Biochimica et Biophysica Acta. 1009 (1989) 61-69 Elsevier

61

BBAEXP 91991

Characterization of an inhibitor of protein synthesis initiation from mouse erythroleukemia cells

M i c h a e l Bader a n d T h o m a s F. Sarre

Institut f~r Biologie I!!. Freiburg (F. R. G.)

(Received 7 June 1989)

Key words: Protein synthesis; In vitro translation: mRNA binding: Inhibition: (Mouse erythroleukemia cell)

This s~ ly describes the partial purification of a translational inhibitor from mouse erythroleukemia (MEL) cells. It [s present in MEL cells induced to erytlu'oid differentiation and in uninduced cells in approximately equal amounts. The inhibitor blocks initiation but not elongation of in vitro protein synthesis in the rabbit reticulocyte lysate and in exh'acts prepared from induced or uninduced MEL cells. Nuclease-resistance, heat-sensitivi~, and the chromatographic be- haviour of the inhibitor indicate that it is a protein with a relative molecular mass of approx. (45-70) • 1 0 3. The inhibitor has no e |F-2a phosphory|ating activity and does not affect the formation of the ternary complex [elF-2 • GTP- Met- tRNAt] nor the binding of Met-tRNAf to the 40 S ribosomal subunit. The inhibitor interferes with the binding of mRNA to the 43 S preinitiation complex, independent of the presence of the mTGTP cap of the mRNA.

Introduction

Regulation of gene expression on the level of protein synthesis is a well-established phenomenon in mam- malian cells [1-4], but there are only few cases in which the factors involved, their mechanism(s) of action and the physiological role of the regulation are known.

The absence of haemin or the presence of double- stranded RNA in the rabbit reticulocyte lysate leads to the activation of two distinct translational inhibitors which act by phosphorylating the a-subunit of elF-2, the initiation factor which catalyzes the binding of Met-tRNA r to the 40S ribosomal subunit [2,4,5]. A second well-documented case of translational inhibition is the so-called restriction activity observed in poliovirus-infected cells. In this system, activation of a cellular proteinase leads to the degradation of a factor necessary for cap recognition and to the exclusive trans- lation of uncapped (poliovirus) mRNA [6-9].

Abbreviations: MEL, mouse e~th~oleukemia; elF, eukaryotic protein synthesis initiation factor; CT.AB, ce~ylttimethylammoniem bromide; G DPCP, guanyl(/L-t-methylene)diphosphonate; TCA, trichloroacetic acid; SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel e|ectro- phorcsis; Met-tRNA f, eukaryotic initiator methionyl-tRNA; a.u., aro bitrary unit.

Correspondence: T. Sarre, lnstitut for Biologie !!!, Sch~inzlestr. 1, D-7800 Freiburg, F.R.G.

The present study deals with the investigation of translational control mechanisms in mouse erythro- leukemia cells (MEL cells). These cells have been previ- ously used for investigations on protein synthesis, since they offer the possibility to study two different erythroid differentiation states [10-18]. Normal MEL cells exhibit the features of an undifferentiated transformed ceil. After induction by a variety of chemical agents, how- ever, the cells synthesize giobin mRNA, haemoglobin and other proteins which are specific for erythroid cells. This differentiation process is accompanied by a reduc- tion of cell growth, cell size, overall RNA content and by a decrease of overall protein synthesizing activity [18-20]. The latter has also been observed in cell-free protein synthesis systems prepared from induced and uninduced MEL cells [18], Thus, the question arises as to whether or not a translational inhibitor is involved in the process of differentiation. Several studies report inhibitors of protein synthesis from uninduced, but none from induced MEL cells [11-13,15].

Here we report the partial purification and charac- terization of a translational inhibitor from induced MEL cells which interferes with the binding of mRNA to the 43 S preinitiation complex of protein synthesis. This inhibitory activity is also present in cell-free extracts from undifferentiated MEL cells, but not from mouse myeloma cells, a transformed cell line of non-erythroid

origin.

0167-4781/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

62

Materials and Methods

Materials Tissue culture sc, lutions were from GIBCO (Wies-

baden, F.R.G.). Hexamethylenebisacetamide, CTAB, Mops, anisomycin, haemin, 2-mercaptoethanol and phosphocellulose were obtained from Sigma (Dei- senhofen, F.R.G.), DEAE-cellulose (DE-52) from Whatman (Maidstone, U.K.), L-amino acids, strepto- mycin and yeast sodium nucleinate from Merck (Darmstadt, F.R.G.), edeine from Calbiochem (Frank- furt, F.R.G.), dithiothreitol and sodium cacodylate from Roth (Karlsruhe, F.R.G.), penicillin from Serva (Heidelberg, F.R.G.), creatine phosphate, creatine phos- phokinase (EC 2.7.3.2), ATP, GTP, GDPCP and calf- liver tRNA from Boehringer (Mannheim, F.R.G.), T4- RNA-ligase, Sephadex G25, G100 and G200 from Pharmacia (Freiburg, F.R.G.) and tobacco mosaic virus RNA, L-[aSS]methionine (1000 Ci/mmol), [y-32P]ATP (3 Ci//~mol), and cytidine 3',5'-[5'-32P]bisphosphate (3000 Ci/mmol) from Amersham Buchler (Braunsch- weig, F.R.G.).

Cell culture Mouse erythroleukemia cells (line B8) were a gener-

ous gift of Dr. W. Ostertag (Heinrich Pette Institut fiir experimentelle Immunologie, Hamburg, F.R.G.). Cells were grown and induced with 4.5 mM hexamethylene- bisacetamide as described [18]. 3 days after induction, 80-90~ of the cells were in the induced state as de- termined by benzidine staining [21].

Ceil-free extracts and in vitro protein synthesis Cell-free extracts of M EL cells were prepared as

described [18] with or without treatment of the cells with high salt prior to lysis. Conditions for in vitro protein synthesis were the same as described previously [18].

Rabbit reticulocyte lysate was prepared following the method of Jackson and Hunt [22] and used for in vitro protein synthesis of endogenous mRNA under the con- ditions described for MEL-cell extracts [18]. Alterna- tively, it was treated with micrococcal nuclease for the translation of exogenous mRNA [22].

Analysis of the in vitro synthesized proteins was performed either by TCA precipitation after alkaline hydrolysis or by SDS-PAGE as described [18] with the exception that 12~ (w/v) polyacrylamide gels were used instead of gradient gels.

Preparation of poliovirus RNA Poliovirus (strain LSc) was a gift of Dr. T. Monn-

heimer (Institut ffir Virologie, Freiburg, F.R.G.). RNA was isolated from the virion-suspension containing 0.5~ (w/v) SDS by adding the same volume of hot (60 o C) phenol saturated with 20 mM ~£ris-HCl (pH 6.0)/150

mM NaCI/5 mM EDTA/0.5% (w/v) SDS. The phenol phase was extracted twice with water and the aqueous phases were combined and extracted twice with chloro- form/isoamyl alcohol (24:1, v/v). RNA was precipi- tated by adding 2 vol. of ethanol at - 2 0 o C. The RNA pellet was collected by centrifugation, washed with 70% (v/v) ethanol and dissolved in water.

Purification of the inhibitor Fractions were tested for translational inhibition in

the in vitro protein synthesis system from induced MEL cells [18]. One arbitrary unit (1 a.u.) of inhibitory activ- ity was defined to represent that amount which leads to a 50~ reduction of [3SS]methionine incorporation in proteins in a 20 #l assay.

Cell-free extracts were prepared from MEL cells without KC! treatment prior to the lysis as described [18] and centrifuged either in 5 mi aliquots in a Ti50 rotor at 200000 x g for 3 h or in 1 ml aliquots in a TL100 rotor at 460000 × g for 45 rain. The resulting postribosomal supernatant was applied to a Sephadex G25 column (volume: at least 10 x the load volume) equilibrated with buffer A (10 mM Hepes-KOH (pH 7.5)/0.1 mM EDTA/2 mM 2-mercaptoethanol/10 mM KCI). The column was eluted with the same buffer and the void-volume fraction was applied to a DEAE-cel- lulose column (volume: 1 ml per 5 mg of loaded pro- tein) equilibrated with buffer A. The column was exten- sively washed with buffer A and protein was eluted in a stepwise manner with buffer A containing 100, 200, 300 and 500 mM KCI, respectively. The fractions eluted with 100 mM KC! were combined and applied to a phosphocellulose column (volume: 1 mi per 5 mg of loaded protein) equilibrated with buffer A containing 100 mM KCI. After washing the column with the same buffer, protein was eluted by adding sequentially buffer A containing 200, 300, 500 and 1000 mM KCI. The combined fractions of each step were precipitated by adding crysta!lin~ ammonium sulfate to a final satura- tion of 70~. Proteins were collec~.ed by centrifugation, resuspended in buffer A and dialyzed overnight against the same buffer. The concentrated i00 mM KCI frac- tion from phosphocellulose was applied to a MonoQ column (1 ml) equilibrated with buffer A and protein was eluted by a linear gradient from 10 to 500 mM KC! in buffer A. The inhibitory activity eluted with 175 mM KCI.

Gel-filtration for relative molecular mass determination Gel-filtration was performed using Sephadex G100

and G200. The columns (26 x 0.7 cm) were equilibrated with buffer A. 300/~l protein fractions were loaded and the columns were eluted with the same buffer at a flow rate of 0.1 ml/min. 400 #! fractions were collected and aliquots of each fraction were either tested for protein- synthesis inhibition or applied to SDS~polyacrylamide

gels. For calibration a solution of 20 ~g fl-galactosidase (/14', = 540- 103), 40 ~g phosphorylase B (185- 10~), 35 /tg bovine serum albumin (68. 103), 40 / tg ovalbumin (45. 103), 40 /~g carboanhydrase (29-10 a) and 35 /~g myoglobin (18.10 a) in 300 #l buffer A was applied to the columns. The elution volume of each protein was determined by analysis of the Coomassie staining pat- tern of the SDS-polyacrylamide gels. The elution volume of fl-galactosidage was taken for the void volume.

Labelling of Met-tRNA! and mRNA Met-tRNAf was labelled according to the method of

Lenz and Baglioni [23]. The 300 ttl assay, containing 0.5 mg of calf liver tRNA, 1 mCi of [3SS]methionine, 30/~i of E. coli aminoacyl-tRN/-, synthetases (a gift of Dr. G. Igloi from our Institute), 2.5 mM ATP, 2 mM dithio- threitol, 10 mM Mg(OAc) 2 and 100 mM sodium cacodylate (pH 6.9), was incubated for 10 min at 37 * C. The assay was applied to a 1.7 ml DEAE-cellulose column equilibrated with 10 mM KOAc (pH 4.6)/250 mM KCI. The column was washed extensively with the same buffer to remove all proteins, ATP and unbound radiolabelled methionine. The tRNA was eluded by adding 10 mM KOAc (pH 4.6)/1 M KCi. Fractions containing more than 2000 cpm//~! were combined and the RNA was precipitated by adding 2 vol. of ethanol at - 2 0 0 C . The pellet was collected by centrifugation, washed twice with 70% (v/v) ethanol, resuspended in 20 mM KOAc (pH 4.6)/2 mM dithiothreitol and stored at - 8 0 ° C .

Globin-mRNA, purified as described by Zeichner and Stern [24], was a gift of Dr. M. GiSrlach from our Institute and labelled according to the method of En- gland et al. [25]. 7 ~g of giobin-mRNA were incubated with 50 ittCi of cytidine 3',5'-[5'-32P]bisphosphate, 0.1 mM ATP, 10 milts of T4-RNA-ligase, 50 mM Hepes- KOH (pH 7.8), 20 mM MgC! 2, 3.5 mM dithiothreitol, 10% (v/v) dimethylsulfoxide in a 30/L! assay at 4°C for 5 h. Then 3/ t l of 3 M NaOAc (pH 4.8) and 33/ti of isopropanol were added and the RNA was precipitated overnigh~ at - 2 0 o C. The pellet was colleted by centrif- ugation, washed three times with 70% (v/v) ethanol and dissolved in water. Intactness of the RNA was checked on a 7 M urea-containing denaturing polyacrylamide gel according to Maniatis et al. [26].

elF-2a phosphorylation assay elF-2 was purified from rabbit reticulocytes as de-

:.,~cribed [18]. 10 ~1 phosphorylation assays contained 20 mM Tris-HCl (pH 7.2), 2 mM MgCI 2, 100 mM KCI, 30 /tM ATP including 1/tCi [~,-32P]ATP and the amounts of protein fractions as indicated and were incubated for 15 rain at 30°C. Proteins were separated on 12% SDS- pcflyacrylamide gels and the dried gels were exposed for autoradiography using Fuji RX X-ray films.

63

Svcrose density gradient centrifugation analysis 60 /tl in vitro protein synthesis assays, containing

either -asS-labelled Met-tRNAf (400000 cpm; 0.08 pmol) or .~2 P-labelled giobin-mRNA (100 000 cpm; 0.02 pmoi), were incubated for 15 rain at 30°C and then im- mediately layered onto 5 mi 15-40% (w/w~ linear sucrose gradients, prepared as described [18], and centrifuged in a SW65 rotor at 160000 × g and 4°C for 4 h in the case of Met-tRNA, and 5 h in the case of mRNA. The content of the tube was withdrawn from the top by pipetfiag 200/~! fractions. Gradient fractions containing 32P-labelled globin-mRNA were mixed with 5 ml Bray scintillation cocktail and counted in a Beck- man LS 7000 counter. Fractions containing -asS-labelled Met-tRNAf were either treated in the same way or precipitated by addin~ sequentially 0.5 ml of a 0.2% (w/v) yeast nucleinate solution in 0.5 M NaOAc (pH 5.2), and 0.5 mi of 3% (w/v) CTAB in the same buffer. The RNA precipitate was collected by filtering the suspension through Whatman G F / C filters. The filters were washed several times with a 0.0001% (w/v) yeast nucleinate soluti¢l~ and dried. A 0.5% (w/v) diphenyl- oxazole solution ;n toluene was added and the filter- bound radioactivity was measured in a scintillation counter.

For calibration, parallel gradients were run with 10 A26 o units of MEL cell-free extract or rabbit reticu- locyte lysate and analysed for the distribution of ultra- violet absorbing material by withdrawing the content of the tube from the top and pumping it through a flow cell of a recordinF:, spectrophotometer. Absorbance was measured at 260 nm.

Results

Partial purification of the translational inhibitor We have recently shown that protein synthesis ceases

during differentiation of MEL cells [18], but to date only studies concerning inhibitory activities for protein synthesis in uninduced cells have been reported [11-13,15]. Our observation prompted us to look for a putative inhibitory activity in cell-free extracts (S 10) from induced MEL cells using the following protocol (for details see Meateriais and Methods).

The ribosomal material was pelleted and the result- ing supernatant (S 200) was applied to a Sephadex G25 column. The void-volume fraction contained an inhibi- tory activity for in vitro translation. For further purifi- cation this fraction was applied to a DEAE-cellulose column. Protein was eluted from this column in a stepwise manner at i00, 200, 300 and 500 mM KCI, respectively, and the fractions of each step were com- bined. The proteins of each pool were concentrated by ammonium sulfate precipitation. The DEAE-cellulose fraction, which is eluted with 100 mM KCI, contained the inhibitory activity (180 a.u./mg protein; Table !).

64

TABLE !

Purification of the inhibitor

The inhibitory activity of purified fractions was determined in 20/~i in vitro protein synthesis assays containing 0.8 Az60 units of extract from induced MEL cells which were incubated for 60 rain at 30 o C. One arbitrary unit (a.u.) of inhibitory activity was defined to represent the amount which leads to 50~ inhibition of [ 3SS]methionine incorporation in proteins in these assays. Contr :J incorporation (0~ inhibition) was equivalent to 2 pmol of methionine.

Fraction Total activity Specific activity Purification Recovery (a.u.) (a.u./mg protein) (fold) (~)

Sephadex G25; void volume DEAE-cellulose; 100 mM KCI Phosphocellulose-unbound MonoQ; 175 mM KCI

3100 50 1 100 1800 180 3.6 58 1500 250 5 48

200 2000 40 6.5

For most of the experiments described below this pre- paration was used, The fraction eluted with 200 mM KCi also showed a slight but significant inhibitory activity (spec. act.: 60 a.u./mg protein), which is due either to the translational repressor described by Cimadevilla et al. [11,12] or to a small amount of the inhibitor which is eluted with 100 mM KCI. For further purification, the combined fractions eluted with 100 mM KCI were directly applied to a phosphocellulose column at 100 mM KCi. Protein was eluted with 200, 300, 500 and 1000 mM KC. The combined fractions of each step were concentrated by ammonium sulfate pre- cipitation. The inhibitor was found in the unbound (100 mM KCi) fraction. This fraction was applied to a MonoQ column and protein was eluted by a linear gradient from 10 to 500 mM KCI. The inhibitory activ- ity is eluted with 175 mM KCI. This fraction still contained a heterogeneous population of proteins, as tested by SDS-PAGE and silver staining, but further purification attempts were accompanied by the loss of the inhibitory activity (data not shown). Table 1 sum- marizes the purification of the inhibitor.

We used the same purification protocol starting with uninduced MEL cells and surprisingly also detected the inhibitory activity in these cells in the same fractions. An estimate indicates that it is present in approximately equal amounts as in induced cells (data not shown).

Properties of the inhibitor The inhibitory activity is insensitive to digestion with

micrococcal nuclease but sensitive to incubation at 95 ° C (data not shown), which suggests that the inhibitor is a protein.

For determination of its relative molecular mass, 300 pl (1 mg) of the unbound fraction from phospho- cellulose were applied to a Sephadex G200 column. The inhibitory activity is eluted with an asymmetrical distri- bution, the maximum of activity being found in frac- tions 10 and 11 (Fig. 1). The same asymmetrical elution profile is observed with the proteins used as calibration standards as depicted in Fig. 1 (filled triangles). Corre- sponding to the calibration, the relative molecular mass

of the inhibitor was estimated to be (45-70). 103 . This result was confirmed by chromatography on a Sephadex G100 column (data not shown).

The inhibitor blocks initiation o.f protein synthesis The inhibitor is active in the haemin-supplemented

rabbit reticulocyte lysate (data not shown) as well as in cell-free protein synthesis systems from induced (Table I) and uninduced MEL cells. A time-course of in vitro protein synthesis of MEL-cell extracts in the presence of the inhibitor shows a biphasic kinetic suggesting that

c o

o

O k-

C

1000

80.0

600

4 0 . 0

2nO

O0

--~ I ~- T ~ - r - T ' T - r - K - - T - - T - - r 1 - - ~ T - - T - T - T " ~ - - ' T - - r - -

~ m S l B # V m i i m ~ - -

, I__j I ~___L_. _L_.L. I_ , I_a l _.L l_ _ a __L_ a_ I , 2 4 6 8 lO 12 14 16 18 20

Frc]ction Number

Fig. 1. Relative molecular mass determination of the inhibitor by Sepha~dex-G200 chromatography. 1 mg of the phosphocellulose- unbound fraction was subjected to gel-filtration on a Sephadex G200 column equilibrated with buffer A. 11 /zl of each collected column fraction were assayed for its influence on in vitro protein synthesis in 20/~! asays containing 0.8 Az¢,o units of cell-free extract from unin- duced MEL cells prepared after high-salt treatment as described [18]. I00~ represents the amount of [ 3SS]methionine (equivalent to 4 pmol) incorporated in TCA-precipitable material during incubation for 60 min at 3 0 ° C in the presence of 11/zl buffer A. 5/zM edeine inhibited to 16.2~. The elution profiles (as filled triangles) and the relative molecular masses (M r × 10 - J ) of the proteins used as calibration

standards are shown.

100.0

C: 0

o ~

0 BO.O 0

o

• ~- 60.0

o u 40.0

o

20.0

0.0 2 4 6 8

~ul of inhibitor f rac t ion

Fig. 2. Inhibition of in vitro translation of extracts from differentially pretreated MEL cells. Extracts from uninduced MEL cells, which were either not pretreated (closed symbols) or preincubated in high-salt medium prior to lysis (open symbols) were prepare and assayed for in vitro protein synthesis as described il8l. 20 lai assays contained 0.8 A26 o units of cell-free extract and the indicated amounts of the inhibitory fraction eluted with 100 mM KCI from DEAE-cellulose. in the absence (circles) or presence of 5 laM edeine ~squares). After incubation at 3 0 ° C for 60 rain, 5 lal samples were analyzed for [35Slmethionine incorporation in TCA-pr~ipitable material as de- scribed [18]. 10070 represents the amouat of radioactivity incorporated in the absence of inhibitors (equivalent to 8 pmol of methionine with extract from non pretreated cells and 4 pmol with extract from

pretreated cells).

the inhibitor blocks initiation of translation (data not shown). The effect of increasing amounts of the inhibi- tor (100 mM KCI fraction from DEAE-cellulose) on in vitro protein synthesis of two different cell-free systems from uninduced MEL cells supports this result (Fig. 2). When MEL cells are treated with high salt prior to the preparation of the extract, in vitro protein synthesis is due to a high portion of (re)initiation [18]. The transla- tional activity of this kind of extract is decreased to more than 80% by the inhibitor from MEL cells, as it is by the inhibitor of initiation cdeine. In extracts from non-pretreated cells the translational activity is based to a large extent on the elongation of preexisting nascent peptide chains; in this cas~, the MEL inhibitor reduces in vitro protein synthesis by only about 30%. In both cases, the inhibition does not exceed the inhibition mediated by edeine. The addition of the inhibitor to the in vitro translation system in the presence of edeine does not result in a significantly additional inhibition. These results indicate tha~t the edeine-insensitive elonga- tion step of protein synthesis is not impaired by the inhibitor but that it specifically affects the initiat'~on

65

rate, as does edeine. Gel-electrophoretic analysis of the in "itro synthesized proteins shows that the label of all polypeptides is reduced to the same extent (data not shown), indicating that the block of initiation is identi- cal for all mRNAs rather than restricted to distinct mRNAs.

As initiation of protein synthesis is a rather complex sequence of events, the question arises as to which step is affected by the inhibitor.

elF-2~ phosphorylation and ternary complex formation In a variety uf mammalian cells protein synthesis is

regulated by the phosphorylation of the small (a-) sub- unit of elF-2 [2,4]. Since MEL cells contain an elF-2a- kinase activity in the postribosomal supernatant [46,47], DEAE-ceiluiose fractions were tested for kinase activity in the absence (Fig. 3, lanes 1-5) or presence of purified elF-2 (Fig. 3, lanes 6-10). No elF-2ot-kinase activity could be observed in lhe inhibitor containing 100 mM KC! fraction (lane 7), but could be found in the 300 mM and 500 mM KCI fraction (lanes 9 and 10).

In order to test whether the inhibitor directly blocks the formation of the ternary complex [e|F-2. Met- tRNAf-GTP], the formation of this complex was

M r x 10 -3

116 , ,~ 9 4

68"-*,

4 5 " ~ i̧ ~̧̧ ¸̧ ~ i ®

~ a

I 2 3 4 5 6 7 8 9 10

Fig. 3. elF-2~ phosphorylation activity in DEAE-cellulose fractions. 10 pl phosphorylation assays contained 2 lai of the fractions eluted from the DEAE-cellulose column with 10 mM (lanes I and ~)~ 100 mM (lanes 2 and 7), 200 mM (lanes 3 and 8), 300 mM (lanes 4 and 9) or 500 mM (lanes 5 and 10) KCI in the absence (lanes 1-.-5) or presence (lanes 6-10) of 1 lag of elF-2. After incubation at 3 0 ° C for 15 min the samples were subjected to SDS-PAGE and autotadiog- raphy. The autoradiograph is shown. The position of the a-subunit of

e lF-2 is marked.

66

f= '==t ul L - - - *

A 18000.0

16000.0

14000.0

12000.0

10000.0

8000.0

6000.0

4000.0

2000.0

0.0

I I I I I I " I I I I I I I I

40S 80S T T

J

• I , I , I l I , I , I ! , I , I i I I • I R I

2 4 6 8 10 12 14 16 18 20 22 24 26 28

Fraction Number

30

B

5000.0

mRNA 40S 80S °T T T

4000.0

E (3. U

3000.0 n

2000.0

1000.0

0.0 2 4 6 8 I 0 12 14 16 18 20 22 24 26 28

Fraction Number Fig. 4. Binding of Met-tRNA f and mRNA to preinitiation complexes. Ce~-free extracts from uninduced high-salt treated MEL cells (panel A) or rabbit reticulocytes (panel B) were incubated under in vitro protein synthesis conditions as described []8] in the presence of either [ 35S]Met-tRNA f (panel A) or 32P-labelled 81obin-mRNA (panel B) and analyzed on 15-40~ (w/w) sucrose gradients. After centrifugation for 4 h (panel A) or 5 h (pane; B), fractions were collected from the top of the gradient. The amount of radioactivity in each fraction is shown. The positions of the isolated 81obin mRNA (panel B), the 40 S and 80 S peak ir~ parallel 8radients as monitored by ultraviolet absorption arc indicated. In addition, the assays contained: in panel A, no addition (n), 0.4 mg/ml anisomycin (A},, 3 Fg of the inhibitor fraction eluted from MonoQ with ]75 mM KCi (®); in

panel B, ]0 mM GDPCP ([3), 50 Fg of the inhibitor fraction eluted from DEAE-cellulose with ]00 mlVl KGI (®) or both (o).

67

studied using a modified filter-binding assay [271. The inhibitor does not bind Met-tRNAf whether GTP is present or not and has no effect on the binding of Met-tRNAf to elF-2 in the absence or presence of GTP (data not shown). This indicates that the first step in protein synthesis initiation is not affected by the inhibi- tor.

Binding of Met-tRNA¢ to the 40 S ribosomal subunit During unimpaired protein synthesis the ternary

complex binds to the 40 S ribosomal subunit. In order to study this step, protein synthesis extracts from either rabbit reticulocytes (data not shown) or MEL cells were incubated under in vitro translation conditions in the presence of [3SS]methionine-charged Met-tRNA f. The formation of [35S]Met-tRNA clabelled preinitiation complexes was analysed on 15-40% (w/w) sucrose gradients by precipitation with CTAB. Fig. 4A shows the effect of the inhibitor on the distribution of Met- tRNAf in sucrose gradients. Without inhibitor the major portion of labelled methionine (from the Met-tRNAf) is incorporated into protein (which is TCA-, but not CTAB-precipitable) and is thus accumulated in the top region of the gradient (data not shown). A small portion of [35S]Met-tRNAf is found in the top and 40 S region (open squares). Anisomycin, an inhibitor of the elonga- tion step of translation causes the accumulation of the 35S-labelled Met-tRNAf in the 40 S region, containing 43 S or 48 S preinitiation complexes, and less but significantly in the 80 S initiation-complex region of the gradient (open triangles). The partially purified inhibi- tor from MEL-cells (MonoQ 175 mM KCI fraction) caused an increase of labelled Met-tRNAf in the 40 S region of the gradient. No radioactivity could be found in the 80 S region (closed circles). These data indicate that the inhibitor blocks a step after the binding of Met-tRNAf to the 40 S subunit, i.e., the binding of the mRNA to the 43 S preinitiation complex or the subse- quent joining of the 60 S subunit, whilst 80 S initiation complexes - once formed - proceed into unimpaired protein synthesis elongation (marked by the disap- pearance of labelled Met-tRNAf in the 80 S region of the gradient). In order to exclude any unspecific effects due to the depletion or deacylation of labelled Met- tRNAf, we repeated this experiment with a shorter incubation time (5 rain). No obvious differences could be observed (data not shown).

Binding of mRNA to the 43 S premitiation complex To evaluate the effect of the inhibitor on the subse-

quent step of protein synthesis initiation (i.e., the bind- ing of mRNA to 43 S preinitiation complexes), 32p. labelled rabbit globin-mRNA was incubated under in vitro protein synthesis conditions in extracts from MEL cells (data not shown) or rabbit reticulocytes, which were subsequently analysed on 15-40% (w/w) sucrose

gradients. In the absence of inhibitor, 70% of the labelled globin mRNA is found in the polysomal pellet of the gradient (data not shown), which indicates an unim- paired utilization of the labelled mRNA. In the pres- ence of the inhibitor (DEAE-cellulose 100 mM KCi fraction: Fig. 4B, closed circles), the globin mRNA remains in the top region of the gradient, where it is also found when it is loaded as pure RNA on a parallel gradient (data riot shown). T[:is finding suggests that the inhibitor interferes with the binding of mRNA to the 43 S preinitiation complex and not with peptide chain termination. In the latter case, an accumulation of lhe globin mRNA in the polysomal pellet of the gradi- ent would be observed. The specific inhibition of the binding of mRNA to the 43 S preinitiation complex by the inhibitor was confirmed by an additional experi- ment using GDPCP, an unhydrolyzable GTP analogue, which inhibits the joining of the 60 S ribosomal submlit during the formation of the 80 S initiation complex [28,29]. As expected, GDPCP alone (10 raM, 200-fold molar excess over GTP) causes the accumulation of the labelled mRNA in the 40-50 S region of the gradient (Fig. 4B, open squares), i.e., the region of the 48 S preinitiation complexes. In the presence of both GDPCP and the inhibitor from MEL cells (open circles), the radioactivity in the 40-50 S region of the gradients is

Mr × 10 .3

180 . ~

116 , -~

8 4 , . ~

58. , ,~

4 8 , ~

37- ,~

27, , . .

1 2 3 4 5

Fig. 5. Analysis of proteins synthesized by nuclease-treated rabbit reticulocyte lysate. 20/~l assays containing 12 #l of nuclease-treated rabbit reticuilocyte iysate were incubated under in vitro protein ~jnthesis conditions as described [22] and contained: no addition (lane 1), 0.5 ~tg tobacco mosaic virus RNA (lanes 2 and 3), 1 pg poliovirus RNA (lanes 4 and 5), buffer A (lanes 2 and 4) or 9 ~g protein of the inhibitor fraction eluted from DEAE-cellulose with 100 mM KCI (lanes 3 and 5). After incubation at 30 °C for 60 rain, 5 ~! samples of each assay were subjected to SDS-PAGE with subsequent autoradiography. The autoradiograph is shown. The position of the artifactu~a! band described by Jackson and Hunt [22] is marked by an

arrowhead.

68

markedly reduced and accumulates in the region of free mRNA. This result provides further evidence that the inhibitor from MEL cells blocks protein synthesis ini- tiation one step before the joining of the 60 S subunit, Le., the binding of mRNA to the 43 S preinitiation complex.

Translation of capped and uncapped mRNAs Inhibition of binding of mRNA to the 43 S preini-

tiation complex has been held responsible for several physiological blocks of translation [8,30-32]. One of the best characterized examples is the shut-off of host-cell protein synthesis following poliovirus infection [8]. The inhibitor which is active in poliovirus-infected cells has been shown to be a cellular proteinase which is activated by a viral protein and cleaves the 220 kDa subunit of eIF-4F responsible for the recognition and binding of capped mRNA. Thus, no capped mRNA can be effi- ciently translated, while the translation of the uncapped poliovirus-RNA remains unaffected. The cellular pro- teinase has a molecular mass [8] and chromatographic properties [33] comparable to the inhibitor described here. In vitro protein synthesis in the nuclease-treated rabbit reticulocyte lysate in the presence of ;he inhibitor from MEL cells, however, shows no discrimination be- tween capped and uncapped mRNAs (Fig. 5). It blocks the translation of uncapped poliovirus RNA (lanes 4 and 5) and of capped tobacco mosaic virus mRNA (lanes 2 and 3).

Discussion

This paper describes the partial purification and characterization of a translational inhibitor present in the postribosomal supernatant of uninduced and in- duced MEL cells. Molecular mass determination (Fig. 1), heat sensitivity and RNase-resistance indicate that it is a protein with a relative molecular mass in the range (45-70) - 103 . The inhibitor blocks protein synthesis ini- tiation (Fig. 2) by affecting the binding of mRNA to the 43 S preinitiation complex (Figs. 4A and B). Trivial explanations like an unspecific proteinase activity of the inhibitor could be excluded by the observation that it specifically affects initiation but not elongation of pro- tein synthesis (Fig. 2). An unspecific RNase activity could be ruled out by a direct assay using 32p-labelled globin mRNA and denaturing gel electrophoresis (data not shown) and by the finding that the localization of globin mRNA on sucrose gradients is not changed by inhibitor treatment (Fig. 4B). Increasing concentrations of ATP in the in vitro protein synthesis assays did not diminish the inhibitory activity (data not shown), thus ruling out the possibility that its action is due to an ATPase activity that would impair the binding of mRNA to the 43 S preinitiation complex as well [29,34].

There are several reports concerning translational inhibitors from MEL cells [11-13,15]. Stringer and Friend [15] found an inhibitory activity in crude ex- tracts from uninduced but not from induced MEL cells. However, their cell-free protein synthesis system was not shown suitable to distinguish between inhibition of initiation or elongation of protein synthesis. FLC-in- hibitor B, an inhibitor described by Pinphanichakarn et al. [13], affects protein synthesis initiation, but is pre- sent in the ribosomal salt-wash fraction of uninduced MEL cells. This property as well as its relative molecu- lar mass (214-103), its chromatographic behaviour and mechanism of action exclude identity with the inhibitor described here. Cimadevilla et al. [11,12] reported the partial purification of a translational inhibitor from the postribosomal supernatant of uninduced MEL cells which inhibits a step of protein synthesis initiation following the binding of MET-tRNAf to the 40 S ribosomal subunit. Though, in this respect, it resembles the inhibitor described here, its chromatographic be- haviour is slightly different, i.e., it is eluted earlier from Sephadex G200 and at a higher salt concentration from DEAE-cellulose. Since CimadeviUa et al. do not show detailed purification data, the identity of both activities remains in question.

The binding of mRNA to the 43 S preinitiation complex involves, besides ATP, a set of initiation fac- tors concerned with the recognition of the mTGTP cap of the mRNA and with the unwinding of the 5' leader region [29,34-36]. Furthermore~ eukaryotic mRNA is associated with a number of proteins the role of which in translation is unknown [37-41]. Since the inhibitor did not show ATPase activity (see above) nor in- fluenced the formation of the ternary complex [eI F-2- GTP" Met-tRNAr] or the stability of the 43 S preini- tiation complex (Fig. 4A), we focused our interest on the factors involved in mRNA recognition. One exam- ple of severely impaired mRNA recognition is the so- called restriction activity, which is activated in poliovirus-infected cells and leads to the exclusive trans- lation of uncapped mRNA [6-9,32,33]. Since, however, the translation of both capped and uncapped mRNA is affected by the inhibitor from MEL cells (Fig. 5), a similar activity or mode of action involving the mTGTP cap is rather unlikely. The mechanisms of initiation for the two kinds of mRNA appear to be considerably different [42,43]. Therefore, the inhibitor should affect the action of factors involved in both processes, i.e., the 40 S ribosomal subunit, the mRNP, eIF-4A, 4B [42,43] and perhaps elF-2 [44]. At present, we are not able to pinpoint the target factor of the inhibitor from MEL cells.

Since the translational inhibitor described here is present in uninduced and induced MEL cells, its physiological role is rather obscure. It has been pos- tulated that the inhibition of protein synthesis observed

during mitosis is due to a block in the mRNA-binding step of initiation [30,45]. Since the MEL cells used in this study represent unsynchronized ceils, they might contain a certain portion of mitotic ceils. Fhus, we have purified the inhibitory activity, following the same pro- tocol, from MEL cells arrested in mitosis by colchicine treatment. However, neither an increase in inhibitory activity nor any additional activity could be observed (data not shown). Furthermore, the inhibitor described here is not present in mouse myelo~,~i cells, a trans- formed cell line of non-erythroid origin (data not shown). Since this might indicate a cell-type specific role of the inhibitor, it leads us back to our previous observation that, in vivo, overall protein synthesis is markedly reduced during the differentiation of MEL cells, though globin synthesis is unimpaired and the content of overall poly(A) + mRNA is unchanged [18]. Further experiments will be necessary to show whether or not the inhibitor of mRNA-binding exists in unin- duced MEL cells in an inactive (proinhibitor) state and is activated during differentiation and, artifactually, during the preparation of cell-free extracts from unin- duced MEL cells.

Acknowledgements

This work was supported by a grant from the Deutsche Forschungsgemeinschaft to Dr. K. lii|se (Hi 188/3-4), whose advice and comments we gratefully acknowledge. We thank Dr. M. GiSrlach, G. Schulze and Dr. W. Michaike for helpful ciriticism during the work and the preparation of the manuscript.

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