62 Biochimica et Biophysica A cta 868 (1986) 62-70 Elsevier

BBA 91628

R e d u c e d tu rnover of the e longa t ion f ac to r E F - I • r i b o s o m e compl ex a f te r

t r e a t m e n t with the p ro te in syn thes i s inhibi tor II f r om bar ley seeds

Lars N i l s son a K a t s u h i k o A s a n o b,., Bir te Svensson b, F l e m m i n g M. Pou l sen b

and O d d N y g ~ r d a

a Department of Cell Biology, The Wenner-Gren Institute, Unioersity of Stockholm, Biology E5, S-106 91 Stockholm (Sweden) and b Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Fej 10,

DK-2500, Copenhagen Valby (Denmark)

(Received 9 April 1986)

Key words: Elongation cycle; Elongation factor; Protein synthesis; Translation inhibitor; (Rabbit reticulocytes)

The effect of the protein synthesis inbibitor II from barley seeds (Hordeum sp.) on protein synthesis was studied in rabbit reticulocyte lysates. Inhibitor treatment of the lysates resulted in a rapid decrease in amino acid incorporation and an accumulation of heavy polysomes, indicating an effect of the inhibitor on polypeptide chain elongation. The protein synthesis inhibition was due to a catalytic inactivation of the large ribosomal subunit with no effect on the small subparticle. The inhibitor-treated ribosomes were fully active in participating in the EF-l-dependent binding of [t4C]phenylaianyl-tRNA to poly(U).programmed ribo- somes in the presence of GTP and the binding of radioactively labelled EF-2 in the presence of GuoPP[CH2]P. Furthermore, the ribosomes were still able to catalyse peptide-bond formation. However, the EF-I- and ribosome-dependent hydrolysis of GTP was reduced by more than 40% in the presence of inhibitor-treated ribosomes, while the EF-2- and ribosome-dependent GTPase remained unaffected. This suggests that the active domains involved in the two different GTPases are non-identical. Treatment of reticulocyte lysates with the barley inhibitor resulted in a marked shift of the steady-state distribution of the ribosomal phases during the elongation cycle as determined by the ribosomal content of elongation factors. Thus, the content of EF-1 increased from 0Jt8 mol/mol ribosome to 0.71 mol/mol ribosome, whereas the EF-2 content dropped from 0.20 mol/moi ribosome at steady state to 0.09 mol/mol ribosome after inhibitor treatment. The data suggest that the inhibitor reduces the turnover of ribosome-bound ternary EF-1- GTP. aminoacyl-tRNA complexes during proof-reading and binding of the cognate aminoacyl-tRNA by inhibiting the EF-l-dependent GTPase.

Introduction

Eukaryotic protein synthesis elongation is a cyclic mechanism mediated through the elonga-

* Present address: Applied Bioscience Laboratory, Kirin Brewery Co, Ltd, 2-2, Soujamachi 1 chome, Maebashi-shi, 371 Japan.

Abbreviations: GuoPP[CH2]P, guanosine 5'-[fl,'t-rnethylene] tripliosphate; EF, elongation factor. Correspondence address: Dr. Odd Nyg/trd, Department of Cell Biology, The Wenner-Gren Institute, Biology ES, University of Stockholm, S-106 91 Stockholm, Sweden.

tion factors EF-1 and EF-2 [1,2]. EF-1 forms a ternary complex with aminoacyl-tRNA and GTP [3,4]. This complex exclusively associates with ribosomes having peptidyl-tRNA in the P-site [5,6]. After selection of the cognate aminoacyl-tRNA by a proof-reading mechanism involving an EF-1- catalysed hydrolysis of GTP, the correct amino- acyl-tRNA is bound to the ribosomal A-site [4,5]. This step is followed by the peptide-bond forma- tion, resulting in a ribosome having the peptidyl- tRNA in the A-site [1,2]. EF-2 in complex with

0167-4781/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

GTP binds to these A-site ribosomes [6], thereby mediating the translocation of the nascent peptide from the A-site to the P-site [6-8]. This reaction is coupled to the EF-2-dependent hydrolysis of GTP [1,2].

Several elongation inhibitors have been isolated from plants and plant seeds [9,10]. Most of the so far characterized plant-derived inhibitors seem to inhibit the elongation process by inactivating the ribosome [11,12]. Translational inhibitors derived from molds such as a-sarcin, restrictocin and mitogillin act as very specific ribonucleases by cleaving a single phosphodiester bond near the 3'-end of the 28 S ribosomal RNA [13,14]. In contrast, the plant-derived inhibitors do not seem to possess any ribonuclease activity [12,15]. At- tempts to demonstrate modification of ribosomal proteins after treatment with cytotoxins such as ricin have also been unsuccessful [12]. The mecha- nism by which these plant inhibitors inactivate the translational apparatus is still unknown. We have previously described the isolation and preliminary characterization of three closely related protein synthesis inhibitors from barley seeds [15,16]. No RNA degradation was observed in the presence of the barley-derived proteins, indicating that the inhibitors do not belong to the a-sarcin group of ribonucleases [15].

In the present work we have studied the effects of the barley protein synthesis inhibitor II on the different partial reactions involved in the transla- tion. The inhibitor was shown to inhibit the pro- tein synthesis elongation step by a catalytic in- activation of the large ribosomal subunits. This inactivation results in a marked decrease in the ribosome- and EF-l-dependent GTPase activity. In a complete translational system from rabbit reticulocytes the inhibitor causes a shift in the steady-state distribution of the ribosomal phases during the elongation cycle as demonstrated by a reduced ribosomal content of EF-2 and a con- spicuous increase in the content of EF-1.

Materials and Methods

Chemicals. [14C]Leucine (57 Ci/mol), [35S] methionine (1450 Ci/mol), [14C]valine (290 Ci/mol), [14C]phenylalanine (522 Ci/mol), [y- 32p]GTP (13.8 Ci/mmol) and [14C]NAD+ (266

63

Ci/mol) were from Amersham International, U.K. GTP, GuoPP[CH2]P, Met-Val and ribonuclease A were from Sigma Chemical Co., St. Louis, MO. Micrococcus nuclease was from Boehringer Mann- heim, Mannheim, F.R.G. Cronex lo'dose mammography X-ray film was from DuPont De Nemours & Co Inc., Wilmington, DE. Poly(U) was obtained from Miles Laboratories, Elkhart, IN. Nitrocellulose filters HAWP 0.45 #m were from Millipore, Bedford, MA. Diphtheria toxin was a generous gift from Dr. M. Tiru, The Na- tional Bacteriological Laboratories, Stockholm, Sweden.

Preparation of reticulocyte lysates and rat lioer ribosomal subunits. Rabbit reticulocyte lysates were prepared as previously described [17] and treated with micrococcal nuclease as described by Pelham and Jackson [18]. The ribosomal content of the lysates was determined by centrifugation through discontinuous sucrose gradients composed of I ml of 0.5 M sucrose in buffer A (20 mM Tris-HCl (pH 7.6)/10 mM 2-mercaptoethanol/100 mM KCI/5 mM Mg(CH3COO)2 ) superimposed on 1 ml 1.0 M sucrose in the same buffer. After centri- fugation for 170 min at 257000 × gay, the pellets were rinsed with 0.25 M sucrose/70 mM KC1/30 mM Hepes-KOH (pH 7.6)/2 mM Mg(CH 3- COO)2/1 mM dithiothreitol, resuspended in the same buffer and the absorbance at 260 nm was determined.

Rat liver ribosomal subunits were prepared as previously described [19].

Preparation of protein synthesis inhibitors from barley and Ricinus communis. The protein synthesis inhibitor II from barley seeds was prepared as previously described [15]. Ricinus toxin was pre- pared from the seeds of R. communis as described by Olsnes et al. [20].

Preparation of t25I-labeiled EF-1 and EF-2. Elongation factors EF-1 and EF-2 were prepared from rat liver as described by Moon et al. [21] and Nilsson and Nyghrd [22], respectively. The purity of the factors was analysed as previously de- scribed [23]. The factors were labelled with 1251 according to Bolton and Hunter [24].

Preparation of globin mRNA and [14C]Phe- tRNA. Globin mRNA was prepared from isolated reticulocyte polysomes by phenol extraction [25] and repeated oligo(dT)cellulose chromatography

64

[26]. Rat liver [14C]Phe-tRNA was prepared as previously described [27].

Protein synthesis. The effect of the protein synthesis inhibitors was determined in reticulocyte lysates supplemented with creatine phospho- kinase, haemin, creatine phosphate, KC1, Mg (CH3COO)2, 19 unlabelled amino acids, 50 /~M [~4C]leucine and inhibitors as indicated. After in- cubation the reaction mixtures were placed on filter paper discs and precipitated with trichloro- acetic acid and extracted as described [17]. The polysomal distribution after incubation with the barley inhibitor II was analysed on 4 ml linear 0.5-1.8 M sucrose gradients as previously de- scribed [17].

For determination of the translational activity of inhibitor-treated ribosomal subunits, isolated rat liver 40 S and 60 S subunits were incubated in the presence of the protein synthesis inhibitor. Control subunits were incubated without inhibi- tor. After 5 min at 30°C the subunits were sep- arated from the inhibitor by chromatography on Sepharose 4B columns (6 × 70 mm) equilibrated in 70 mM KCI/30 mM Hepes-KOH (pH 7.6)/2.5 mM magnesium ace ta te /5 mM 2-mercapto- ethanol. Fractions containing 150 /xl were col- lected and the absorbance at 260 nm was de- termined. The subunit-containing fractions were pooled and the ribosomal activity was determined in a fractionated translation system containing globin mRNA, reticulocyte initiation factors and the complementary subunit [28].

Determination of ribosome-bound EF-1 and EF-2. Micrococcal nuclease-treated reticulocyte lysates supplemented with increasing amounts of 125I- labelled EF-1 were incubated in the presence of unlabelled leucine as described above. The ribo- somes were pelleted by centrifugation through dis- continuous sucrose gradients, carefully rinsed with buffer and the ribosome-associated radioactivity was determined as previously described [23].

For determination of ribosome-bound EF-2, purified reticulocyte polysomes were washed with 0.5 M KC1 and the EF-2 content of the salt wash fraction was analysed by incubation with di- phtheria toxin and [14C]NAD+ as previously de- scribed [17].

For formation of the EF-2- r ibosome-GuoPP [CHz]P complex, 125I-labelled EF-2 was in-

cubated together with isolated 40 S and 60 S subunits and GuoPP[CH2]P as previously de- scribed [29].

Formation of the dipeptide Met-Val. Micrococcal nuclease-treated reticulocyte lysates were in- cubated in the presence of 30 /~g globin mRNA, 450 nM [35S]methionine, 2.3 #M valine and barley protein synthesis inhibitor II at a molar ratio of inhibitor to ribosome of 0.44. Control lysates were incubated in the absence of inhibitor. After 10 min at 30°C the reaction mixtures were layered on top of a discontinuous sucrose gradient com- posed of 2.3 ml 0.35 M sucrose in (25 mM KC1/20 mM Tris-HC1 (pH 7.6)/2 mM MgC12/0.1 mM E D T A / 5 mM 2-mercaptoethanol) superimposed on 1 ml 1.11 M sucrose in the same buffer and the ribosomes were pelleted by centrifugation at 234000 ×gay for 180 min. The ribosomal pellets were carefully rinsed with distilled water and re- suspended in water. The RNA was hydrolysed by incubation for 30 min at 30°C in the presence of 0.1 M NaOH. The released amino acids and peptides were acetylated with 1% (v/v) acetic acid anhydride in the presence of 1 M triethanolamine, pH 8.0, for 1 h at room temperature. The samples were acidified by the addition of HC1 and ex- tracted three times with 500 ~1 ethyl acetate. The extracted acetylated amino acids and oligopep- tides were lyophilised, redissolved in methanol, and the amino acids and oligopeptides were sep- arated by thin-layer chromatography on silica gel plates using isoamyl a lcohol /pyr id ine /wate r / triethylamine (50 : 50 : 35 : 2) as solvent. Acetyl [14C]valine, acetyl[35S]methionine and acetyl-Met- Val were run in parallel as standards (Rv acetylvaline 0.58, acetylmethionine 0.63, acetyl- Met-Val 0.70). The radioactive spots were identi- fied by autoradiography. Acetyl-Met-Val was detected by staining with ninhydrin.

EF-l-dependent binding of aminoacyl-tRNA to ribosomes. The EF-l-catalysed binding of [14C] Phe-tRNA to ribosomes was determined in reac- tion mixtures containing, in final volumes of 100 #1, 73.5 mM KC1, 23 mM Tris-HC1 (pH 7.6), 3.2 mM MgCI/, 5 mM 2-mercaptoethanol, 0.2 mM GTP, 20 /tg poly(U), 25 pmol [14C]Phe-tRNA (5750 cpm), 25 pmol 40 S subunits, 25 pmol 60 S subunits and EF-1 as indicated. The subunits were preincubated at 30°C for 5 min in the presence or

absence of the ba r ley p ro te in synthesis inhibi tor . The inh ib i to r - to - r ibosome ra t io was 0.12. Af te r incuba t ion for 5 min at 30°C, the reac t ion was t e rmina ted by the add i t i on of 1 ml ice-cold buf fe r (100 m M KC1, 20 m M Tris-HC1 (pH 7.6) and 5.5 m M MgC12). The incuba t ion mixtures were f i l tered th rough Mi l l ipore H A W P 0.45 # filters, dr ied , and the rad ioac t iv i ty was de te rmined .

Determination of the EF-1- and EF-2-dependent GTPase activities. The r ibosome- and fac tor-de- pe nden t G T P a s e ac t iv i ty was de t e rmined in reac- t ion mixtures con ta in ing 100 m M KC1, 20 m M Tris-HC1 ( p H 7.6), 5 m M MgC12, 7 m M 2- mercap toe thano l , 40 # M [y-32p]GTP, 25 pmol 40 S subuni ts and 25 pmol 60 S subunits . F o r the E F - l - d e p e n d e n t hydrolys is , the reac t ion mixtures were supp lemen ted with 20 #g poly(U) , 25 p m o l P h e - t R N A and 100 pmol EF-1. F o r de t e rmina t ion of the E F - 2 - d e p e n d e n t G T P a s e act ivi ty the in- cuba t ion mixtures con ta ined 50 pmol EF-2. The r ibosoma l subuni t s were p re - incuba ted for 5 min at 30 o C in the presence or absence of the p ro te in synthesis inhibi tor . The G T P a s e act ivi ty was de- t e rmined by incuba t ion for 10 min at 37°C. The re leased [32p]phosphate was ex t rac ted as phos- p h o m o l y b d a t e , wi th b e n z e n e / i s o b u t a n o l ( 1 : 1 ) [22] and the rad ioac t iv i ty was de te rmined .

R e s u l t s and D i s c u s s i o n

Effect of the inhibitor on the ribosomal activity Prote in synthesis inh ib i tors such as abrin, r icin

and tr i t in have been shown to inact iva te r ibo- somes [11,12]. In o rde r to see if the ba r ley inhib i - tor in ter fered with r ibosomes, the t rans la t iona l ac t iv i ty of inh ib i to r - t r ea ted 40 S and 60 S r ibo- somal subuni ts was c o m p a r e d with that of non- t rea ted subunits . As seen in Tab le I, inh ib i tor - t rea ted r ibosomes were less eff icient in ca ta lys ing the i nco rpo ra t ion of a m i n o acids in to prote in . The net inh ib i t ion ob t a ined in the f rac t iona ted system was much lower than tha t observed in the lysate sys tem (Table II). In the la t ter system, the major - i ty of r ibosomes are a r ranged in polysomes , while mos t of the t rans la t ion in the f r ac t iona ted sys tem was pe r fo rmed b y p r o g r a m m e d monosomes . Thus, inac t iva t ion of a single r ibosome on a po lysome could arrest the t rans la t ion of the whole po lysome [30]. Fu r the rmore , the t rans la t iona l eff iciency of

65

TABLE I

EFFECT OF THE BARLEY PROTEIN SYNTHESIS IN- HIBITOR II ON THE ACTIVITY OF ISOLATED RIBO- SOMAL SUBUNITS

Isolated 40 S and 60 S ribosomal subunits were incubated with the barley protein synthesis inhibitor at 30°C for 5 min at molar ratio of inhibitor to ribosome of 0.1. Control samples were incubated in the absence of inhibitor. The subunits were separated from the inhibitor by column chromatography on Sepharose 4B. The protein synthesis reaction mixtures con- tained, in final volumes of 30 ~!, 70 mM KC1/30 mM Hepes- KOH (pH 7.6)/2.5 mM magnesium acetate/5 mM 2- mercaptoethanol/50 #M spermine/1 mM ATP/0.4 mM GTP/20 mM creatine phosphate/1.25 ~g creatine phos- phokinase/30 #M of all L-amino acids except leucine/3 #M [14C]leucine/1.22 pmol 40 S subunits/1.22 pmol 60 S sub- units/0.3 ~g globin mRNA/17.5 ~g pH 5 enzymes/20 #g reticulocyte initiation factors. Incubation was for 40 min at 37°C.

Ribosome pre-treatment mol [~4C]leucine % of 40 S 60 S incorporated/mol control

ribosomes

Control control 6.6 100 Inhibitor inhibitor 5.1 77 Control inhibitor 4.9 74 Inhibitor control 7.1 108

the f rac t iona ted sys tem was much lower than that observed in the lysates, ind ica t ing that the t rans- la t ion was l imi ted by factors o ther than the sub-

TABLE 1I

EFFECT OF RICINUS TOXIN AND BARLEY PROTEIN SYNTHESIS INHIBITOR II ON THE PROTEIN SYNTHE- SIS IN RABBIT RETICULOCYTE LYSATES

Reticulocyte lysates prepared according to Pelham and Jack- son [18] were supplemented with creatine phosphokinase, haemin, creatine phosphate, KCI, Mg(CH3COO)2 , 19 un- labelled amino acids and [14C]leucine. The protein synthesis inhibitors were added as indicated. Incubation was for 20 min at 30°C.

Inhibitor mol/mol [14C]Leucine % of ribosomes incorporated control

(103 cpm)

Control - 50.2 100 Barley 1.9.10- 4 46.2 92

1.9-10 - 3 36.5 73 1.9.10 -2 11.2 22

Ricin 1.8-10- 5 23.7 47 1.8-10 -4 16.5 32 1.8.10- 3 7.0 14

66

units. By cross-testing treated and control sub- units it was shown that only the 60 S ribosomal subunit was sensitive to the inhibitor, whereas the small ribosomal particle was unaffected. These results strongly suggest that the barley inhibitor inactivates the large ribosomal subunit.

Many of the plant-derived protein synthesis inhibitors have been shown to act catalytically [11,12]. In order to determine whether the barley translational inhibitor was able to inactivate the ribosome at a sub-stoichiometric ratio, the protein synthesis inhibition in the reticulocyte lysate was correlated with the ratio of inhibitor to ribosome. As little as 1 mol inhibitor per 500 mol ribosomes was sufficient to produce an approx. 25% inhibi- tion of the protein synthesis (Table II). At higher concentrations the protein synthesis ceased almost completely. Despite the catalytic action, the barley inhibitor was significantly less efficient than ricin (Table II).

The protein synthesis inhibition caused by the barley inhibitor was accompanied by an accumu- lation of heavy polysomes (Fig. 1). Polysome stabilisation has been described as a characteristic feature of protein synthesis inhibitors interfering with the elongation cycle [12,17,31]. Thus, the observed accumulation of polysomes indicates that the barley inhibitor blocks translation during the elongation cycle.

Alteration of the distribution of the different phases of the elongation cycle

At steady-state translation in non-treated re- ticulocyte lysates approx. 35% of the ribosomes can be isolated complexed to EF-1 (Fig. 2), whereas approx. 20% are in the pre-translocation phase containing EF-2. The remaining ribosomal population is puromycin-sensitive, suggesting that they participate in selection of the cognate aminoacyl- tRNA. EF-1 . GTP complex in the complete lysates [23]. After barley inhibitor treat- ment, the steady-state distribution was drastically shifted. The content of EF-1 was measured by an isotope dilution technique [23]. Regression analy- sis of the data in Fig. 2 showed that the ribosomal content of EF-1 increased to 71%, suggesting that the barley inhibitor did not interfere with the binding of the ternary EF-1 -GTP-aminoacy l - tRNA complex. This is further supported by the

2 -

I -

o

4 "

j J f l

2 4 ml

i j

\ 2 4

Fig. 1. Accumulation of heavy polysomes as a result of barley protein synthesis inhibitor II treatment. Reticulocyte lysates (200 /~1 containing 52 pmol ribosomes) were incubated as previously described [17]. After 20 min at 30°C 30-/~1 samples were put on filter paper discs and the incorporation of [14C]leucine was determined. The remaining samples (170 ~i) were analysed on sucrose gradients as previously described. (A) control; (B) 190/Lmol inhibitor/moi ribosomes; (C) 1.9 mmol inhibitor/mol ribosomes; (D) 19.0 mmol inhibitor/tool ribo- somes.

observation that the EF-l-mediated binding of Phe-tRNA to poly(U)-programmed ribosomes was not affected by the treatment (not illustrated). Binding of the ternary EF-1. G T P . aminoacyl-

67

^ e •

~ 0 . 2

g ,10

" '

aJ

10 20 10 20 10"11/[IZSI] EF-I added (mol -I)

Fig, 2. Determination of endogenous ribosome-bound EF-1. Micrococcal nuclease-treated rabbit reticulocyte lysates (0.4 ml), prepared according to Pelham and Jackson [18], were incubated with or without the barley protein synthesis inhibitor in the presence of globin mRNA and increasing amounts of 125I-labelled EF-1. After 5 min at 30°C the incubation mixtures were layered on discontinuous sucrose gradients and the ribosomes were isolated by centrifugation for 180 min at 257000 ×gav- The binding data were linearised by plotting the reciprocal of the added concentration of radioactive EF-1 against the reciprocal of the recovered ribosome-bound radioactive EF-1. The intercept on the y-axis represents the reciprocal of the total number of ribosome-bound EF-1 [23]. (A) control; (B) barley inhibitor treatment at an inhibitor-to-ribosome ratio of 0.1.

tRNA complex to the ribosome requires an empty A-site, i.e. post-translocation ribosomes [6]. The increased ribosomal content of EF-1 therefore in- dicates an accumulation of post-translocation ribosomes.

The increase in ribosome-bound EF-1 was par- tially compensated for by a marked drop in the ribosomal content of EF-2 from 0.20 tool per mol ribosomes to 0.09 mol EF-2 per mol ribosomes. This reduction was not attributed to a decreased ribosomal affinity for the factor, as the formation of ribosomal complexes between EF-2 and empty reconstituted ribosomes was not affected (not il- lustrated).

EF-2 has been shown to exclusively associate with ribosomes having a peptidyl-tRNA in A-sites [6]. The reduced level of ribosome-associated EF-2 in the barley inhibitor-treated lysates is therefore suggested to be caused by a reduction in available pre-translocation binding sites for EF-2. An accu- mulation of ribosomes in the post-translocation phase of the elongation cycle is consistent with an inhibition of the reactions leading to the forma-

tion of peptidyl-tRNA in the ribosomal A-site. The barley inhibitor would therefore be expected to interfere with the translation process either during peptide bond formation or during the EF- 1-catalysed process.

Peptidyltransferase activity after inhibitor treatment The peptidyltransferase activity can be studied

either by the puromycin-dependent release of P- site-located nascent peptides or by the formation of dipeptides on newly initiated ribosomes [32]. The two first codons in both a and fl globin code for the amino acids methionine and valine, respec- tively [33]. After micrococcal nuclease treatment of reticulocyte lysates and addition of globin m R N A the translating ribosomes should be ex- pected to have a Met-tRNA in their P-sites and a Val-tRNA in their A-sites. If the peptidyltransfer- ase is blocked by the inhibitor treatment no Met- Val-tRNA should be formed on the ribosome. This hypothesis was tested by incubating micro- coccal nuclease-treated lysates with radioactively labelled methionine in the presence of globin

68

mRNA. The ribosomal content of amino acids and oligopeptides was analysed by thin-layer chro- matography. No reduction in Met-Val synthesis could be detected after treatment of the lysates with the barley inhibitor (Fig. 3). We therefore conclude the peptidyltransferase was not affected by the inhibitor. This is in agreement with earlier observations using the puromycin reaction [16].

Effect of the inhibitor on the elongation factor-cata- lysed hydrolysis of GTP

The first step in the post-translocation phase is the EF-l-dependent binding of aminoacyl-tRNA to the ribosomal A-site. As the binding of the ternary EF-1. GTP. aminoacyl-tRNA complex to the ribosomes was not reduced by the barley inhibitor treatment, the results suggest that the inhibitor interferes with the turnover of the ribo- some. EF-1 complex.

During the binding of aminoacyl-tRNA to the ribosomal A-site, EF-1 catalyses the hydrolysis of GTP to GDP, thereby facilitating the dissociation of EF-1 from the ribosome [4-6]. In order to determine whether the barley inhibitor-induced block of the elongation cycle was due to a reduced GTPase activity and EF-1 turnover, we measured the EF-1- and ribosome-dependent hydrolysis of GTP. As seen in Table III, the barley inhibitor-treated ribosomes showed a markedly re- duced EF-l-dependent GTPase activity. EF-1 by itself showed a substantial GTP hydrolysis, while the ribosomes were essentially free from hydroly- sis activity. Neither the EF-1- nor the ribosome- dependent hydrolysis was stimulated by Phe- tRNA. However, in the incomplete system com- posed of EF-1 and ribosomes the GTP hydrolysis was more than additive. The GTPase activity of this system was not influenced by the barley in- hibitor. The hydrolysis in the presence of EF-1 and ribosomes was therefore used as background in determination of the GTPase activity of the complete system.

Inhibition of the EF-l-dependent GTPase ac- tivity in lysates supplemented with the non-cleava- ble GTP analogue GuoPP[CH 2 ]P has been shown to result in the accumulation of post-translocation ribosomes complexed to EF-1 [23]. The results therefore indicate that the barley inhibitor-re- duced decrease of the EF-l-dependent GTPase

A B C D E

ti - , A c - M e t - V a I

O OJ I

Q

Fig. 3. Peptide bond formation following barley protein synthesis inhibitor treatment. Micrococcal nuclease-treated re- ticulocyte lysates were incubated in the presence of globin mRNA and [3SS]methionine and valine. After 10 min at 30°C the ribosomes were pelleted by centrifugation through a sucrose cushion. The pellets were resuspended in water and the RNA was hydrolysed by incubation with ribonuclease A followed by incubation in the presence of 0.1 M NaOH. The released amino acids and peptides were acetylated with 1% (v/v) acetic acid anhydride and extracted with ethyl acetate. The material was analysed by tin-layer chromatography followed by autora- diography. AcetyI-Met-Val was run in parallel. (A) acetyl []4C]valine; (B) extract from control lysate; (C) acetyl[35S] methionine and acetyl[]4C]valine; (D) barley inhibitor-treated lysate; (E) acetyl[35S]methionine. The position of the ninhydrin-stalned acetyl-Met-Val is indicated.

activity leads to a reduced turnover of the ribo- some-bound EF-1 and thereby to an accumulation of EF-l-containing post-translocation ribosomes. Interestingly, the dipeptide formation on barley

inhibitor-treated ribosomes was not significantly reduced despite the inhibited EF-l-dependent GTPase activity and the accompanying accumtda- tion of EF-l-containing ribosomes.

It has been shown that EF-1 and EF-2 bind to the ribosome at the same or partially identical binding sites [6,7,34,35], and a common GTPase center involved in both EF-1- and EF-2-depen- dent GTP hydrolysis has been suggested [36]. However, studies using reactive GTP analogues indicate that the catalytic domain is situated within the elongation factors and not on the ribosome [22,37]. An inactivation of a ribosome-located GTPase center would automatically lead to a reduction of both the EF-1- and EF-2-dependent GTPases. Since the inhibitor had no effect on the

TABLE III

EFFECT OF THE BARLEY PROTEIN SYNTHESIS IN- HIBITOR II ON THE RIBOSOME- A N D EF-1 DEPEN- D E N T GTPase

Isolated 40 S and 60 S ribosomal subuni ts were pre-incubated in the presence or absence of the protein synthesis inhibitor for 5 min at 30°C. The inhibitor-to-ribosome ratio was 0.12. The GTPase activity was determined using duplicate samples of 100 ~1 containing 100 m M KCI /20 m M Tris-HCl (pH 7.6)/5 m M MgC12/7 m M 2-mercaptoethanol /40 # M [y-32 P]GTP/20 #g poly(U)/25 pmol 40 S and 60 S ribosomal subuni t s /25 pmol phe - tRNA/100 pmol EF-1. The released [32P]phosphate was extracted as phosphomolybdate, with benzene/ isobutanol (1 : 1) and the radioactivity was determined. The ternary com- plex-dependent GTP hydrolysis was calculated as the hydrol- ysis observed in the complete system minus the hydrolysis observed in the absence of Phe-tRNA. Values in parentheses represent % of control.

Additions pmol 32p released

h3EF-1 Phe- tRNA ribosomes EF-1. aminoacyl- tRNA. ribosome- dependent

-~" - - - - 4 4

- + - 0

- - control 14 - - inhibitor 12 + + - 39 - + control 4 - + inhibitor 1 + - control 82 + + control 190 108 (100) + - inhibitor 82 + + inhibitor 143 61 (56)

69

TABLE IV

RIBOSOME- A N D EF-2-DEPENDENT GTPase IN THE PRESENCE OF THE BARLEY PROTEIN SYNTHESIS IN- HIBITOR II

Isolated 40 S and 60 S ribosomal subunits were pre-incubated in the presence or absence of the protein synthesis inhibitor for 5 rain at 30°C. The inhibitor-to-ribosome ratio was 0.12. The GTPase activity was determined using duplicate samples of 50 #1 containing 100 m M KCI /20 mM Tris-HCl (pH 7.6)/5 m M M g C I 2 / 7 m M 2-mercaptoethanol /40 # M [V-32p]GTP/25 pmol 60 S and 40 S ribosomal subuni t s /50 pmol EF-2. The released [32p]phosphate was extracted as phosphomolybdate, with benzene/ isobutanol (1:1) and the radioactivity was de- termined. Values in parentheses represent ~ of control.

Additions pmol 32p released

EF-2 ribosomes EF-2- and ribosome dependent

+ - 22 - control 33 - inhibitor 31 + control 811 756 (100) + inhibitor 796 743 (98)

EF-2-dependent hydrolysis (Table IV), the data indicate that the ribosomal domains inducing the EF-1- and EF-2-dependent GTPases are non-iden- tical. Furthermore, the results are in agreement with a factor-located GTPase center.

Acknowledgements

The technical assistance of Mrs. B. Lundberg is gratefully acknowledged. O.N. and L.N. are in- debted to the Swedish Natural Science Research Council for a research grant (B-BU 8463-100).

References

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