery of HSP7O mRNA which occurs when its distribution is altered by puromycin or EDTA treatment is not due to an overall increase in the level of HSP70 mRNA in these samples. Rather, the increase in the recovery of HSP70 mRNA is likely to be due to an increase in the hybridization signal caused by a more homogeneous distribution of the mRNA. These results provide quantitative evidence that the majority of HSP70 mRNA in control reticulocytes is polysome-associated.

Polysomes from Control and Heat Shocked Cells Direct the Synthesis of HSP70 in Vitro-An independent approach to establish that HSP70 mRNA is associated with polysomes in reticulocytes was obtained by in vitro translation "run-off" assays. Polysomes isolated from control and heat shocked cells are used to direct the synthesis of proteins in vitro. If the assays are performed in the presence of the initiation inhibitor aurintricarboxylic acid, the only synthesis that oc- curs is from the elongation of pre-initiated nascent peptides.

Chicken reticulocytes were incubated at 37 or 43 "C for 30 min; a post-mitochondrial supernatant was prepared and cen- trifuged through a 1.4 M sucrose cushion to pellet the poly- somes. The polysome-containing pellet was resuspended and used to program protein synthesis in a rabbit reticulocyte translation extract. Cells from the same reticulocyte prepa- ration were either labeled with [35S]methionine or processed for RNA purification as described. Fig. 4A shows the typical

TABLE I1 Recovery of HSP70 m R N A f r o m sucrose gradients

Units Recovery

%

Total" 700 [loo1 Sum of gradient fractionsb

Control 389 56 EDTA 485 69 Puromycin 648 93

"A portion of cell lysate was deproteinized by treatment with Proteinase K and phenol, precipitated with ethanol, and resuspended in water. Serial dilutions of the RNA were spotted onto nitrocellulose and hybridized to the chicken hp70 gene (pC1.8). The relative levels of hybridization were determined by scanning densitometry; the total number of units were determined by the slope of the line fitting the data points in the linear region of analysis.

Gradient fractions were spotted onto nitrocellulose and hybrid- ized to the chicken h p 7 0 gene; the levels of hybridization were determined by scanning densitometry.

Translation Elongation Control of HSP70 Synthesis 14583 A R C Hsp70-n globm -

R N A polysomes " + + " + + P " + + " + +

R N A Polysomes

-""""- "

A T A

C HS E l 2 3 4 5 6 7 8 I" W V O l " Y l I l 0 8 " V l l l U

rabbvl Chocken

E 1 2 3 4 5 0 7 8

FIG. 4. Polysome-directed in vitro translation. Chicken retic- ulocytes were incubated at 37 or 43 "C for 30 min; a post-mitochon- drial supernatant was prepared and centrifuged through a 1.4 M sucrose cushion to pellet the polysomes. The polysome-containing pellet was resuspended and used to program protein synthesis in rabbit ( B ) or chicken (C) reticulocyte translation extract in the presence or absence of the initiation inhibitor aurintricarboxylic acid (ATA). Cells from the same preparation were also labeled with [3sS] methionine or processed for purification of total poly(A)' RNA. A, in uivo pattern of protein synthesis of control and heat shocked cells from the same preparation from which polysomes were made. B, in uitro translations in rabbit reticulocyte lysates were programmed as follows: lane E, no RNA added. Lanes I and 3, poly(A)' RNA from control cells; lanes 2 and 4, poly(A)' RNA from heat shocked cells; lanes 5 and 7, polysomes from control cells; lanes 6 and 8, polysomes from heat shocked cells. Translation reactions in lanes 3 , 4 , 7, and 8 were performed in the presence of aurintricarboxylic acid. C, in vitro translations in control chicken reticulocyte lysates were programmed as described in B.

10-fold induction of HSP70 synthesis in vivo upon heat shock. When RNA isolated from these cells is used to program protein synthesis in vitro, similar amounts of HSP7O are synthesized (Fig. 4B, lanes 1 and 2), thus confirming our previous observation. Addition of aurintricarboxylic acid to the translation extract abolishes translation of purified RNA (Fig. 4B, lanes 3 and 4 ) , indicating that HSP70 mRNA translation in vitro does not escape the inhibitory effects of aurintricarboxylic acid. The translation products directed by isolated polysomes (Fig. 4B, lanes 5-8) are enriched for the synthesis of large proteins, characteristic of run-off systems in which initiation is limited. Moreover, recovery of large mRNAs is much greater in our polysome preparations; for example, approximately 50% of HSP70 mRNA in the cell can be recovered in the polysome pellet, whereas only 10 to 20% of (?-globin mRNA is recovered under these conditions (data not shown). Consequently, this assay is not useful for com- paring the abundance of different sized mRNAs, such as HSP7O and (?-globin mRNAs. However, we can compare the levels of the same mRNA in different polysome samples.

Polysomes from both control and heat shocked cells direct the synthesis of HSP70 both in the absence (Fig. 4B, lanes 5 and 6) and in the presence (Fig. 4B, lanes 7 and 8) of aurintricarboxylic acid. Therefore, HSP70 mRNA must be associated with polysomes in both control and heat shocked cells in a functional manner, that is, associated with a nascent peptide. The nearly exclusive synthesis of HSP7O directed by polysomes isolated from heat shocked cells (Fig. 4B, lanes 6 and 8) is likely to be due to a poor recovery of non-heat shock mRNAs, a consequence of heat shock-induced polysome dis- aggregation, as observed in Fig. 3E.

To avoid possible complications that might occur in trans- lating polysomes isolated from chicken reticulocytes in a heterologous rabbit reticulocyte lysate, we examined the translation of HSP70 mRNA in chicken reticulocyte trans-

lation extracts. A cell-free extract was made from control chicken reticulocytes. The optimal concentrations of KC1 and MgC12 for translation activity were determined, and the lysate was shown to support translation for a t least 60 min (data not shown). The lysate was made mRNA-dependent by treat- ment with micrococcal nuclease and programmed with puri- fied polyadenylated RNA or polysomes isolated from control and heat shocked chicken reticulocytes as described above. Nuclease-treated extracts from control chicken reticulocytes support the synthesis of HSP70 directed by purified mRNA isolated from control or heat shocked reticulocytes (Fig. 4C, lanes I and 2) or by polysomes isolated from chicken reticu- locytes (Fig. 4C, lanes 5-8). These results reveal that the repression of HSP7O synthesis detected in uivo is not main- tained in uitro even in a homologous translation system. Similar results were obtained using a lysate made from heat shocked chicken reticulocytes (data not shown).

To test the possibility that nuclease treatment might have destroyed a discriminatory component of the lysate, we ex- amined the proteins synthesized in a non-nucleased lysate from control chicken reticulocytes that was not supplemented with added RNA or polysomes. The lysate tested under these conditions is also permissive for the translation of HSP70 mRNA (data not shown). Therefore, we conclude that the translational regulatory mechanisms that operate in intact chicken reticulocytes are too subtle to be readily duplicated in uitro. Nevertheless, the translation of HSP70 in control chicken reticulocyte extracts reveals that the inefficient trans- lation of HSP70 in control cells in uivo is not due to the lack of a necessary translation factor.

HSP70 mRNA Is Released from Polysomes in Pactamycin- treated Cells-The inefficient translation of HSP70 mRNA in control cells despite its polysome association may indicate that the elongation of HSP70 nascent peptide is blocked for most HSP70 mRNAs, perhaps with only a small fraction of the mRNA escaping this block to give the low level of HSP70 synthesis observed in control cells. Alternatively, the ineffi- cient translation of HSP70 mRNA may be due to a decreased rate of elongation of HSP70 nascent peptide. To distinguish between these two possibilities, we examined the distribution of HSP70 mRNA on polysomes of cells treated with the initiation inhibitor pactamycin. If any HSP70 mRNA is com- pletely arrested in elongation, it should remain polysome- associated even after extended incubation in the presence of pactamycin.

Cells were incubated a t 37 or 43 "C for 30 min and then further incubated a t 37 "C in the presence of 0.1 pM pacta- mycin, a concentration that inhibits greater than 99% [35S] methionine incorporation into protein. At various times after incubation, cells were removed for polysome analysis as de- scribed above. Incubation of cells for 10 min in the presence of pactamycin causes polysome disaggregation (Fig. 5 , A and F ) , consistent with the known effect of pactamycin on initi- ation (Colombo et al., 1966; MacDonald and Goldberg, 1970; Lodish et al., 1971; Vbzquez, 1979). We analyzed the distri- bution of HSP70 mRNA on polysomes of pactamycin-treated cells. When control cells are incubated in the presence of pactamycin, the distribution of HSP7O mRNA gradually shifts to the post-polysomal pool over a 30-min period (Fig. 5, B-E) . Therefore, HSP70 mRNA is not completely arrested in elongation. Rather, the nascent peptide is elongating slowly. In heat shocked cells, pactamycin causes HSP70 mRNA to redistribute to monosomes within 10 min (Fig. 5, G J ) . Although this assay is not sensitive enough to define the elongation rates of HSP70 nascent peptide in control and heat shocked cells, it provides support for the model that the

14584 Translation Elongation Control of HSP70 Synthesis

~ pacta

I I

I C O N O ’ p a c

C O N l O ‘ p a c . 11 HS 10’pac .

C O N 2 0 ’ o a c . 11 ‘ H S 2 0 ’ o a c

5 10 15 2 0 5 10 15 2 0 f r a c t i o n no.

FIG. 5. Release of HSP70 mRNA from polysomes in the presence of pactamycin. Reticulocytes were incubated at control or heat shock temperatures for 30 min. Pactamycin was added to 0.1 PM and incubation was continued for 10, 20, or 30 min. A post- mitochondrial supernatant was prepared for sucrose gradient analy- sis. Gradient fractions were denatured with formaldehyde, spotted onto nitrocellulose, and hybridized to the chicken h p 7 0 gene (pC1.8). A and F, absorbance profiles of polysomes from untreated cells (solid line) or cells incubated with pactamycin for 10 min (dashed line). The direction of sedimentation is from right to left. B-E, distribution of HSP70 mRNA on polysomes of control cells after 0, 10,20, and 30 min of incubation in the presence of pactamycin. G-J, distribution of HSP70 mRNA on polysomes of heat shocked cells after 0,10,20, and 30 min of incubation in the presence of pactamycin.

elongation rate of HSP70 nascent peptide is slower in control cells relative to its rate in heat shocked cells.

DISCUSSION

We have previously established that the heat shock-induced synthesis of HSP70 in chicken reticulocytes is controlled at the translational level. Although HSP70 mRNA levels in- crease less than 2-fold after 30 min of heat shock, HSP70 synthesis increases greater than 10-fold (Banerji et al., 1984). Here we demonstrate that HSP70 mRNA is polysome-asso- ciated yet translationally repressed in non-heat shocked cells.

Our conclusions are based on the following data: (i) HSP70 mRNA cosediments with large polysomes during sucrose gra- dient velocity sedimentation; (ii) EDTA or puromycin treat- ment causes the release of HSP70 mRNA from polysomes; and (iii) polysomes from both control and heat shocked cells direct the synthesis of HSP70 in vitro. Although the rate of HSP70 synthesis in control cells is repressed relative to its mRNA level, the polysome-associated HSP70 mRNA is not completely arrested in elongation. The results of experiments using protein synthesis inhibitors that disrupt polysomes suggest that ribosomes associated with HSP7O mRNA are engaged in peptide bond formation and translocation, albeit at a reduced rate in control cells relative to the rate in heat shocked cells. Heat shock relieves this repression and allows HSP70 mRNA translation to proceed at levels according to its mRNA abundance.

Translational regulation at the level of elongation has also been implicated in the repression of non-heat shock protein synthesis during heat shock of Drosophila cells (Ballinger and Pardue, 1983) and during treatment of HeLa cells with amino acid analogues (Thomas and Mathews, 1982, 1984). It is not clear whether the mechanism that represses HSP7O mRNA translation in control chicken reticulocytes is similar to that which represses the translation of control mRNAs in heat shocked Drosophila or amino acid analogue-treated HeLa cells. In those studies, both heat shock and non-heat shock mRNAs were polysome-associated, but only the heat shock mRNAs were translated efficiently. The results described here differ in that control chicken reticulocyte HSP70 mRNA is translationally repressed yet polysome-associated, whereas in heat shocked chicken reticulocytes the polysomes disaggre- gate (Fig. 3E) and globin mRNA shifts to the post-polysomal pool (data not shown). Thus, there are at least two transla- tional control mechanisms in chicken reticulocytes; one re- sults in the translational repression of polysome-associated HSP70 mRNA in control chicken reticulocytes, and the other inhibits globin mRNA translation initiation in heat shocked chicken and rabbit reticulocytes (Bonanou-Tzedaki and Arn- stein, 1976; Mizuno, 1975). The repression of globin synthesis in heat shocked reticulocytes resembles the repression of control protein synthesis in heat shocked HeLa cells, in which polysomes disaggregate and non-heat shock mRNAs shift to the post-polysomal pool (DeBenedetti and Baglioni, 1986; Duncan and Hershey, 1984; McCormick and Penman, 1969; Theodorakis and Morimoto, 1987).

Another possible interpretation of our data is that HSP70 is synthesized efficiently but rapidly degraded in control cells. We view this possibility as extremely unlikely for the follow- ing reasons. First, we detect no differences in the levels of accumulated HSP70 between control and heat shocked cells either by Coomassie Blue staining of gels (Morimoto and Fodor, 1984) or by Western blot analysis.’ Thus, if a HSP70- specific protease is operating in non-heat shocked reticulo- cytes, it would have to distinguish between newly synthesized and accumulated HSP70. Second, we find no evidence of rapid turnover of newly synthesized HSP70 in non-heat shocked cells in pulse-chase experiment^.^ Third, if there is a protease that rapidly degrades newly synthesized HSP70 in control cells, its activity is not apparent in cell-free extracts made from control cells (Fig. 4C).

The major unresolved problem concerns the mechanism of control of HSP70 synthesis at the elongation level. It is unlikely that the repression of HSP70 synthesis in control cells is due to limiting translational components (e.g. rare

R. I. Morimoto, unpublished results. N. G. Theodorakis, unpublished results.

Translation Elongation Control of HSP70 Synthesis 14585

tRNAs or elongation factors) because HSP70 mRNA trans- lates with equal efficiency in control chicken reticulocyte extracts, as well as in heterologous wheat germ or rabbit reticulocyte extracts (Banerji et al., 1984). Alternatively, a factor in control cells may prevent the elongation of HSP70 nascent peptide by binding to that peptide or its mRNA, in an analogous manner to the elongation arrest caused by the signal recognition particle (Walter and Blobel, 1981). This factor may dissociate during preparation of polysomes or crude lysates, perhaps due to dilution or unfavorable ionic conditions. Another possibility is that a stable secondary structure in the mRNA may prevent the efficient transit of ribosomes on HSP70 mRNA. However, this model does not explain the efficient translation of HSP70 mRNA in vitro nor does it explain the apparent requirement of transcription for the heat shock-induced translation of HSP70 mRNA (Banerji et al., 1984).

Our data suggest the possibility that a factor present in control reticulocytes inhibits the translation of HSP70 mRNA. Heat shock would then cause a change in the activity or subcellular localization of this factor, allowing HSP70 synthesis to occur. We suggest that HSP70 can interact with its mRNA or nascent peptide, if all other cellular targets for HSP70 have been saturated. This situation might easily occur in reticulocytes, since they have high levels of HSP70 (Mori- mot0 and Fodor, 1984). In most cells, high cytoplasmic con- centrations of HSP70 might cause the rapid decay of HSP70 mRNA; however, reticulocytes, being teIminally differen- tiated, may not have a mechanism for the specific degradation of HSP70 mRNA. (Indeed, preliminary evidence suggests that HSP70 mRNA is long-lived in these cell^.^) The high concen- tration of HSP70 in reticulocytes might hinder the transit of ribosomes along its mRNA. When the cell is heat shocked, HSP7O migrates to the nucleus and nucleolus (Lewis and Pelham, 1985; Velazquez and Lindquist, 1984; Welch and Feramisco, 1984), thereby being unable to interact with its mRNA in the cytoplasm and allowing HSP70 mRNA to be efficiently translated. If transcription is blocked, however, HSP70 might not interact with its nuclear target, perhaps because those targets are transcription or splicing complexes no longer present in transcriptionally arrested cells. Thus the apparent requirement of transcription for the efficient trans- lation of HSP70 mRNA during heat shock (Banerji et al., 1984) might be to supply targets for HSP70, thereby freeing its mRNA for translation. The autoregulation hypothesis for the regulation of HSP70 mRNA translation in control retic- ulocytes presented here shares several features with the hy- pothesis proposed by DiDomenico et al. (1982) for the regu- lation of HSP70 synthesis in Drosophila cells recovering from heat shock. In both cases, a high concentration of HSP70 is proposed to influence the synthesis of HSP70, either by destabilizing the mRNA in Drosophila cells or by inhibiting its translation in reticulocytes.

The translational regulation at the elongation level ob- served in this study is unlikely to be a general form of hsp70 regulation; for example, in the human cell line 293, which constitutively expresses HSP70 under non-heat shock condi-

tions (Nevins, 1982; Wu et al., 1985), HSP70 mRNA is trans- lated efficiently and according to its mRNA abundance (Theo- dorakis and Morimoto, 1987). The studies presented here have revealed a form of post-transcriptional regulation of hsp70 expression that may be specific to reticulocytes. Fur- thermore, the reticulocyte-specific form of h p 7 0 regulation appears to be lineage-specific. Erythroid cells of the primitive lineage constitutively synthesize HSP70; however, they are not heat shock-responsive at either the transcriptional or translational levels (Banerji et al., 1987). We suggest that the translational regulation of HSP7O synthesis in chicken retic- ulocytes represents a cell-type specific form of regulation, perhaps acquired during terminal differentiation.

Acknowledgments-We are grateful to L. Dumas and members of his laboratory for the generous use of their gradient fractionation equipment, to Dr. Richard Keene of the Upjohn Company for the pactamycin, and to T. McClanahan for critically reading the manu- script.

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