THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemista, Inc.

Vol. 262, No. 21, Ieaue of July 25, pp. 10164-10170,1987 Printed in U.S.A.

The Isolation and Characterization from Rabbit Reticulocytes of Two Forms of Eukaryotic Initiation Factor 2 Having Different &Polypeptides*

(Received for publication, August 4, 1986)

Jaydev N. Dholakia and Albert J. WahbaS From the Department of Biochemistry, The University of Mississippi Medical Center, Jackson, Mississippi 39216-4505

We have isolated from the high salt wash of rabbit reticulocyte ribosomes two forms of the polypeptide chain initiation factor 2 (eIF-2) which differ with re- spect to their &subunit, GDP content, and sensitivity to M$+ in ternary (eIF-2*GTP-Met-tRNAf) and binary (eIF-2-GDP) complex formation. The form of eIF-2 eluting first from a cation exchange (Mono S, Phar- macia) column has a &subunit of lower molecular weight (eIF-2(BL)) and a more acidic PI value than the form eluting at a higher salt concentration (eIF-2WH)). These two forms of eIF-2 &polypeptides are also de- tected in reticulocyte lysates when the proteins are resolved by two-dimensional isoelectric focusing-do- decyl sulfate polyacrylamide gel electrophoresis fol- lowed by immunoblotting. The peptide mapping of the isolated &subunits after limited proteolysis by papain, pancreatic protease, a-chymotrypsin, or Staphylococ- cus aureus VS protease further demonstrates that the two forms of &subunits are not the product of a non- specific proteolytic action that occurred during the purification procedure, but rather reflects the exist- ence in vivo of both forms of eIF-2. The GDP content of eIF-2WL) and eIF-2&) is approximately 0.85 and 0.22 mol of GDP/mol of eIF-2, respectively. The Kn for GDP of eIF-2(BL) was lower (2.2 X lo-’ M) than that of eIF-2(BH) (6.0 X lo-’ M). In the presence of 1 mM Mg2+, the activities of eIF-2(bL) and eIF-2VH) in forming a binary and a ternary complex are inhibited 90 and 25‘70, respectively. The extent of Mg2* inhibi- tion and its reversal by the guanine nucleotide ex- change factor is directly proportional to the amount of GDP bound to eIF-2. No inhibition by Mg2+ is observed when eIF-2-bound GDP is removed by alkaline phos- phatase. In the presence of the guanine nucleotide ex- change factor, both forms of eIF-2 are equally active in ternary complex formation, and the complex formed is quantitatively transferred to 40 S ribosomal sub- units.

The eukaryotic polypeptide chain initiation factor 2 (eIF- 2)’ plays a significant role in the regulation of protein synthe-

* This work was supported in part by United States Public Health Services Grant GM-25451. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed Dept. of Biochem- istry, University of Mississippi Medical Center, 2500 N. State St., Jackson, MS 39216-4505.

The abbreviations used are: eIF-2, the eukaryotic polypeptide chain initiation factor 2; eIF-2(@,), the form of eIF-2 eluted at lower salt concentration and containing lower molecular weight &subunit as compared to the form eIF-2(@H); eIF-2(BH), the form of eIF-2 eluted at higher salt concentration and containing higher molecular weight @-subunit as compared to the eIF-2(pL) form; GEF, guanine nucleotide exchange factor; FPLC, fast protein liquid chromatography.

sis in mammalian cells (1). The first two steps in polypeptide chain initiation in eukaryotes involve the formation of a ternary complex containing equimolar amounts of eIF-2, GTP, and Met-tRNAf, and the selection of a message in the formation of a 40 S initiation complex. In the presence of a 60 S ribosomal subunit and other initiation factors, an 80 S initiation complex is formed, thereby setting the stage for polypeptide chain elongation. Several laboratories purified eIF-2 from rabbit reticulocytes as a complex of three non- identical polypeptides with subunit molecular weights in the range of 57,000-52,000 for the y-subunit, 52,000-48,000 for the @-subunit, and 38,000-35,000 for the a-subunit (1-6). In addition to differences in the reported molecular weights of the individual polypeptides, there are conflicting reports re- garding the number and stoichiometry of the different poly- peptide chains (2-9). Meyer et al. (6) observed multiple forms of rabbit reticulocyte eIF-2 having different y-subunits. On the other hand, eIF-2 preparations from pig (7) and calf (8) liver and rabbit reticulocytes (9) were isolated as having only two polypeptides, with the 6-subunit not being present. Such heterogeneity in the composition of eIF-2 subunits can either be attributed to the anomalous behavior of the subunits in different gel systems (4, 6) or it may be due to the failure of conventional purification techniques to separate different forms of eIF-2 present in lysates. In this paper we report the isolation of two different forms of the reticulocyte factor which differ with respect to the 6-subunit, GDP content, and sensitivity to Mg2+ in binary (eIF-2. GDP) and ternary (eIF- 2 . GTP. Met-tRNAf) complex formation. A preliminary ac- count of this work was presented (10).

EXPERIMENTAL PROCEDURES

Materials [%]Methionine, [3H]GDP, and [Y-~’P]ATP were purchased from

Du Pont-New England Nuclear, and GTP and GDP were obtained from Boehringer Mannheim. GTP was further purified by chroma- tography on a Pharmacia FPLC polyanion SI column. Nucleoside diphosphate kinase, pancreatic protease (type l), papain, a-chymo- trypsin, Staphylococcus aureus V8 protease, Escherichia coli alkaline phosphatase, and various protease inhibitors were obtained from Sigma; the proteins used as molecular weight markers were from Bio- Rad; peroxidase-conjugated rabbit anti-goat IgG was from ICN, and ampholytes were from LKB (Ampholine, pH 3.5-10). Reticulocyte lysate preparations were purchased from Green Hectares, Oregon, WI, and DEAE-cellulose (DE52) and phosphocellulose (P-11) were from Whatman. The FPLC system as well as the Mono S and Mono Q columns were from Pharmacia. Preparations of [W]Met-tRNAr (11) and reticulocyte 40 S ribosomal subunits (12) were the same as previously described. Goat anti-rabbit reticulocyte eIF-2 antibodies and casein kinase I1 from rabbit skeletal muscle were generously provided by Martin Gross (Department of Pathology, University of

10164

Rabbit Reticulocyte eIF-2 Having Two Different /3-Subunits 10165

Chicago, Chicago, IL) and Erwin Reimmann (Department of Bio- chemistry, Medical College of Ohio, Toledo, OH), respectively.

Methods Purification of the Polypeptide C h i n Initiation Factors-All pro-

cedures were performed at 4 "C except for chromatography on FPLC columns, which was performed at room temperature.

Preparations of eIF-2 were purified from the 0.5 M KC1 wash of reticulocyte ribosomes by a modification of the previously described method (2). The 0.5 M KC1 ribosomal wash fraction (1500-2000 mg of protein) was dialyzed overnight against buffer A (20 mM Tris-HC1, pH 7.5, 100 mM KC1, 1 mM dithiothreitol, 50 p M EDTA, 0.3 mM phenylmethylsulfonyl fluoride, 10% (v/v) glycerol) and was applied to a DEAE-cellulose column (2.5 X 37 cm) previously equilibrated with the same buffer. The column was washed at 30 ml/h with 500 ml of buffer A. The protein fractions containing eIF-2 activity were eluted with buffer A containing 300 mM KC1 and were dialyzed for 18 h against 2 liters of the same buffer. This material (100-150 mg) was applied to a phosphocellulose column (2.5 X 6 cm) previously equilibrated with buffer A. The column was washed with 2 volumes of the equilibrating buffer and the adsorbed proteins were successively eluted with 50 ml of buffer A containing 400 and 650 mM KCl. The fractions containing eIF-2 activity (10-20 mg) eluted with 650 mM KC1. They were then pooled, dialyzed for 18 h against 2 liters of buffer A, and applied to a Mono Q (anion exchanger) column (5 x 50 mm) previously equilibrated with the same buffer. The column was washed (60 ml/h) with 2 ml of buffer A and the adsorbed proteins were eluted with three consecutive linear gradients; 5-ml gradient, 100-250 mM KC1; 20-ml gradient, 250-500 mM KCl; and a 5-ml gradient, 500-1000 mM KCl. Individual fractions (0.5 ml) containing eIF-2 activity, which eluted with 310-325 mM KCl, were pooled and dialyzed for 8 h against 2 liters of buffer A. This eIF-2 preparation (3-6 mg) was then applied to a Mono S (cation exchanger) column (5 X 50 mm) previously equilibrated with buffer A. After washing the column (60 ml/h) with 2 ml of the same buffer, the proteins were eluted with three consecutive linear gradients in buffer A; 2-ml gradient, 100-200 mM KCl; 16-ml gradient, 200-400 mM KCI; and a 5-ml gradient, 400-1000 mM KC1. Fractions of 0.5 ml were collected. The elution profile of eIF-2 activity from the Mono S column is illustrated in Fig. 1.

The guanine nucleotide exchange factor was purified as described earlier (13) and was freed of eIF-2 by chromatography on a Mono Q column. The purified preparation consisted of five polypeptides of M, 82,000, 65,000, 55,000, 40,000, and 34,000.

Assays-Binary (eIF-2.GDP) and ternary (eIF-2.GTP.Met- tRNAf) complex formation and nucleotide exchange were assayed as described earlier (2). To determine the KO for GDP of the two forms of eIF-2, the binding of [8-3H]GDP was analyzed by nitrocellulose filtration assays in the presence of 1.5 pg of factor, 1 mM M$+, and GDP over the concentration range of 1-800 nM (2, 14). Met-tRNAf binding to rabbit reticulocyte 40 S ribosomal subunits was performed as previously described (11). Where indicated, eIF-2 was phosphoryl- ated by rabbit skeletal muscle casein kinase I1 (15). The limited proteolytic digestion of eIF-2 (30 pg) for the degradation of the @- subunit was performed (3) with pancreatic protease (50 ng). The amount of GDP bound to eIF-2 was determined as described by Bagchi et al. (5). After treatment with alkaline phosphatase (16) to remove eIF-2-bound GDP, the factor was again applied to a Mono Q column.

Gel Electrophoresis-Dodecyl sulfate-polyacrylamide gel electro- phoresis was carried out in two different slab gel systems (4). In system I, the concentration of acrylamide was 5% in the stacking gel and 10% in the resolving gel. The acrylamide to bisacrylamide ratio was 37:l (w/w) in both stacking and resolving gels. In system 11, the acrylamide concentration in the stacking and resolving gels was 5 and 15%, respectively, with an acrylamide to bisacrylamide ratio of 37:l for the stacking gel and 167:l for the resolving gel. The gels were stained with 0.1% Coomassie Brilliant Blue R-250 and destained in an aqueous solution containing 10% methanol and 7% acetic acid. The gels were enclosed in a plastic bag and exposed to Kodak XR-5 film at -70 "C for autoradiography. Densitometer tracings of the Coomassie Brilliant Blue-stained gels were made on a Transidyne 2510 densitometer. Two-dimensional electrophoresis of the purified eIF-2 was carried out as described by O'Farrell (17) in which the polypeptides were separated in the first dimension by isoelectric focusing and in the second dimension by dodecyl sulfate-polyacryl- amide gel electrophoresis (system 11).

The technique described by Duncan and Hershey (18) to fraction- ate cell lysate proteins by two-dimensional gel electrophoresis fol- lowed by immunoblotting to identify eIF-2(8) was used with the following modifications. The reticulocyte lysates were made 9 M in urea and the protease inhibitors, phenylmethylsulfonyl fluoride, pep- statin, leupeptin, and chymostatin, were added prior to electropho- resis to a final concentration of 0.5 mM. The ampholytes were used at a final concentration of 6% in isoelectric focusing gels. The second- dimension dodecyl sulfate-polyacrylamide gel electrophoresis was carried out as in system 11, and the electrophoresis was continued for 60 additional min at 30 mA after the tracking dye (bromphenol blue) electrophoresed out of the gel. Due to the high concentrations of globin in the reticulocyte lysates, these modifications were essential in order to achieve sharp focusing and a better separation of the eIF- 2 @-subunits. Goat anti-rabbit reticulocyte eIF-2 and peroxidase- conjugated rabbit anti-goat IgG were used as primary and secondary antibodies, respectively. Immunoreactive spots were visualized by using methanolic 4-chloro-1-napthol.

In situ peptide mapping of the @-subunits of eIF-2 by limited proteolysis was carried out as previously described (19) except that system I1 dodecyl sulfate-polyacrylamide gel electrophoresis was em- ployed. The @-subunit (approximately 3 pg) of either eIF-2(oH) or eIF-2(pL) was cut out from the first dodecyl sulfate-polyacrylamide gels and was re-electrophoresed in the absence or presence of the various proteases. The peptides were visualized by silver staining (20).

RESULTS

Isolation of Two Forms of eIF-2"The use of FPLC ion exchange column chromatography has allowed us to purify eIF-2 from rabbit reticulocyte lysates to apparent homoge- neity with 80% recovery of the initial eIF-2 activity (Table I). Two forms of eIF-2 were obtained after chromatography of the Mono Q preparation (step 4, Table I) on a Mono S column (Fig. 1). The individual fractions of eIF-2 eluting from the Mono S column were examined for eIF-2 activity by ternary complex formation and for subunit content by dodecyl sulfate- polyacrylamide gel electrophoresis. The fractions of eIF-2, eluting at a lower (215 mM KC1) salt concentration contained a lower molecular weight P-subunit and were less active in the presence of M$+ as compared to those eluting at a higher (265 mM KC1) salt concentration. The peak fractions of these two forms of eIF-2, fractions 27-29 and 33-36 (Fig. I), were pooled separately and subsequently will be referred to as eIF- 2(PL) and eIF-2(PH), respectively. The purification of eIF-2 by this procedure was repeated 10 times and an identical elution pattern from the Mono S column was observed.

Isoelectric Points and Molecular Weights-Both forms of eIF-2 were analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis in system I and system I1 as described under "Methods." It is evident from Fig. 2 that each form of eIF-2 consists of three polypeptides. As described earlier (4, 6), the order of migration of the eIF-2 polypeptides when separated

TABLE I Purification of reticulocvte eIF-2

protein cific activity activity cation Recovery Total Purifi- Total

rng rng units' -fold % units/

protein 1. 0.5 M KC1 wash 1,716 10 17,160 1 100 2. DEAE-cellulose 135 115 15,525 11.5 90 3. Phosphocellulose 15 1,000 15,000 100 87 4. Mono Q 5 2,840 14,200 280 83 5. Mono S eIF-2(PL) 1.6 3,970 6,350 400 37 6. Mono S eIF-2(Bw) 1.9 3.910 7.430 390 43

One unit of eIF-2 activity is defined as 1 pmol of [35S]Met-tRNAf bound under standard assay conditions.

10166 Rabbit Reticulocyte eIF-2 Having Two Different @-Subunits

FIG. 1. Elution of reticulocyte eIF-2 from Mono S . eIF-2 was chro- matographed on the Mono S column as described under "Methods." The AzW elution profile of protein with the linear KC1 gradient is presented in B. An ali- quot (10 pl) of each fraction was ana- lyzed for subunit composition by dodecyl sulfate-polyacrylamide gel electropho- resis in system I ( A ) and an aliquot of 1 pl was assayed for ternary complex formation activity (C) in the presence (M) or absence (M) of 1 mM MgZ+.

h ""1 I \

0.2 a

by dodecyl sulfate-polyacrylamide gel electrophoresis system I is a , @, and y, with the a-subunit migration being most rapid (Fig. 2.4). The order of migration of @- and y-subunits is reversed (Fig. 2B) when eIF-2 is subjected to electrophoresis in system I1 (3, 4, 6). In order to confirm these results, both forms of eIF-2 were incubated with [T-~~PIATP and casein kinase 11, which phosphorylated the @-subunit in eIF-2(@,) and eIF-2(PH) to the same extent. These results demonstrate that it is indeed the @- and not the y-subunit that differs in these two forms of eIF-2. The @-subunits of the two prepara- tions differ in molecular mass by 1,000 daltons (Fig. 2). This molecular weight difference is observed in both gel electro- phoresis systems. Polypeptides of M, 45,000-55,000 are better resolved in gel electrophoresis system I1 and hence the differ- ence between the two @-subunits is more obvious when eIF-2 is subjected to electrophoresis in this system (Fig. 2B). The molar ratios of the a/@/y subunits of eIF-2(PL) and eIF-2(PH)

W, x1~-3

200

116 93

68

45

31

21

14

n 4 I

350 9

s 300 - E

0 Y

- 250

200

Fraction Number

were 1.0/0.92/0.96 and 1.0/0.88/0.95, respectively, as calcu- lated from densitometric (A595) tracings of the Coomassie Brilliant Blue-stained gels shown in Fig. 2, A and B. Thus, each form of eIF-2 has one copy each of three nonidentical polypeptides. The electrophoresis of eIF-2 in system I does not always separate the PH-subunit from the y-subunit and often, due to such poor resolution, eIF-2 appears to have only two subunits or appears to be partially deficient in the @- subunit. This is illustrated in Fig. 2.4, lune 1. The molecular weights and isoelectric points of the two @-subunits differ when a mixture of eIF-2(PL) and eIF-2(PH) is subjected to isoelectric focusing in the first dimension and dodecyl sulfate- polyacrylamide gel electrophoresis in the second dimension (Fig. 2C, Table 11). The lower M, @-subunit is more acidic (PI 5.6) than the higher M , @-subunit (PI 6.0). A difference of 0.4 pH unit between the PI values of PL- and &-Subunits is also observed when the two forms of eIF-2 are subjected to two-

Rabbit Reticulocyte eIF-2 Having Two Different &Subunits 10167

4 4 I I 7 6 5 4

PH FIG. 2. Gel electrophoretic analysis of the two forms of eIF-

2. Each form of eIF-2 (2 pg) was electrophoresed in system I ( A ) or system I1 ( B ) as described under “Methods.” A , lune 1 , a mixture of eIF-2&) and ~ I F - ~ ( P H ) ; lane 2, eIF-2(bL); hne 3, eIF-2(&). In B, the eIF-2(PL) (lanes 1 , 2 ) and eIF-2(&) (lanes 3, 4 ) were incubated with [Y-~*P]ATP and 0.1 pg of casein kinase I1 as described under “Meth- ods.” Lanes 1 and 3, Coomassie Brilliant Blue R-250-stained gel; lanes 2 and 4, autoradiogram of lanes 1 and 3, respectively. For C, a mixture of eIF-2(PL) and eIF-2(&) was subjected to isoelectrofocusing (IEF) in the first dimension and dodecyl sulfate-polyacrylamide gel electro- phoresis (PAGE) in the second dimension. On the right is the sepa- ration of the polypeptides by dodecyl sulfate-polyacrylamide gel elec- trophoresis only.

dimensional electrophoresis after phosphorylation by casein kinase I1 (data not presented).

The reticulocyte lysate proteins were fractionated by two- dimensional gel electrophoresis as described under “Meth- ods.” The presence of the two forms of eIF-2 P-polypeptides in the crude lysate preparations was demonstrated by immu- noblotting using goat anti-rabbit eIF-2 (Fig. 3). These anti- bodies, which were raised against purified eIF-2, react strongly to the p-subunit and weakly to the a-subunit of eIF-2. They also react with a few other proteins which are presumably present as contaminants in the antigen preparation. The 7-

TABLE I1 eIF-2. PHIGDP binary complex and eIF-2. GTP. PSlMet-tRNAf

ternary complex formation by reticulocyte eIF-2 GEF did not bind [3H]GDP or [35S]Met-tRNAf under these assay

conditions. eIF-2&), 1.65 pmol(0.24 pg); eIF-2(PH), 2 pmol(0.28 pg); and GEF, 0.15 pg, were included in the standard filtration assays in the absence or the presence of 1 mM M e as indicated.

[3H]GDP bound [%]Met-tRNA, bound Factors added eIF-2(BL) eIF-2(BH) eIF-2(8~) ~IF-~(BH)

-M$+ +Mg+ -M$+ +M$+ -M$+ +M$+ -M$+ + M e pmol P m l

None 1.01 0.1 1.05 0.76 1.00 0.1 0.98 0.73 GEF 1.34 1.33 1.20 1.15 0.98 0.97 1.0 0.98

subunit of eIF-2 (21, 22) is very basic (PI 8.9) and is not detected in the isoelectric focusing method employed in the present studies (18).

Limited Proteolytic Degradation of the P-Subunit of eIF-2- The P-subunit of the reticulocyte eIF-2 is very susceptible to proteolytic degradation and this results in the appearance of eIF-2 with only a- and y-subunits (3). To characterize the protease sensitivity of the two forms of the @-subunit in our eIF-2 preparations, limited proteolytic digestion of eIF-2(PL) (Fig. a), ~ I F - ~ ( P H ) (Fig. 4B) , and a mixture containing both forms of eIF-2 (Fig. 4C) was carried out as described by Das et al. (3). It is evident from the results illustrated in Fig. 4 that both forms of the P-subunit are equally sensitive to protease digestion. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis was performed to characterize further the two eIF-2 P-sub- units. Papain, pancreatic protease, a-chymotrypsin, and S. aurezu V8 protease were utilized as previously described (19). The apparent M, difference between the two P-subunits was also observed between their respective peptides (Fig. 5 ) . This conclusively demonstrates that the two forms are not the product of a nonspecific proteolytic action that occurred dur- ing purification, but rather reflects the existence in vivo of both forms of eIF-2.

Characterization of the Two Forms of eIF-2-The two forms of eIF-2 differ with respect to the sensitivity to M$+ in both binary and ternary complex formation (Table 11). Upon ad- dition of 1 mM M$+, the activity of eIF-2(PL) is reduced 90% and that of eIF-2(PH) is reduced 25% in both binary and ternary complex formation. When analyzed for the content of bound GDP, eIF-2(&) and ~ I F - ~ ( P H ) contain 0.85 and 0.22 pmol of GDP per pmol of eIF-2, respectively. The affinity of eIF-2(PL) for GDP is higher (KD 2.2 X lo-’ M) than that of eIF-2(PH) (KO 6.0 X lo-’ M) as determined by Scatchard plot analysis. Therefore, the extent of M$+ inhibition is propor- tional to the GDP content of the eIF-2 preparation. The inclusion of GEF in the reaction mixture results in the rever- sal of Mg2+ inhibition of binary and ternary complex forma- tion (Table 11). In the absence of M P , GEF has no effect on ternary complex formation with either form of eIF-2. Similar amounts of the binary (eIF-2.GDP) complex are formed by both forms of eIF-2 in the absence of Mg2+ (Table 11). This binary complex is stable when 1 mM M e is included in the second stage of the nucleotide exchange reaction, and both forms of eIF-2. GDP require GEF for the exchange of bound GDP for GTP (data not presented). The addition of M e does not result in the inhibition of activity when eIF-2(PL) and eIF-2(PH) are freed of bound GDP by alkaline phospha- tase treatment (data not presented). Under these conditions, the activities of both forms are identical in binary and ternary

10168 Rabbit Reticulocyte eIF-2 Having Two Different P-Subunits

IEF PQ

81 E

FIG. 3. Immunoblot analysis of rabbit reticulocyte lysates. The retic- ulocyte lysate proteins (800 pg) were re- solved by isoelectric focusing/dodecyl sulfate-polyacrylamide gel electrophore- sis as described under “Methods,” elec- trophoretically transferred to nitrocel- lulose paper, and sequentially reacted with goat anti-eIF-2 antibodies followed by peroxidase-conjugated secondary an- tibodies. The reacting spots correspond- ing to eIF-2(8~) and eIF-2(PL) are la- beled.

A B C

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

J P H . -PI. X Y

FIG. 4. Protease digestion of eIF-2. The limited proteolytic digestion of eIF-2(PL), eIF-2(&), and a mixture of both forms was carried out as described under “Methods.” Lanes in A and B represent 2.5 pg of eIF-2(PL) and eIF-2(BH), respectively, and the lanes in C have 2.5 pg of each form of eIF-2, with 5 ng of protease in each lane. Lane I , native eIF-2; lanes 2-6, protease-treated samples incubated for 5, 10, 15, 20, and 30 min, respectively.

complex formation either in the presence or absence of M$+. These results conclusively demonstrate that the inhibition of eIF-2 activity in the presence of M$+ is due to bound GDP. The difference observed in the electrophoretic mobility of the @-subunit of the two forms of the factor is not altered when either an eIF-2. [3H]GDP binary complex is formed prior to electrophoresis or bound GDP was removed by alkaline phos- phatase treatment. Approximately 90% of the ternary com- plex formed by either e1F-2(@~) or eIF-2(/3~) is transferred to 40 S ribosomal subunits (Table 111).

DISCUSSION

We have isolated from reticulocyte lysates two forms of eIF-2 which differ with respect to their @-subunit, GDP con- tent, and sensitivity to M P . Both forms of the factor have one copy each of three nonidentical polypeptides. The a- and y-subunits of the two forms are similar but the @-subunit of eIF-2(BL) has a lower molecular weight and a more acidic isoelectric point than that of eIF-2(BH). The @-subunit of reticulocyte eIF-2 was identified unequivocally by its specific phosphorylation by casein kinase I1 (4,15,23). The difference

observed between the migration of the two @-subunit forms in one- and two-dimensional gel electrophoresis is identical whether the two eIF-2 forms were subjected to electrophoresis either before or after phosphorylation by casein kinase 11. This suggests that the observed difference in the migration and, hence, M, and PI values, is not simply due to the phosphorylation status of the @-subunit. One form of the factor, eIF-2(PL), is more negatively charged and this is con- sistent with its elution pattern from the Mono S (cation exchanger) column. The a-subunits of the two forms of eIF- 2 migrated together as a single spot on two-dimensional gel electrophoresis and the PI value of 5.1 is in agreement with earlier reports (6, 21, 22).

The technique employed in the present study to demon- strate the presence of the two forms of @-subunits of eIF-2 in the reticulocyte lysate was previously used to detect in uiuo covalent modifications of eukaryotic initiation factors (24, 25). Previous observations indicated that two forms of eIF-2 may exist (22). Isoelectric focusing of the factor in the absence of urea and separation of the polypeptides in the second dimension by dodecyl sulfate-polyacrylamide gel electropho- resis yielded two forms. The more acidic form had three

Rabbit Reticulocyte eIF-2 Having Two Different P-Subunits 10169

A

B

Papain Protease trypsm V 8 Pancre. Chymo-

H L H L H L H L H L

Pancre. Protease V 8

L H L H L H

FIG. 5. Peptide maps of the &subunit of the two forms of reticulocyte eIF-2. The &subunits of eIF-2(&) ( H ) and eIF-2(PL) ( L ) were cut out from the dodecyl sulfate-polyacrylamide gel and the limited proteolysis and the “in situ” peptide mapping was carried out as described under “Methods” by its re-electrophoresis in the absence or presence of either 50 ( A ) or 100 ( B ) ng of papain, pancreatic protease, a-chymotrypsin, or S. aureus V8 protease (V8) as indicated.

TABLE I11 40 S initiation complex formation by reticulocyte eIF-2

eIF-2(PL), 1.65 pmol (0.24 pg); eIF-2(&), 2 pmol (0.29 pg); and GEF, 0.15 pg, were included as indicated in the standard reaction mixture which contained 2 mM M$+ as described (13).

Factors [35S]Met-tRNAt bound added eIF-2(BL) eIF-Z(Bd

P W l

0.83 0.86 None 0.02 GEF

0.6

subunits and the other had only M , 52,000 and 37,000 poly- peptides (22). When the latter form of eIF-2 was subjected to conventional denaturing isoelectric focusing, three polypep- tides were revealed, suggesting that the 0- and y-subunits had not been separated earlier in the second dimension. The difference observed between the two forms was not attributed to the phosphorylation of eIF-2 but to other modifications

(21). Meyer et al. (6) also reported that the 0- and y-subunits co-migrate in the Laemmli system. These authors observed multiple forms of eIF-2, but it was unclear whether this heterogeneity existed in vivo or resulted from in uitro proteo- lytic degradation (6). Our results of the limited proteolysis of the two forms of eIF-2 (Fig. 4) or of the isolated 0-subunits (Fig. 5) clearly demonstrate that the two forms are not the product of a nonspecific proteolytic action that occurred dur- ing the purification procedure, but rather reflect the existence in uiuo of both forms of eIF-2.

At present there are conflicting reports regarding the sub- unit composition of eIF-2 (2-9). The anomalous behavior of the subunits in different gel electrophoresis systems and the failure of conventional purification techniques to separate the different forms of eIF-2 have added to this confusion. The use of FPLC column chromatography has simplified the iso- lation of the two forms of eIF-2 which differ with respect to their GDP content and sensitivity of Mg2+ in ternary and binary complex formation. eIF-2(BL) contains nearly equi- molar amounts of GDP, and in the presence of 1 mM Mg2+ the factor is 90% inhibited in binary and ternary complex formation. This inhibition is reversed by the addition of GEF, which promotes GDP displacement from eIF-2(PL). GDP. The activity of eIF-2(PH) is affected to a lesser extent by the presence of M e , since it contains only 0.2 pmol of GDP per pmol of eIF-2. The activity of this form in binary and ternary complex formation is only slightly stimulated by GEF. How- ever, in the presence of M e , GEF is required for the exchange of bound GDP for GTP with both forms of eIF-2 (data not presented). This suggests that, for the recycling of eIF-2 during polypeptide initiation, both forms have to interact with GEF. It is evident from our results that the activity of either form of eIF-2 in the presence of M e and the stimu- lation of ternary complex formation by GEF is directly pro- portional to the GDP content. Previous reports from other laboratories (16, 26) indicated that 50% of the isolated retic- ulocyte eIF-2 has bound GDP. It is interesting to note that the average KgDP value of the two forms of eIF-2 is 3.1 X lo-* M, which is identical to that previously reported (14). This suggests that both forms of eIF-2 were present in the earlier reticulocyte eIF-2 preparations that were used to determine the dissociation constant for GDP. The greater affinity of eIF-2(PL) for GDP may account for association of this nucleo- tide with the factor. In this paper we provide direct evidence for the existence of two forms of eIF-2 which differ with respect to GDP content.

The a-subunit of eIF-2 seems to serve as a regulatory subunit because its phosphorylation by the heme-controlled repressor inhibits GEF-catalyzed GDP displacement from the eIF-2. GDP binary complex (1, 27). However, the precise role of the eIF-2 subunits in ternary complex formation is not fully understood, and evidence as to which subunit is respon- sible for nucleotide binding is conflicting. In experiments with eIF-2 subunits isolated under denaturing conditions, the a- subunit was found to retain partially the ability to bind GDP (21). By affinity labeling experiments, Kurzchalia et al. (28) demonstrated that the y-subunit of eIF-2 binds GTP, but recently it was reported that only the 0-subunit may be cross- linked with azidobenzoyl-GTP (29). By analyzing the nucleo- tide and amino acid sequences of the eIF-2 subunits, Ernst et al. (30) also observed that the 0-subunit has a potential guanine nucleotide-binding site. The two forms of eIF-2 we isolated differ in the 0-subunit and GDP content, suggesting that the 0-subunit may be involved in regulating the amounts of GDP bound to eIF-2. Whether the subunits of eIF-2 dis- sociate as a consequence of its catalytic function in initiation

10170 Rabbit Reticulocyte eIF-2 Having Two Different @-Subunits

(31) and whether the P-subunit plays a role in recycling of the factor (32, 33) are important questions to be answered. The existence of the crucial intermediates for this proposed mode of recycling of eIF-2 during its catalytic activity remains to be established (34).

Recent studies (35-37) have indicated that conformational changes of proteins, due either to enzymatic covalent modi- fications (35) or ligand binding (36,37), result in a change in the apparent molecular weight as detected by dodecyl sulfate- polyacrylamide gel electrophoresis. The Ca2+-free form of the calcium vector protein was reported to be more acidic and migrates on dodecyl sulfate-polyacrylamide gel as a lower molecular weight polypeptide (37). Although in vitro GDP binding to eIF-2 does not alter its migration in dodecyl sulfate- polyacrylamide gel electrophoresis, it is possible that two forms of eIF-2 observed in the present study may represent different conformational forms during polypeptide initiation. The first step in polypeptide initiation is the transfer of eIF- 2 . GTP. Met-tRNAf ternary complex to 40 S ribosomal sub- units. Recent reports (38, 39) suggest that, upon 80 S initia- tion complex formation, GTP is hydrolyzed and eIF-2.GDP is transferred to 60 S ribosomal subunits, where it interacts with GEF. Since eIF-2 .GTP is not a stable complex, the purification of eIF-2 associated with 40 S ribosomal subunits may yield eIF-2 free of bound guanine nucleotide (eIF-2(PH). eIF-2. GDP is a stable binary complex and may represent the other form, eIF-2(PL). For its prokaryotic counterpart, the conformation of prokaryotic polypeptide initiation factor 2 changes upon binding GTP or GDP (40). The binding of GTP stimulates the binding of the initiation factor to 30 S subunits and depresses its binding to the 50 S subunits, whereas GDP binding has the opposite effect (40). A conformational differ- ence between elongation factor Tu. GTP and elongation fac- tor Tu. GDP and a similar change in the conformation of elongation factor G depending upon the nature of the bound ligand (GDP or GTP) have also been reported (41). Whether the conformation of eIF-2 is altered during initiation, either upon GTP or GDP binding or its interaction with GEF, remains to be established.

Acknowledgments-We wish to thank Charles L. Woodley for critical reading of this manuscript and Anthony Jones and Usha Verma for their excellent technical assistance.

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