Biochimica et Biophysica Acts, 390 ( 1 9 7 5 ) 8 2 - - 9 3 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 9 8 2 6 7

A DISSOCIATION FACTOR FROM EMBRYOS OF XENOPUS L A E VIS

M. D E C R O L Y * a n d M. G O L D F I N G E R

Ddpartement de Biologie Moldculaire, Universitg libre de Bruxelles, 67, rue des Chevaux, 1640 Rhode St. Genese (Belgium) ( R e c e i v e d O c t o b e r 21st, 1 9 7 4 )

Summary

A dissociating factor has been extracted from the ribosomal KCI wash and from the cytosol of developing embryos of Xenopus laevis. No dissociating activity could be detected in the KCI wash of ribosomes from full grown oocytes and unfertilized eggs. As in bacteria, the activity of the dissociation factor seems to be correlated with the rate of protein synthesis suggesting a physiological role of the dissociation factor. The possibilitythat the dissociation factor might be one of the components which limits the rate of protein synthesis in the oocytes is discussed.

Introduction

It is well established that, until the blastula stage, protein synthesis is mainly controlled at the translationallevel in amphibian egg [1]. The mechanisms which control the progressive utilisation of the stored messenger R N A molecules, which have already been synthesized during oogenesis, are stillunknown. The presently accepted view is that, in both bacteria and eukaryotic cells, dissociation of the ribosome monomers into subparticles is a prerequisite for the initiation of protein synthesis [2--4]. Factors with dissociatingactivityon ribosomes have been found in Escherichia coli [5], Bacillus stearothermophilis [6], yeast [7], rat liver [8], reticulocytes [9] and ascitescells [10]. Moreover, in bacteria, it has been demonstrated that (1) the dissociating activity is parallel to the rate of protein synthesis [6,11] and (2) that the dissociatingactivityis a property of initiationfactor IF3 [12--14]. In eukaryotic cells,the identity between the dissociatingfactor (DF) and the initiationfactors remains to be established [15] ; but, at least in ascitescells * Maftre d e r e c h e r c h e $ au F o n d s N a t i o n a l d e la R e c h e r c e s c i e n t i f l q u e . Abbreviation: DF, dissociation factor,

83 [10], it has been f o u n d that the initiation factor (IF3) required for the translation of the natural m R N A has a dissociating activity. The fact that the D F supply regulates the concentration of ribosomal subunits which are required for the initiation of protein synthesis [5,16] has led us to investigate if, during embryonic development, the dissociating activity is, as in bacteria, parallel to the rate of protein synthesis. In the present work, we report on the identification of a dissociating factor which can be extracted from ribosomes of developing embryos. Our results further show that no dissociating activity could be detected in the KC1 wash of ribosomes isolated from full grown oocytes and unfertilized eggs, where protein synthesis is rather low. Materials and Methods

Materials. Eggs of Xenopus laevis were obtained as previously described [ 1 7 ] . The stages were defined according to the table of Nieuwkoop and Faber [ 1 8 ] . To obtain a large quantity of full grown oocytes, free of follicle cells, the ovaries of three females were placed in a solution of collagenase 0.1% in Barth's m e d i u m modified according to Merriam [19] for 15 h at 20°C. Full grown oocytes were selected and rinsed several times in Barth's medium. Preparation of the ribosomes used for DF extraction. The medium used for the preparation of ribosomes contained: 1 0 - 2 M Tris. HC1 (pH 7.6), 1.5 • 10 -3 M MgC12,10 -2 M KC1 and 5 • 10 -3 M mercaptoethanol. After being washed several times in the above mentioned buffer, the eggs or embryos were homogenized in one volume of this buffer. Two centrifugations of 10 min at 13 500 rev./min in the Sorvall centrifuge were carried o u t in order to eliminate pigment, yolk and mitochondria. 2.5 ml of supernatant corresponding to a b o u t 2 g of wet weight, were layered onto 15--30% (w/v) sucrose gradients in the Tris. HC1/MgC12/KC1 buffer without mercaptoethanol, and centrifuged 5 h at 22 500 rev./min in the Spinco centrifuge (rotor SW 25) at 4°C. The gradients were displaced from the b o t t o m and continuously monitored at 254 nm in a Isco flow cell. The fractions corresponding to subunits, monosomes and polysomes were pooled together and centrifuged 3.5 h at 65 000 rev./min (Spinco rotor 65) at 4°C. The ribosomal pellets were stored in liquid N2 and used for D F extraction when a b o u t 40 mg of pooled ribosomes had been obtained. (1 g (wet weight) of eggs corresponds to a b o u t 1 mg of ribosomes). Preliminary experiments have shown that, if the ribosomes are not purified on a sucrose gradient, the ribosomal KC1 wash extract is heavily contamined with RNAase. Preparation of free ribosomes. The postmitochondrial supernatant of the developing e m b r y o s was treated with puromycin at a final concentration of 2 • 10 -3 M for 30 min at room temperature. The supernatant diluted with an equal volume of 10 -2 M Tris • HC1/1.5 • 1 0 - 3 M MgC12/10 - 2 M KC1/5 • 10 -3 M mercaptoethanol buffer was layered onto 5 ml of 0.5 M sucrose containing the same buffer and centrifuged 2.5 h at 105 000 X g (Spinco rotor 40) at 4°C. For the isolation of ribosomes from oocytes and unfertilized eggs, the puromycin treatment was omitted since they contain a great majority of free ribosomes [201.

84

Extraction of the dissociating [actor from ribosomes. The ribosomes, after purification on a sucrose gradient were suspended in: 0 . 5 M KC1, 10-2M Tris • HC1, (pH 7.6), 10-2M mercaptoethanol, at a concentration of 5 mg/ml [211. The suspension was clarified by a low speed centrifugation (10 min 10 000 rev./min Sorvall centrifuge); the supernatant was stirred during 0.5 h at 0°C and then centrifuged during 1.5 h at 65 000 rev./min (Spinco rotor 65). The supernatant of this centrifugation is referred as the "KC1 ribosome extract". This KC1 extract was then made 0.1 M with respect to Tris • HC1 (pH 7.8), brought to 30% saturation with a m m o n i u m sulfate and left during 30 min at 4 ° C. The small precipitate was discarded by low speed centrifugation. The 30% supernatant was brought to 70% saturation in the same way and centrifuged. When the crude extract was used, the precipitate was dissolved in a small volume of buffer containing: 1 0 - : M Tris-HC1 (pH 7.4), 10-' M KC1, 3 • 10 -4 M MgCI:, 5 • 10 -3 M mercaptoethanol [9]. After dialysis against the same buffer during 3 h (3 changes), the protein concentration was measured by the method of Lowry et al. [22] or estimated by ultraviolet absorption using the formula: 1.45 X A2 s 0,m -- 0.74 × A: ~ 0 nm = mg/ml. If the c o n t e n t of protein was lower than 1 mg/ml, the solution was concentrated by ultrafiltration and immediately used. In most experiments, the KC1 ribosomes extract was purified on DEAE~cellulose as described by Scheier and Staehelin [23] for initiation factors. The column was eluted stepwise with 0.12, 0.3 and 0.4 M KCI; the bulk of DF activity was eluted at the 0.3 M step. These fractions were pooled and concentrated as described for the crude extract. Extraction of the dissociating factor from the supernatant. Soluble enzymes were prepared from the supernatant of the crude ribosomes after centrifugation by the m e t h o d of Allen and Schweet [24]. The supernatant was brought to 40% and then 70% saturation with a m m o n i u m sulfate. The precipitate obtained at 40% saturation was discarded; the 70% precipitate was purified on DEAE~ellulose, as described for the extraction of DF from ribosomes. A DF was eluted at the 0.3 M KC1 concentration. DF assays. Isolated ribosomes were suspended in the 10 -2 M Tris • HC1/ 10 -1 M KC1/3 • 10 -4 M M g C 1 2 / 5 • 10 -3 M mercaptoethanol buffer and the suspension clarified by low speed centrifugation. An aliquot of 50 #1, containing 0.2 A260nm unit of ribosomes was mixed with 100 #1 of buffer containing 0.3 mg/ml of bovine albumin {Fraction V} and DF, as specified in the legends of the figures. The samples were incubated as specified, transferred to an ice bath, and fixed with glutaraldehyde in order to prevent the dissociation of the free ribosomes by hydrostatic pressure [25]. The samples were layered on cold 15--30% (w/v) linear sucrose gradients containing 10 -2 M Tris. HCI/10 -1 M KC1/3 • 10 -4 M MgC12/5 • 10 -a M mercaptoethanol buffer and spun for 90 min at 47 000 rev./min (Spinco rotor SW 50.1). The gradients were monitored for absorbance through a flow cell fitted in a Gilford spectrophotometer. The absorbance profiles were quantitated by cutting o u t the peaks and weighing the paper. The dissociation ratio was determined as follows: dissociation ratio = (40-S + 60-S peak area)/(40-S + 60-S + 80-S peak area). Preparation, injection and labelling of oocytes. Large oocytes of X. laevis

85 were isolated from the ovaries with watchmaker's forceps; 40 oocytes were injected with either 50 nl of Ringer (control) or 50 nl of D F in a Ringer solution at a concentration of 500 /~g/ml. Prior the injection, the D F eluted from the DEAE~ellulose column was dialyzed overnight against Ringer medium. Immediately after injection, the oocytes were incubated for 1 h in 5 ml of L-[3-3H]phenylalanine (1 /zCi/ml, 1 C i / m m o l ) a t room temperature. After incubation, the oocytes were washed in label-free medium; groups of 8 oocytes were frozen and then homogenized in 1 ml of 0.15 M NaC1. After the removal of a 100/zl for measurement of precursor uptake, 1 ml of trichloroacetic acid 10% was added and left 30 min in an ice bath. The mixture was centrifuged (30 min at 1400 X g) and the pellet was washed three times with 5% trichloroacetic acid and 0.1% unlabelled phenylalanine. The pellet was dissolved in 0.5 ml of Soluene (Packard) and counted in a Packard scintillation spectrometer. Results

Dissociating activity o f the KCl wash from ribosomes o f developing embryos (stage 33--34) on ribosomes extracted at the same stage It has been shown that bacterial [26] or reticulocyte DF [9] act only on free ribosomes. In preliminary experiments, the ribosomes extracted from the developing embryos were treated with puromycin before they were tested with the KC1 wash of ribosomes. Like others [ 9 ] , we observed a lower stability of the puromycin-treated ribosomes; the background of dissociation (dissociation without DF) was rather high, sometimes more than 30%. Since we have previously shown [20] that, at this stage, the technique used to extract ribosomes gives a b o u t 70% o f free ribosomes (ribosomes not complexed with m R N A or peptidyl-tRNA), in most of the experiments, the puromycin treatment was O2[

A

B

2 0 " / . disscx:iation

8 0 "/o d i s s o c i a t i o n

80S

E c o

40S

60S

80S

0.1

J I 1

, ,

I 2

| 3

| ,I | i I 4 5 0 1 2 Distance from top of tube (ml)

I 3

I 4

5

Fig. I. D i s s o c i a t i o n o f 8 0 - S r i b o s o m e s e x t r a c t e d f r o m d e v e l o p i n g e m b r y o s (stage 33--34) by DF e x t r a c t e d from ribosomes of e m b r y o s at the same stage. 0.2 A 2 6 0 n m unit of r i b o s o m e s w e r e i n c u b a t e d 1 5 m l n at 25~C in 10 -2 M T%is • HCI/10 -I M KCI/3 • 10 --4 M MgCI2/5 • 10 -3 M mercaptoethanol buffer. A, w i t h o u t DF; B, with 50 /~g of DF purified o n DEAE-cellulose (fraction eluted at 0.3 M KCI). 80-S ribosomes were n o t t r e a t e d w i t h p u r o m y e i n . T h e p e r c e n t a g e o f dissociation~ d e t e r m i n e d f r o m t h e areas of the peaks, is indicated.

86

omitted. Fig. 1 shows a typical dissociation pattern obtained by incubation of untreated ribosomes with partially purified DF (purified on DEAE-cellulose, see Materials and Methods). 70% of dissociation is observed. The same dissociation patterns are obtained with the crude product, except that a precipitation of the ribosomes sometimes occurs. This might be due to the presence of a contaminant. An incubation at 30°C even during 5 min, gives a too high background of dissociation. The incubation was thus carried out at a lower temperature.

Dissociating activity o f the DF extracted from developing embryos on ribosomes extracted from unfertilized eggs or full grown oocytes Fig. 2 shows that the DF extracted from developing embryos is able to dissociate ribosomes extracted f r o m unfertilized eggs. Exactly the same profiles were obtained with ribosomes extracted from full-grown oocytes. This lack of specificity is not surprising, since the DF of reticulocytes is able to dissociate E. coli ribosomes [9]. Fig. 2 also shows that the reaction is dependent on the a m o u n t of DF: 78% of dissociation is obtained with 66/~g o f DF and only 30% with 22/~g o f DF in the same conditions. The time course of dissociation of the 80-S monomers by DF is summarized in Fig. 3. The reaction is linear during 15 rain. The kinetics of the reaction with reticulocytes or ascites cells DF are much faster, being complete in about 1 rain at 37°C [9,10]. In yeast [7], the dissociation ratio was found to reach a plateau after 20 min incubation at 30°C. This difference between the different materials in the kinetics of the reaction may be due to the fact that we are n o t working with a submaximal concentration of DF and t h a t the incubation temperature is lower in our exa2 8 % dissociation

44 % dissociation

5 f

°'0

l

I

i

2

i

Distance from top of tube (ml)

87 Q2 C

D

5 8 % dissociation

78% dissociation

E 0.1

C'

1

I

i

I

0

1

2

3

/

l

4 5 Distance f r o m top of tube (ml)

(221 E 35% dissociation

o

0.1.

l

I

I

I

0

1

2

3

i . 4

i 5

Distance from top of tube (ml) Fig. 2. D i s s o c i a t i o n o f 80-S u n f e r t i l i z e d eggs r i b o s o m e s b y t h e D F e x t r a c t e d f r o m r i b o s o m e s o f d e v e l o p i n g e m b r y o s . 0.2 A 2 6 0 n m u n i t o f r i b o s o m e s w e r e i n c u b a t e d in 10 -2 M Tris • HC1/10 -1 M KC1/3 • 10 -4 M M g C I 2 / 5 • 1 0 - 3 M m e r c a p t o e t h a n o l b u f f e r at 2 5 ° C . A , D , E , t h e i n c u b a t i o n w a s carried o u t for 15 r a i n ; B,C the t i m e o f i n c u b a t i o n was 5 a n d 1 0 rain, r e s p e c t i v e l y . A, w i t h o u t D F ; B, C, D, w i t h 66 ~tg o f D F ; E , w i t h 2 2 Dg o f D F .

periments. Moreover, DF has not been purified enough to say whether the reaction is stoichiometric or not. As found for DF factors extracted from other materials [ 9 , 1 0 , 2 6 ] , the dissociating activity is very sensitive to the Mg 2÷ concentration. At 5 • 10 -3 M MgC12, no dissociation is observed even with a large amount of DF. The dissociating activity is destroyed by heating DF during 5 rain at 80°C, suggesting that DF is a protein.

88

80

6C r-

4O O

0

5

1

15

Time (rain) Fig. 3. T i m e c o u r s e o f d i s s o c i a t i o n o f t h e 8 0 - S m o n o m e r s b y t h e D F e x t r a c t e d f r o m d e v e l o p i n g e m b r y o s . S a m e e x p e r i m e n t a l c o n d i t i o n s as d e s c r i b e d o n t h e l e g e n d o f Fig. 2. T h e d i m ~ e i a t i o n o f t h e c o n t r o l h a s been subtracted.

Effect of the KCI wash extracted from ribosomes of oocytes and unfertilized eggs on ribosomes of various stages of development N o dissociatingactivity could be detected in the KCI wash from ribosomes of oocytes or unfertilized eggs. With 50 ~g of D F purified as the KCI wash of ribosomes from developing embryos, the dissociation ratio has the same value as the control (Fig.4). With the KCI wash of oocytes ribosomes, same profiles are obtained whatever was the origin of the tested ribosomes (unfertilizedeggs, oocytes or developing embryos). If larger amounts are added, a decrease of the 80-S ribosomes is observed; but there is no formation of subunits peaks. These experiments do not allow us to say whether the factor is absent or inactivated. Immunological tests are needed in order to answer that question.

Presence of a dissociating factor in the supernatant of developing embryos. It has been reported that initiation factors are present in the c y t ~ l of several mammalian tissues [21,27]. Although, the identity of the dissociating factor with one of the initiation factors of the eukaryotic cells is not proved, it was interesting to see whether a DF is present in the cytosol of oocytes and embryos.

89 0.2 A 18 % dssociation

B 1B % dissociation

o.1

0

J

S

~ 0

I.

f ,

1

1

,L

i

2 3 Distance f r o m top of tube (ml)

i

i

4

5

(22

C

o 0.1 e~

J

I

I

3 Distance from top of tube (ml)

s

Fig. 4. E f f e c t o f t h e KC1 w a s h o f r i b o s o m e s f r o m u n f e r t i l i z e d eggs o n r i b o s o m e s o f u n f e r t i l i z e d eggs. 0.2 A 2 6 0 n m u n i t r i b o s o m e s w e r e i n c u b a t e d 1 5 rain at 2 5 ° C in 1 0 -2 M Tris • H C I / I O- I M KC1/3 • 10 -4 M M g C I 2 / 5 • 1 0 -3 M m e r c a p t o e t h a n o l b u f f e r . A, w i t h o u t KCI w a s h ; B, w i t h 50 #g o f KC1 w a s h ; C, w i t h 1 0 0 #g.

Fig. 5 shows that this is the case: a dissociating factor is present in the post-ribosomal supernatant of developing embryos. Table I shows that this D F dissociates ribosomes of unfertilized eggs as well as those of developing embryos. Our attempts to extract a D F from the cytosol of oocytes were negative, but only the fraction where the D F of developing embryos elutes was studied. Since a D F was not detected in the KC1 wash of ribosomes of oocytes and unfertilized eggs, it was possible that at these stages D F is n o t linked to the ribosomes, b u t present in the supernatant. But our results do n o t give support to that hypothesis.

90 0.2

A 28% dissociation

B 52% dissociotion

15

o.1

f o

Distonce fPom'top of tube (ml) 0.2 c

76 % dissoci~ion

E

o.1

°o

;

Distance from t o p of tube (ml)

Fig. 5, D i s s o c i a t i o n o f 8 0 - S u n f e r t i l i z e d eggs r i b o s o m e s b y a D F p r e s e n t in t h e c y t o s o l o f t h e d e v e l o p i n g e m b r y o s . 0 . 2 A 2 6 0 n m u n i t o f r i b o s o m e s w e r e i n c u b a t e d f o r 1 5 r a i n a t 2 5 ° C . A, c o n t r o l ; B, w i t h 1 3 5 , g o f D F ; C, w i t h 1 9 0 / ~ g o f D F . TABLE I Protein extracted from the post-ribosomal supematant of the developing embryos was added to 0.2 A260 nm unit of ribosomes of unfertilized eggs or of stage 33--34 incubated during 15 min at 25°C; the samples were then cooled, fixed with glutaraldehyde and layered on sucrose gradient for dissociation measurement. Stage

Protein added (~g)

Dissociation (%)

U n f e r t i l i z e d eggs

0 135 195

28 52 76

Developing embryos

0 200

40 83

91 TABLE n INJECTION OF DF FROM RIBOSOMES OF DEVELOPING EMBRYOS INTO OOCYTES T h e c o n c e n t r a t i o n o f t h e D F w a s 5 0 0 m g / m l ~ 5 0 nl w e r e i n j e c t e d i n t o e a c h o o c y t e . I m m e d i a t e l y a f t e r t h e i n j e c t i o n , t h e o o c y t e s w e r e i n c u b a t e d a t r o o m t e m p e r a t u r e d u r i n g 1 h in t h e p r e s e n c e o f L - [ 3 - 3 H ] p h e n y l a l a n i n e (1 /~Ci/ral, i C i / m m o l ) .

Control injected w i t h 5 0 nl of Ringer

Injection of 5 0 nl o f D F in R i n g e r

7427 cpm 8695 cpm 7938 cpm

12334 cpm 12841 cpm 11881 cpm

Effects o f the DF on protein synthesis when it is injected into oocytes As shown in Table II, after the injection of DF into oocytes one observes an increase of 60% incorporation of L-J3 -3 H] phenylalanine into the proteins. This stimulation is rather important since the amount of factor injected represents less than 0.5% of the ribosomes weight of the oocytes; in living reticulocytes, the amount of factors present in the KC1 wash of ribosomes, represents about 5% of the weight of ribosomes. In order to verify the physiological state of the oocytes injected with the DF, they have been placed in a solution of progesterone at the concentration of 1 pg/ml; the maturation was induced like in the controls injected with 50 nl of Ringer without DF. Still, it is a preliminary result; the injection has been done with the oocytes of one female, at one concentration. We do not know if the injection of the DF provokes the maturation. Discussion

To explain the low rate of protein synthesis in full-grown amphibian oocytes or in unfertilized sea urchin eggs, two main mechanisms have been proposed: one involves the inactivation of stored messenger RNA, the other the inhibition of ribosomes function (for a general review see ref. 28). In both materials, it has been shown that a stabilization of the subunit association cannot be the mechanism by which protein synthesis is repressed at certain stages of development [20,29]. On the other hand, if the 9-S globin mRNA from reticulocytes is injected into oocytes, at low RNA concentration, haemoglobin is synthesized; but, injection of large amounts of mRNA saturates the translational capacity of the oocyte. One can calculate that, at saturation, only 10% of the ribosomes present in the oocyte are engaged in protein synthesis [30]. This finding implies that the overall rate of protein synthesis is limited by some other factor than the mRNA [30]. The present paper shows that a dissociating factor can be isolated from the KC1 wash of 80-S ribosomal particles or from the supernatant of developing embryos. This dissociating factor present analogies with the dissociating factors

92 isolated from other materials: the dissociation is proportional to the amount of DF and is very sensitive to the Mg 2+ concentration; the dissociating activity is destroyed b y heating. But the more important fact is that, as in bacteria, the activity seems to be correlated with the rate of protein synthesis of the DF. The fact that no dissociating factor was found in oocytes, where protein synthesis is repressed, leads us to suggest that it might be one of the components which limit the rate of protein synthesis in the oocytes. A ribosomes associated translation inhibitor has been found in E. coli [ 3 1 ] , sea urchin eggs [32,33] and during the differentiation of Blastocladiella emersonii [34]. A correlation might exist between this inhibitor of translation and the fact that no dissociating factor could be detected at stages where protein synthesis is rather low. The preliminary experiments of injection of DF into oocytes, indicate that the DF stimulates the incorporation of phenylalanine to an extent of 60%, if they are confirmed, they would suggest that DF plays a physiological role. Crude KCI washes of o o c y t e ribosomes and of reticulocytes have already been injected together with haemoglobin m R N A in living oocytes [ 3 5 ] . When protein synthesis is limited by some other c o m p o n e n t than m R N A , that is to say at the saturation level, the pattern of protein synthesis is altered in favor of o o c y t e proteins with the o o c y t e ribosomes KCI wash and of haemoglobin with the reticulocyte ribosomes KC1 wash; b u t the effect is quite small, this small effect observed is not a strong objection against our suggestion that DF might control the rate of protein synthesis for the following reasons. In the case of the oocytes, our experiments have shown that if the ribosomes are n o t purified on sucrose gradients before the extraction b y KCI, the supernatant is contaminated by RNAase. Furthermore, a dissociation factor is n o t detectable at that stage. The small effect obtained with the KC1 wash from reticulocyte ribosomes is of d o u b t f u l significance since the crude KC1 wash was injected. It is possible that some contaminant not tolerated by the o o c y t e could interfer; furthermore, since the D F activity has n o t been tested, its activity might have been lost. At least in our material, the DF is very unstable. Many points remain to be settled: the DF activity at intermediate stages, particularly before gastrulation, should be measured. The localization of the D F on ribosomes particles is u n k n o w n since subunits and 80-S monomers were not separated. The effect of the D F extracted at various stages of embryonic development, on the translation of an exogenous m R N A should be studied. Finally, we do n o t k n o w whether there is a correlation between D F and the initiation factors involved in the recognition of different types of m R N A .

Acknowledgement We thank the " F o n d s de la Recherche fondamentale collective" for financial support {Convention 10.224).

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