Purification of protein synthesis initiation factor eIF-3 from rat liver microsomes by affinity chromatography on rRNA-cellulose

Biochimica et Biophysica Acta, 697 (1982) 263-269

263

Elsevier BiomedicalPress BBA 91079

PURIFICATION OF PROTEIN S Y N T H E S I S INITIATION FACTOR elF-3 F R O M RAT LIVER M I C R O S O M E S BY AFFINITY C H R O M A T O G R A P H Y ON rRNA-CELLULOSE ODD NYG.~RD a AND PETER WESTERMANNb a Department of Cell Physiology, The Wenner-Gren Institute, University of Stockholm, S-113 45 Stockholm (Sweden) and b Central Institute of Molecular Biology, Academy of Sciences of the G.D.R., Department of Cell Physiology, 1115 Berlin-Buch (Germany)

(Received January 5th, 1982)

Key words: Initiation factor," rRNA cellulose," Protein synthesis," Affinity chromatography," (Rat liver microsome)

An efficient four-step procedure is described for preparing highly purified polypeptide chain initiation factor elF-3 from rat liver microsomal saltwash. The method involves fractionation with ammonium sulfate between 25-40% saturation (0°C) followed by affinity chromatography on rRNA-cellulose, DEAE-cellulose chromatography and sucrose density gradient centrifugation, elF-3 is eluted from the affinity column at a KCI concentration of 0.18 M. The purification is 10-times and the recovery of activity better than 85%. In the sucrose gradients, elF-3 sediments as a 15 S particle indicating a total mass of 650000 Da. The purified elF-3 is highly active in stimulating globin synthesis in a fractionated translation system. Factor elF-3 contains eight subunits with molecular weights ranging from 40000 to II0000. Seven of the subunits are present in one copy per elF-3, whereas the factor contains two copies of one subunit. The isoelectric points of the factor subunits range from 5.5 to 7.3 with most of the polypeptides being acidic.

Introduction Protein synthesis initiation in eukaryotes requires at least seven different initiations factors, elF-l, elF-2, elF-3, elF-4A, elF-4B, elF-4C and elF-5 [1,2]. These factors have been purified from the 0.5 M KC1 ribosomal wash from rabbit reticulocytes and Krebs ascites cells [3-6]. The structurally most complex initiation factor is elF-3. It is a multi-subunit assembly with a total mass of approx. 700000 Da [2,5,7]. The factor has been suggested to inhibit the association of 40 S and 60 S ribosomal subunits [8] and is absolutely essential for the mRNA binding [1,9]. The large number of elF-3 subunits may be essential for the multiple functions suggested for the factor [7]. Some of the initiation factors, including elF-2 Abbreviations: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. 016%4781/82/0000-0000/$02.75 © 1982 ElsevierBiomedical Press

and elF-3, have been characterized as RNA-binding proteins [10]. Due to this property, affinity chromatography on rRNA-cellulose columns has proved to be a very efficient step in the purification of elF-2 [11]. In the present experiments we have used the ability of elF-3 to attach to RNA in the purification of highly active elF-3 from rat liver microsomes. The purification procedure involves ammonium sulfate fractionation, affinity chromatography on rRNA-cellulose, DEAE-cellulose chromatography and sucrose density gradient centrifugation. A fractionated globin-synthesizing system, completely dependent on added elF-3, was developed and used to follow the purification.

Materials and Methods Materials

Creatine phosphate,

creatine phosphokinase

264

(EC 2.7.3.2), ATP, GTP, dithiothreitol, puromycine, Hepes, spermine and amino acids were from Sigma Chemical Co., St. Louis, MO, U.S.A. L[14C]leucine (330 Ci/mol) was from Amersham International Ltd., Buckinghamshire, U.K. DEAE-cellulose DE-52 and cellulose CF-I 1 were from Whatman Biochemicals Ltd., Maidstone, U.K. Ampholine pH 3.5-9.5 was from LKB, Bromma, Sweden.

elF-3 0 10

"~ 0.5

I

Methods Preparation of ribosomal subunits. Rat liver polysomes were prepared as described by Schreier and Staehelin [12] and suspended in 30 mM KC1/20 mM Tris-HCl, pH 7.6/2mM MgCI2/14 mM 2-mercaptoethanol at a concentration of 80100 A260 units/ml. Isolated polysomes, 80 A260 units, were incubated for 30 min at 37°C in 1.2 ml of 0.5 M KC1/20 mM Tris-HC1, pH 7.6/3 mM MgCI2/ll mM 2-mercaptoethanol/2mM puromycine/2mM GTP [13]. The incubated mixtures were layered onto 33 ml of 10-40% (w/v) linear sucrose gradients containing 0.35 M KCI/20 mM Tris-HC1, pH 7.6,/3 mM MgCI2/10 mM 2mercaptoethanol. The samples were overlayered with 0.5 ml of 30 mM KC1/20 mM Tris-HC1, pH 7.6/2 mM MgC12/14 mM 2-mercaptoethanol. The gradients were centrifuged for 70 min at 50000 rev./min in a Sorvall TV850 vertical rotor (DuPont Instruments, Newtown, CT, U.S.A.) and monitored at 260 nm. The separated subunit fractions were collected and pelleted by centrifugation for 15 h at 107000 × gay" The pellets were suspended in 0.25 M sucrose/70 mM KCI/30 mM Hepes pH 7.6/2mM MgCI2/5 mM 2-mercaptoethanol at a concentration of 60-80 A260 units/ml and were stored at - 7 0 ° C until used. Preparation of elF-3 deficient reticulocyte initiation factors. Crude reticulocyte initiation factors were prepared from anemic rabbits as described previously [14]. The 0.5 M KC1 ribosomal wash obtained from six rabbits was applied to a 10 ml DEAE-cellulose column equilibrated with Buffer A (20 mM Tris-HCl, pH 7.6/14 mM 2-mercaptoethanol/10% (v/v) glycerol) containing 0.1 M KCI and 0.1 mM EDTA. The column was washed with the same buffer until no protein was detected in the effluent by monitoring the absorbance at 280 nm. The bound material was eluted with Buffer A

!

10 20 F:aetion number

30

Fig. 1. Preparation of elF-3-deficient initiation factors. Rabbit reticulocyte initiation factors purified by DEAE-cellulose chromatography were dialysed against Buffer A containing 0.35 M KCI and 0.1 mM EDTA and applied to 11 ml linear 10-30% (w/v) sucrose gradients in the same buffer. After centrifugation for 16 h at 200000× gav the gradients were monitored at 280 nm. The top fractions (indicated with a bar) containing all initiation factors except elF-3 were pooled and concentrated with ammonium sulfate.

containing 0.35 M KCI and 0.1 mM EDTA. Protein-containing fractions were pooled, concentrated with ( N H 4 ) 2 S O 4 a t 70% saturation (0°C) and dialysed against Buffer A containing 0.35 M KC1 and 0.1 mM EDTA. After dialysis for 10 h, the material was applied to a 10-30% (w/v) linear sucrose gradient in the same buffer. The gradient was centrifuged at 200000 × g,v for 16h, monitored at 280 nm, and 0.38 ml fractions were collected (Fig. 1). The top fractions containing total initiation factors minus eIF-3 were pooled and concentrated by dialysis against 50 mM Tris-HC1, pH 7.6/0.5 mM EDTA/14 mM 2-mercaptoethanol containing ( N H 4 ) 2 S O 4 a t 70% saturation (0°C). Gradient fractions containing eIF-3 were pooled and concentrated separately. Protein precipitates were collected by centrifugation at 9000 × g,v for 10 min, dissolved in Buffer A containing 0.1 M KCI and 0.1 mM EDTA and dialysed against the same buffer. The dialysed initiation factors were stored in small aliquots at - - 7 0 ° C until used. Preparation of globin mRNA and rat liver pH 5 enzymes. Globin mRNA was prepared from isolated reticulocyte polysomes by phenol extraction [15] and repeated oligo d(T)-cellulose chromatography [16]. RNA not bound to oligo d(T)-cellulose was used for preparing ribosomal RNA-cellulose.

265 Rat liver pH 5 enzymes were prepared as described by Falvey and Staehelin [17] with minor modifications [ 14]. Preparation of ribosomal RNA-cellulose. The preparation of rRNA-cellulose was as previously described [ 11,18,19]. Briefly, cellulose powder was washed with ethanol and dried. Ribosomal RNA was dissolved in Buffer B (50 mM Tris-HC1, pH 6.85/0.1 M NaCI/1 mM EDTA) and mixed with the dried cellulose powder to a slurry containing 20 mg rRNA, 5 g cellulose, 35% (v/v) ethanol and 65% (v/v) Buffer B. After 60 min stirring at room temperature the rRNA-cellulose mixture was precipitated with ethanol and filtered under reduced pressure. Portions of the mixture, 0.6 g, were suspended in 20 ml ethanol and 0.2 ml 1 M magnesium acetate and irradiated with ultraviolet light (254 nm) under constant stirring. After 60 rain irradiation the cellulose was washed with ethanol and dried. Unbound rRNA was removed by extensive washing of the cellulose in Buffer A containing 0.8 M KC1.

Determination of initiation factor elF-3 activity. The assay of elF-3 activity was based on the translation of globin mRNA in a fractionated protein-synthesizing system containing a preparation of reticulocyte initiation factors deficient in elF-3, but otherwise complete (Fig. 1). The incuba-

TABLE I DEPENDENCE OF THE GLOBIN-SYNTHESIZING SYSTEM ON PURIFIED elF-3 The incubation mixtures contained, in final volumes of 30 #1, 70 mM KC1/30 mM Hepes, pH 7.6/2.5 mM magnesium acetate/5 mM 2-mercaptoethanol/50 #M spermine/l mM ATP/0.4 mM GTP/20 mM creatine phosphate/l.25 #g creatine phosphokinase/30 #M of all L-amino acids except leucine/3/,tM [t4C]leucine/3/,tg 40 S subunits/7.5 #g 60 S subunits/0.3 #g globin mRNA/8.6 #g pH 5 enzymes and factors as indicated. Incubation was for 40 min at 37°C. i n i t i a t i o n

Total initiation factors minus elF-3 (#g)

eIF-3 (#g)

-

-

4.5 4.5

0.5 0.5

[ 14C]leucine incorporated (pmol) 0.15 1.84 0.48 15.85

tion mixtures contained, in final volumes of 30 #1, 70 mM KC1/30 mM Hepes, pH 7.6/2.5 mM magnesium acetate/5 mM 2-mercaptoethanol/50 #M spermine/1 mM ATP/0.4 mM GTP/20 #M creatine phosphate/1.25 #g creatine phosphokinase/30 /~M of all L-amino acids except leucine/3/~M L-[14C[leucine/3/~g 40 S subunits/7.5 #g 60 S subunits/0.3 #g globin mRNA/8.6 #g pH 5 enzymes/4.5 /~g eIF-3 deficient initiation factors/eIF-3 as indicated. After incubation for 40 min at 37°C the mixtures were placed on filter paper discs, precipitated with trichloroacetic acid and extracted as described [20]. As seen in Table I the globin mRNA translation in the presence of the elF-3 deficient initiation factor preparation was completely dependent on the addition of eIF-3. Polyacrylamide gel electrophoresis. Molecular weight estimation of eIF-3 subunits was made in 400 mm long and 1 mm thick 7-12% (w/v) gradient SDS-polyacrylamide slab gels [21]. Carbonic anhydrase, ovalbumin, bovine serum albumin, phosphorylase B, fl-galactosidase and myosine were used as molecular weight markers. Determination of factor subunit stoichiometry was made in the SDS-polyacrylamide gels as described [22]. For isoelectric focusing 13 #g elF-3 (10 #1) in Buffer A containing 0.1 M KCI and 0.1 mM EDTA, was diluted with 40 #1 10.6 M urea, 2.5% (w/v) Ampholine pH 3.5-9.5 and 3.1% (v/v) Nonidet P40. Samples were analysed on slab gels containing 8.5 M urea/2% (w/v) Ampholine pH 3.5-9.5/2.5% (v/v) Nonidet P40/4.5% (w/v) acrylamide/0.12% (w/v)bisacrylamide [23]. The electrode buffers were 1.0M NaOH and 1.0M H3PO4 for the cathode and anode, respectively. Protein determination. Protein concentrations were determined as described by Bradford [24] using bovine serum albumin as standard. Results

Purification of elF-3 Rats of 120g body weight were used without fasting. After decapitation the livers were quickly removed and squeezed into 2.5 vol. cold 0.27 M sucrose/30 mM KC1/35 mM Tris-HC1, pH 7.6/5 mM MgCI2/10 mM 2-mercaptoethanol by

266 4

,o 06"

o

~

04-

02-

o o.

3;

3 ,

10

20 30 Fraction number

40

50

Fig. 2. Affinity chromatography on rRNA-cellulose. Rat liver microsomal 0.5 M KC1 wash was precipitated with ammonium sulfate at 25-40% saturation (0°C). The dialysed material (184 mg protein) was applied to a 1.5x15 cm rRNA-cellulose column equilibrated in Buffer A containing 0.1 M KC1. Bound proteins were eluted with a 250 ml linear 0.1-0.5 M KCI gradient in Buffer A. The activity of the collected fractions in stimulating the globin translation system was determined (O O). - Absorbance at 280 nm. - . . . . . Concentration of KC1.

use of a tissue press. Liver material from 30 rats were homogenised by the use of a Dounce homogenizer [25], and the microsomal 0.5 M KC1 wash was prepared as described [11]. The crude microsomal saltwash was fractionated with ammonium sulfate [3]. Material precipitating between 25-40% saturation at 0°C was collected and dialysed against Buffer A (20 mM Tris-HC1, pH 7.6/14 mM 2-mercaptoethanol/10% (v/v) glycerol) containing 0.1 M KCI and 0.1 mM EDTA. Proteins remaining aggregated after dialysis were removed by centrifugation at 2500 X gay for 5 min. Solubilised proteins were applied to a 1.5 X 15 cm rRNA-cellulose column equilibrated with Buffer A containing 0.1 M KC1. The column was washed with the same buffer until no protein was detected in the effluent. Bound proteins were eluted with a 250 ml linear 0.1-0.5 M KC1 gradient in Buffer A at a rate of l m l / m i n (Fig. 2). Fractions, 3 ml, were collected and the eIF-3 activity determined in the globin translation system. Active fractions, eluting at approx. 0.18 M KC1, were pooled and concentrated by precipitation with ammonium sulfate at 60% saturation (0°). The precipitated proteins were collected by centrifugation for 10 min at 10000 X g,v, dissolved in Buffer A containing 0.1 M KC1 and 0.1 mM EDTA and dialyzed against the same buffer. Dialyzed material was applied to a 20 ml DEAE-cellulose column equilibrated with Buffer

A containing 0.1 M KC1 and 0.1 mM EDTA. The column was washed with the same buffer until no protein appeared in the effluent. Bound material was eluted with Buffer A containing 0.25 M KC1 and 0.1 mM EDTA and precipitated with ammonium sulfate as described above. The precipitate was dissolved in Buffer A containing 0.35 M KC1 and 0.1 mM EDTA and dialysed against the same buffer. Samples, 0.4 ml, containing approx. 3 mg protein, were applied to 11 ml linear 10-30% (w/v) sucrose density gradients in Buffer A containing 0.35 M KC1 and 0.1 mM EDTA and centrifuged for 16 h at 200000 X g,v- The gradients were monitored at 280 nm and 0.38 ml fractions collected (Fig. 3). Consecutive fractions were tested for eIF-3 activity in the globin translation system. Active fractions were pooled and concentrated by dialysis against 50 mM Tris-HC1, pH 7.6/0.5 mM E D T A / 1 4 mM 2-mercaptoethanol containing ammonium sulfate at 70% saturation (0°C). Precipitated protein was collected as described above, dissolved in Buffer A containing 0.1 M KC1 and 0.1 mM EDTA and dialysed against the same buffer. The purified eIF-3 was stored in small aliquots at - 7 0 ° C until used. Table II summarizes the purification of eIF-3 from 240 g rat liver. The total activity of eIF-3 in the 0.5 M KC1 microsomal wash was not determined because of the presence of inhibitors of the translation system. A total purification, calculated

1.0

5 4x

©

4~

o

3o

o

o.5

10

Fraction

20

30

number

Fig. 3. Sucrose gradient centrifugation of elF-3. Initiation factor eIF-3 purified by rRNA-cellulose and DEAE-cellulose chromatography (5.8 mg protein) was dialysed and centrifuged in a 10-30% sucrose gradient as described in Fig. 1. Activity of the collected fractions was determined in the globin translation system as described in Table I (O O).

267 TABLE II PURIFICATION OF elF-3 Purification step

Total protein (mg)

Specific activity a (units × 10- 3/mg)

Total activity (units× 10 -3 )

Purification (n-fold)

Yield (%)

1. 2. 3. 4.

184.0 16.5 8.3 2.2

0.5 4.9 8.3 26.9

92 81 69 59

1 10 17 54

100 88 75 64

25-40% Ammonium sulfate fraction rRNA-cellulose chromatography DEAE-cellulose chromatography Sucrose gradient centrifugation

a 1 unit of activity is defined as the amount of protein required to stimulate the incorporation of 1 pmol [14C]leucine into protein under standard incubation conditions, see Table I.

from the ammonium sulfate fraction, of more than 50-times and a yield of elF-3 activity better than 60% was routinely obtained. The activity of the purified f~ictor was determined by use of the fractionated globin-synthesizing system. The translation activity of the system was completely depen-

TABLE III SUBUNIT COMPOSITION OF elF-3 Molecular weights of elF-3 subunits were calculated from SDS-polyacrylamide gels (Fig. 4). Isoelectric points for the subunits were calculated from two-dimensional isoelectric focusing-SDS-polyacrylamide gels (Fig. 5). Stoichiometric determinations were made from densitometric tracings of SDSpolyacrylamide slab gels [22]. The values obtained were normalized to polypeptide No. 4. Polypeptide No.

Mr

pl

Stoichiometry

1 2 3 4 5 6 7 8 9

174000 157000 146000 111000 95000 66000 64000 53000 49 500

7.1 7.2 7.3 5.5 6.1 6.3 6.4 5.6

0.15 0.15 0.10 1.00 1.99 0.74 0.98 0.31

6.1

1.73

10 11 12 13 14

49000 46000 42500 40000 25000

5.6 6.6 5.8 4.7

0.43 0.94 0.94 0.40

Probable number of subunits per elF-3

dent on the addition of elF-3 (Table I). Furthermore, the activity was comparable to that of purified reticulocyte elF-3 (data not given).

Physical characterization of elF-3 As can be seen from Fig. 4, the elF-3 activity sedimented as a 15 S particle [26], indicating a total mass of approx. 650000 Da [27]. The elF-3 preparations contained eight major components ranging in molecular weight from 40000 to 111000 (Table III). Seven of these components were present as a single polypeptide. Of the 95000-Da subunit two copies were present per elF-3. The total mass of the factor, based on the relative mass and stoichiometry of the individual subunits, was calculated to be 620000 Da (Table III). The 25000-Da cap-binding protein [28] was a minor component of the elF-3 preparation. The isoelectric point of the individual factor subunits were determined by isoelectric focusing in 8.5 M urea and 2.5% NP40 (Fig. 5). Most of the factor subunits were acidic proteins with isoelectric points ranging from 5.5 to 7.3 (Table III). Discussion

The eukaryotic initiation factors elF-2 and elF-3 are known to be RNA binding proteins [10]. In a previous report we utilized the affinity for RNA in the purification of rat liver elF-2 [11]. The good results obtained with rRNA-cellulose affinity chromatography in the purification of elF-2 encouraged us to try the same type of affinity column in the purification of elF-3. As shown above, elF-3 binds to the rRNA-cellulose and is eluted

268

A B

Mr

4 ..I

5 I

pI

6 i,,

7 I

8 I

000

200 500

66 200

45 000

31 000

Fig. 4. SDS-polyacrylamide slab gel electrophoresis of purified elF-3. Electrophoresis was according to Laemmli [21] in a 7 12% linear acrylamide gel. (A) 12 bag and (B) 6 bag purified factor. The protein markers were myosin, fl-galactosidase, phosphorylase B, bovine serum albumin, ovalbumin and carbonic anhydrase. Fig. 5. Two-dimensional slab gel electrophoresis of purified elF-3. (A) One-dimension isoelectric focusing was in 1.5 mm slab gels containing 8.5 M urea/2% Ampholine pH 3.5-9.5/2.5% Nonidet P40/4.5% acrylamide/0.12% bisacrylamide [23]. The 13 bag sample of elF-3 was applied in 8.5 M urea/2% Ampholine pH 3.5-9.5/2.5% Nonidet P40. After completing the isoelectric focusing the pH gradient was determined with a pH-electrode, and the gel was stained with Coomassie brilliant blue. Two-dimension SDS-polyacrylamide slab gels were as in Fig. 4. The stained isoelectric focusing gel was incubated in SDS-polyacrylamidesample buffer [21] for 15 min at 56°C and applied to the two-dimension gel. (B) A 6 bag sample of elF-3 run in parallel on the SDS-polyacrylamideslab gel.

from the c o l u m n at a KCI c o n c e n t r a t i o n of 0.18 M. Thus, the i n t e r a c t i o n of eIF-3 with the i m m o b i lized r R N A is weaker than that observed for eIF-2 [1 l]. Because of its lower affinity for r R N A - c e l lulose eIF-3 is eluted together with other cont a m i n a t i n g proteins. The net purification in this c h r o m a t o g r a p h i c step is 10-times. This should be c o m p a r e d to the usual 0 . 5 - 3 - f o l d purification ob-

served with ion exchange c h r o m a t o g r a p h y on DEAE-cellulose or phosphocellulose [5,22,29]. As in the case of eIF-2 [l l] the i n t e r a c t i o n of eIF-3 with r R N A seems to be related to the ability of the factor to b i n d to 40 S ribosomal subunits. In the b i n a r y complex 4 0 S - e I F - 3 , the factor is located in close p r o x i m i t y to 18 S r R N A [30]. Chromatography on rRNA-cellulose and

269

DEAE-cellulose is not sufficient for obtaining highly purified eIF-3. The chromatographic steps were therefore combined with sucrose density gradient centrifugation. Due to its large mass, the active factor sediments as a 15 S particle in sucrose gradients (Fig. 3) [26]. The recovery of total eIF-3 activity from 35 rat livers was comparable to that previously reported for reticulocyte eIF-3 starting with 90 anemic rabbits [5]. However, the specific activity of the rat liver factor was more than 20-times higher than that reported for the corresponding reticulocyte eIF-3 (Table II) [5]. SDS-polyacrylamide gel electrophoresis of the purified factor shows eight major components with masses ranging from 40000 to 1110000 Da. The electrophoretic pattern is closely similar to recently published gel patterns of reticulocyte eIF-3 [31]. Seven of the eIF-3 subunits are present as one copy per eIF-3 molecule, whereas the factor contains two copies of the 95000-Da subunit. The content of the cap-binding protein [28] seems to vary with different preparations. This protein has also been found in purified preparations of eIF-4B [2]. The stoichiometry of the 146000-175000 Da polypeptides varies within the different preparations of eIF-3. The content of these proteins also depends on the treatment of the factor prior to electrophoresis (compare Fig. 5A and B). A similar variability observed in reticulocyte eIF-3 has been suggested to result from proteolytic degradation [31]. Whether the variability in the stoichiometry of the high molecular weight peptides is due to protease action or to strong interactions between factor subunits remains to be clarified. However, no difference in activity was observed among the multiple forms of eIF-3 [31]. Thus, the exact role of the subunit variability for the biological functions of eIF-3 remains to be established.

Acknowledgements The skilful technical assistance of Mrs. Birgit Lundberg is gratefully acknowledged. The work was supported by a grant from the Swedish Natural Science Research Council (B0307-100) to Professor Tore Hultin.

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