Proc. Nati. Acad. Sci. USAVol. 76, No. 4, pp. 1741-1745, April 1979Biochemistry

Further studies on the mode of action of the heme-controlledtranslational inhibitor

(polypeptide chain initiation/protein phosphorylation/eukaryotic initiation factor 2 kinase/eukaryotic initiation factor 2stimulating protein/ternary complex formation)

CESAR DE HARO* AND SEVERO OCHOAtRoche Institute of Molecular Biology, Nutley, New Jersey 07110

Contributed by Severo Ochoa, February 5, 1979

ABSTRACT We have isolated [de Haro, C. & Ochoa, S.(1978) Proc. Nat. Acad Sci. USA 75, 2713-2716J a protein factor(eIF-2 stimulating protein, ESP) that is essential for formationof ternary and 40S initiation complexes by the eukaryoticpolypeptide chain initiation factor 2 (eIF-2) at the low concen-trations of eIF-2 present in reticulocyte lysates. The fact thatstimulation of complex formation by ESP is virtually abolishedwhen the small (38,000 daltons) subunit of eIF-2 is phosphoryl-ated by ATP in the presence of eIF-2 kinase (heme-controlledinhibitor, HCI) is consistent with the notion that HCI inhibitstranslation in lysates by blocking the interaction of eIF-2 withESP. Our present work, with highly purified eIF-2 and ESP, hasadditionally established that, unlike phosphorylation of thesmall- subunit, phosphorylation of the middle (52,000 daltons)subunit of eIF-2, which does not lead to translational inhibitionin lysates, does not affect eIF-2-ESP interaction. This providesfurther support for our model of translational inhibition byHC1.

Incubation of the eukaryotic polypeptide chain initiation factor2 (eIF-2) with heme-controlled inhibitor (HCI) and ATPphosphorylates its a subunit and inhibits translation (1-4). Wehave isolated (5, 6) a protein factor eIF-2 stimulating protein(ESP), that, at the low concentrations of eIF-2 in lysates, is es-sential for eIF-2 function-i.e., for formation of the ternarycomplex eIF-2-GTP-Met-tRNAj (Met-tRNAi is the initiatorspecies of eukaryotic methionyl tRNA) that precedes the as-sembly of the 40S initiation complex. ESP has no activity in theabsence of eIF-2. Phosphorylation of the eIF-2 a subunit doesnot prevent formation of ternary or 40S initiation complexes(7, 8) but abolishes the stimulatory effect of ESP (5, 6). It wouldthen appear that, at physiological concentrations of eIF-2,phosphorylation of its a subunit inhibits translation by blockingthe interaction of eIF-2 with ESP. Our earlier observations havenow been confirmed with highly purified eIF-2 and ESP.Moreover, phosphorylation of the 18 subunit of eIF-2, whichdoes not inhibit translation in lysates (9-11), does not affect theeIF-2-ESP interaction. Further experiments (12) indicate thatformation of the ternary complex is preceded by formation ofthe binary complex eIF-2-GTP. ESP stimulates binary complexformation with intact eIF-2 but not with eIF-2 whose a subunithas been phosphorylated.

MATERIALS AND METHODSAssays. eIF-2 and ESP were assayed by ternary complex

formation. Assay A. The samples (50 Al) contained the fol-lowing components added at 0°C in the order listed: 20 mMHepes at pH 7.6, 100 mM KCI, 0.5 mM Mg(OAc)2, 1 mM di-thiothreitol, and eIF-2, without or with ESP, as specified in thelegends. 35S or 3H-labeled Met-tRNAj (as specified) and 22uMThe publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

1741

GTP (GDP-free) were added last. After incubation for 6 minat 30'C, ternary complex formation was measured by Milliporefiltration (6). Assay B. Samples (30 iil) containing 33.3 mMHepes at pH 7.6,20mM KCI, 0.83 mM Mg(OAc)2, 83gM ATP,1.67mM dithiothreitol eIF-2, and, when present, ESP and eIF-2kinase as specified, were preincubated for 2 min at 30°C. Theywere then supplemented with KCI, GTP (final concentrationsas in assay A) and labeled Met-tRNAi, as specified, made up to50Sl, and incubated and processed as in assay A. The finalconcentration of ATP was 50 AM. The effect of ESP was thesame, and was similarly abolished by eIF-2 kinase, when ESPwas added after the preincubation step in assay B. eIF-2 kinasewas assayed routinely by inhibition of ternary complex for-mation (assay B); DE200 eIF-2 (see Table 1, step 2) served asa source of both eIF-2 and ESP. The # subunit of eIF-2 isphosphorylated by enzyme fractions that phosphorylate casein(10, 11); these enzyme(s) will be referred to throughout thispaper as casein kinase. The activity was assayed with casein assubstrate and ['y-32P]ATP as phosphate donor, essentially asdescribed for histone phosphorylation (13), without additionof cyclic AMP (see Table 3).

Other Methods. GDP-free GTP was used throughout. GDPhas higher affinity for eIF-2 than does GTP and is a potent in-hibitor of ternary complex formation (14-16). GTP was purifiedby chromatography on DEAE-Sepharose, essentially as de-scribed by Moffat (17). The presence of 7% GDP in commercialGTP inhibited ternary complex formation by 30%. Calf livertRNA (Boehringer Mannheim) was charged with [35S]methi-onine (New England Nuclear) or [3H]methionine (Amer-sham/Searle) by using an Escherichia coli extract (the kind giftof J. Ofengand) as a source of tRNA ligases. This procedurecharges only the initiator species of eukaryotic tRNAMet (18).[35S]Met-tRNAi of high specific radioactivity (t70,000cpm/pmol) was used in experiments with small amounts ofeIF-2 (2 pmol or less). Protein was determined spectrophoto-metrically or by the procedure of Lowry et al. (19) with bovineserum albumin as the standard.

Preparation of eIF-2 and ESP. These were prepared fromthe ribosomal salt wash of rabbit reticulocyte lysate. All oper-ations were conducted at 0-20C. Ribosomal wash from 450 mlof lysate (Pel-Freez), prepared as described (6), was concen-trated by precipitation with ammonium sulfate at 80% satu-ration. The precipitate was dissolved in 25 ml of buffer A [20

Abbreviations: Met-tRNAj, initiator species of eukaryotic methionyltRNA; eIF-2, eukaryotic initiation factor 2 [small (38,000), middle(52,000), and large (54,000) subunits of eIF-2 are referred to as a, 3,Iand y, respectivelyl; ESP, eIF-2 stimulating protein; HCI, heme-controlled inhibitor (eIF-2 kinase).* Present address: Centro de Biologia Molecular, Universidad Auto-noma de Madrid, Madrid 34, Spain.

t To whom correspondence and reprint requests should be ad-dressed.

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1742 Biochemistry: de Haro andOchoaP

Table 1. Purification of eIF-2

Yield, %Protein, Specific No With

Step Vol, ml mg Units activity* ESPt ESPt

1. High-salt wash, 80% sat. (NH4)2SO4 ppt. (120,gg) 25 1395 6188 4.4 100 1002. DEAE-Cellulose, DE200 (24, g) 14 100 7000 70 114

CM-Sephadex, CM200 (30,ug) 15 90 135 1.53. CM-Sephadex, CM350 (12,ug) 3.75 4.5 1485 330 24

CM350 (1.2,Mg) + CM200 (30 Mg) 4.5t 5400 1200 884. Phosphocellulose, PC (2,ug) 0.25 0.6 900 1500 15PC (0.2 Mg) + CM200 (30 Mg) 0.6§ 2520 4200 41

Assay A. Values in parentheses give the amount of protein actually used for assay. The following amounts of Met-tRNAi (in pmol) were usedper assay: steps 1-3, 3.5; step 4,4. At steps 3 and 4, eIF-2 was assayed both in the absence and in the presence of ESP (fraction CM200). In thelatter case the samples contained 1/10 as much eIF-2 as in the former.* Units/mg of protein (1 unit is the amount of eIF-2 causing the binding of 1 pmol of Met-tRNAj under assay A conditions).t Fraction CM200.t CM350 protein.§ PC protein.

mH Hepes, pH 7.6/0.1 mM EDTA/1 mM dithiothreitol 5%(vol/vol) glycerol] containing 80 mM KCI, and dialyzed over-night against the same buffer (Table 1, step 1). The dialyzedsolution was applied to a DEAE-cellulose (Whatman DE-52)column (1.5 X 16 cm) equilibrated with buffer A containing80 mM KCl and washed with the same buffer until A280 nmdecreased to <0.2; eIF-2 eluted together with ESP with bufferA containing 200 mM KCl (step 2). The solution was appliedto a column (0.6 X 5 cm) of CM-Sephadex C-50 (Pharmacia)equilibrated with buffer A containing 200 mM KCI. This stepseparates eIF-2 (which is retained by the column) from ESPwhich is not retained (fraction CM200). eIF-2 was eluted withbuffer A containing 350 mM KCI (step 3).The CM350 fraction was brought to 200 mM KCl and 10%

(vol/vol) glycerol with buffer A containing 16.7% (vol/vol)glycerol and applied to a phosphocellulose (Whatman P11)column (0.9 X 6 cm) equilibrated with buffer B [20mM Hepes,pH 7.6/0.1 mM EDTA/1 mM dithiothreitol/10% (vol/vol)glycerol] containing 200 mM KCI. Protein was eluted with a

linear KCI gradient from 200 to 800mM KC1 (total vol, 80 ml)and fractions (1 ml) were collected. Fractions 33-44 were

pooled and concentrated by dialysis against buffer B containing100mM KCI and 80% saturated ammonium sulfate. The pre-cipitate was dissolved in 0.25 ml of buffer B containing 100 mMKCI, dialyzed against the same buffer, and stored in small ali-quots in liquid nitrogen (step 4).ESP was purified further from the CM200 fraction (Table

2, step 3). The precipitate obtained between 25 and 55% satu-ration with ammonium sulfate was dissolved in 3 ml of bufferA containing 2 mM rather than 1 mM dithiothreitol and was

dialyzed overnight against 500 vol of the same buffer (step 4).

The step 4 protein was sedimented through a 10-30% sucrosegradient in buffer A (2 mM dithiothreitol) for 20 hr at 40,000rpm in the SW 40 rotor of the Spinco preparative ultracentri-fuge. The gradients (five tubes, each 12 ml) were fractionatedin an Isco gradient fractionator and fractions (0.4 ml) werecollected. ESP activity sedimented as a sharp, symmetric peak(Fig. 1A). Peak fractions (no. 17-20) were pooled and dialyzedfor 3 hr against buffer B containing 20 mM KCl (step 5).The dialyzed solution was applied to a phosphocellulose

(Whatman P11) column (0.6 X 4 cm) equilibrated with thesame buffer. ESP was not retained by this column (step 6). Theeffluent solution was immediately applied to a column (0.9 X5 cm) of DEAE-cellulose (Whatman DE-52) equilibrated withbuffer B containing 20 mM KCI. Protein was eluted with alinear 20-220 mM KCI gradient in buffer A (total vol, 120 ml)and fractions (1.5 ml) were collected. The peak of activityeluted at 150 mM KC1. Peak fractions were pooled and con-centrated by dialysis against 80% saturated ammonium sulfatein buffer B containing 100 mM KC1, dissolved in 0.4 ml ofbuffer B containing 50mM KC1, and dialyzed against the samebuffer (step 7).

Finally, the protein was sedimented through a 15-35%glycerol gradient in buffer B (18 hr at 50,000 rpm) in the SpincoSW-56 rotor. The gradient was fractionated as above andfractions (0.2 ml) were collected (two tubes, each 4.5-ml gra-dient). ESP activity sedimented as a sharp, fairly symmetricpeak (Fig. 1B). Peak fractions (no. 14-16) were pooled andconcentrated on a small DEAE-cellulose column equilibratedwith buffer B containing 50mM KCI and 15% (vol/vol) glyceroland eluted with a small volume of the same buffer containing200 mM KCI (step 8). ESP preparations were stored in smallaliquots in liquid nitrogen.

Table 2. Purification of ESP

SensitivityProtein, Specific to eIF-2 Yield,

Step Vol, ml mg Units activity* kinase,% %

3. CM-Sephadex, CM200 (6,Mg) 15 90 2700 30 54 1004. 25-55% sat. (NH4)2SO4 fraction (8gg) 3 75 2625 35 70 975. Sucrose gradient (6Mg) 12.5 17 1955 115 70 726. Phosphocellulose (1.3 Mg) 13 13 1950 150 87 727. DEAE-Cellulose (1.1,ug) 0.4 3.3 660 200 88 248. Glycerol gradient (0.25 Mg) 1.0 0.3 156 520 86 6

ESP cannot be assayed at steps 1 and 2 prior to separation from eIF-2. Steps 1 and 2 are the same as in Table 1. Assay B was used with 1.2,Mg of CM350 eIF-2 (see Table 1) and 2.2 pmol of Met-tRNA;. Values in parentheses give the amount of protein actually used for assay.* Units/mg of protein (1 unit is the amount ofESP promoting the eIF-2 kinase-sensitive binding of 1 pmol of Met-tRNAi under assay B conditions).

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Proc. Nati. Acad. Sci. USA 76 (1979) 1743

5 A BE 1.2- 1.5 0.6

C

0: 0.8- 1.0 0.4

.0ZZ 0.4 0.5 0.2

CD 0 O LJ L01 10 20 30 0 5 10 15 20

Fraction

0.3

E0.2 C

0q0.1

FIG. 1. Purification of ESP. (A) Surcose gradient centrifugation.(B) Glycerol gradient centrifugation. ESP assay A with 1.2 jg of eIF-2CM350, 2.2 pmol of [3H]Met-tRNAi (9000 cpm/pmol), and 3-jil ali-quots of gradient fractions. -, A28onm; 0, Met-tRNAi binding(pmol).

Preparation of Protein Kinases. Partially purified eIF-2kinase was prepared as outlined (5). Chromatography ofDEAE-cellulose peak III (20) on phosphocellulose equilibratedwith pH 7 buffer A containing 250 mM KCI separates eIF-2kinase (not retained by the column) from casein kinase (re-tained). Casein kinase was eluted with buffer containing 600mM KCI; its specific activity was about 100 pmol/min per Agof protein. Each of the two kinase preparations catalyzed thetransfer of 1 mol of 32P from [y-32P]ATP per mol of eIF-2, buteIF-2 kinase was specific for the a subunit whereas casein ki-nase was specific for the j3 (Table 3).

RESULTSProperties of eIF-2 and ESP. After separation from ESP

(Table 1, step 3), eIF-2 showed a marked decrease in activityunless assayed in the presence of ESP. Under our assay condi-tions, ESP (fraction CM200) increased 3- to 4-fold the activityof CM350 and PC eIF-2 (Table 1). On sodium dodecyl sul-fate/polyacrylamide gel electrophoresis PC eIF-2 displayedthree major bands and some minor ones (Fig. 2A). This prep-aration is estimated to be about 75% pure. The estimated mo-lecular weights of the major bands were 38,000, 52,000, and54,000, corresponding to the a, (,B and y subunits of eIF-2 (cf.ref. 7); The molecular weight of ESP, estimated from glyceroldensity gradient centrifugation analyses in the presence ofsuitable markers, was about 350,000, substantially higher thancalculated earlier (5, 6) from gel filtration data. On disc gelelectrophoresis (nondissociating), step 8 ESP migrated as a

Table 3. Phosphorylation of a and f3 subunits of eIF-2by protein kinases

32P incorporated into subunit,mol/mol eIF-2

Enzyme a ,yeIF-2 kinase 0.99 0.07 0.01Casein kinase 0.06 1.03 0.15

eIF-2 kinase samples (50 gl) contained 25 mM Hepes buffer (pH7.6), 3 mM Mg(OAc)2, 0.15 mM ['y-32P]ATP (6500 cpm/pmol), 6 jgof eIF-2 PC (30 pmol), and 6 jg of eIF-2 kinase. Casein kinase samplesdiffered by having 10 mM Mg(OAc)2, 130 mM KCl, 5 mM di-thiothreitol, and 4 jig of casein kinase. All samples were incubated for8 min at 300C and subjected to sodium dodecyl sulfate/polyacryl-amide gel electrophoresis (see legend to Fig. 2). The a, fl, and y bandsof eIF-2 were sectioned with a razor blade into 1.5-mm-thick slicesand the slices placed into scintillation vials containing 0.4 ml of 30%H202 and heated at 70°C for 3 hr; then, the radioactivity of the gelsuspensions was measured in Aquasol. The radioactivity of controlslices (about 2% of that in the a and f, bands) was subtracted from allsamples.

52;A2_"

-94

- 67

-43

-30

-20

938 - _

A B C2

FIG. 2. Polyacrylamide gel electrophoresis (21) of eIF-2 and ESP.(A and C) In 0.1% sodium dodecyl sulfate/10% acrylamide/0.26%N,N'-methylenebisacrylamide, 4 hr, 4 mA per gel lane. (B) Disc gelelectrophoresis (4% acrylamide/0.1% NN'-methylenebisacrylamide,pH 7.6,6 hr, 4 mA). Gels were stained with 0.2% Coomassie brilliantblue. (A) PC eIF-2 (6 ,jg). (B) Step 8 ESP (30 ,jg). (C) Lanes: 1, step8 ESP; 2, step 3 ESP (each, about 25 jig protein). The front-runningband in B is bromphenol blue.

single, diffuse band (Fig. 2B). On sodium dodecyl sulfate gelelectrophoresis the same preparation showed a prominent band(Mr -90,000) and several minor ones (Fig. 2C, lane 1). Al-though comparison with step 3 ESP (Fig. 2C, lane 2) indicatesconsiderable purification, step 8 ESP does not appear to be pure.The possibility that ESP may consist of four 90,000-daltonsubunits is an attractive but unproved hypothesis. It is evidentfrom Table 2 that purification of ESP removes proteins thatstimulate ternary complex formation nonspecifically. This ef-fect is distinguished from that of ESP in that it is not abolishedby incubation of eIF-2 with eIF-2 kinase and ATP.

Effect of ESP and Protein Kinases on Ternary ComplexFormation. The experiments of Table 4 were carried out withstep 8 ESP and, except for experiment 4, with PC eIF-2. Non-specific stimulation of ternary complex formation (e.g., bybovine serum albumin, experiment 1) may in large part be dueto stabilization of eIF-2, particularly at low concentrations.Experiment 1 also shows that ESP produced considerablestimulation beyond that caused by bovine serum albumin.Experiment 2, in the presence of albumin and a small amount(1 pmol) of eIF-2, may mimic conditions obtaining in lysates.Here ESP produced a 17-fold stimulation of ternary complexformation that was largely abolished (88%) by eIF-2 kinase.Experiment 3, with a large amount (11.2 pmol) of eIF-2, clearlyshows that, without ESP, eIF-2 kinase does not affect ternarycomplex formation (3.10 vs. 3.08 pmol) but largely eliminates(86%) stimulation by ESP. Finally, experiment 4 demonstratesthat, in contrast to eIF-2 kinase, reticulocyte casein kinase [thatphosphorylates the eIF-2 # but not the a subunit (cf. Table 3)]does not modify the ESP effect. This is further documented inFig. 3.

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1744 Biochemistry: de Haro and Ochoa

Table 4. Effect of ESP and protein kinases on ternary complex formation

[35S]- Met-tRNAj bound Sensi-Met- ESP eIF-2 Casein Net due tivitytRNAj, eIF-2, Albumin, (step 8), kinase, kinase, Total, to ESP to ki-pmol pmol* sg Ag Jsg Mg pmol pmol % nase, %

Exp. 11.8 2.0 (PC) 0.04

10 0.1950 0.18

2.5 0.65 0.6110 2.5 0.75 0.56

Exp. 22.0 1.0 (PC) 25 0.01

25 2.5 0.17 0.16 10025 2.0 0.0125 2.5 2.0 0.03 0.02 12 88

Exp. 38.7 11.2 (PC) 3.10

2.2 4.75 1.65 1002.0 3.08

2.2 2.0 3.32 0.24 14 86

Exp. 42.0 1.8 (CM350) 0.03

1.8 0.86 0.83 1001.5 0.03

1.8 1.5 0.13 0.10 12 881.5 0.03

1.8 1.5 0.82 0.79 95 5

Assay A was used for experiment 1; assay B was used for experiments 2 to 4.* Based on a molecular weight of 150,000 and an estimated purity (Table 1 and Fig. 2) of 75% for PC eIF-2 and 25% for CM350eIF-2.

DISCUSSIONThrough the use of highly purified eIF-2 and ESP, this studyprovides evidence that the stimulation of ternary complexformation by ESP is specific. Characteristically, the stimulatoryeffect of ESP is wiped out by phosphorylation of the eIF-2 a

.S 2 0.6

Zn

0.A

0.2

0 1 3 5 7 0 1 3 5 7Time, min

FIG. 3. Effect of ESP on Met-tRNAi binding to eIF-2 (ternarycomplex formation) as a function of time with intact eIF-2 or eIF-2in the presence of eIF-2 kinase and ATP (phosphorylation of the asubunit) (Left) or intact eIF-2 or eIF-2 in the presence of casein kinaseand ATP (phosphorylation of the iB subunit) (Right). Componentsother than GTP and Met-tRNAi were preincubated for 2 min at 301Cfollowed by addition of these reactants and further incubation at thesame temperature for the indicated times. Assay B with 1.8 pmol ofCM350 eIF-2, 40 MuM ATP, 3 pmol of [35S]Met-tRNAi (26,000cpm/pmol), and, when present, 1.8 Mg of step 5 ESP without or with2 Mg of eIF-2 kinase (Left) or 2 pmol of PC eIF-2, 40 MM ATP, 2.2pmol of 135S]Met-tRNAi (67,760 cpm/pmol), and, when present, 2.5,Mg of step 8 ESP without or with 2,ug of casein kinase (Right). (Left)*, eIF-2 without or with eIF-2 kinase; 0, eIF-2 and ESP; *, eIF-2,eIF-2 kinase, and ESP. (Right) *, eIF-2 without or with casein kinase;*, eIF-2 and ESP; *, eIF-2, casein kinase, and ESP.

subunit, but the smaller stimulation caused by other proteinsof the ribosomal salt wash, which are largely removed uponpurification of ESP, is not. Stimulation (1.5-fold) of ternarycomplex formation by Co-EIF-l (3.6,g), a protein (about20,000 daltons) purified from the high-salt wash of rabbit re-ticulocyte ribosomes (22), was also not affected by phos-phorylation of the eIF-2 a subunit and is probably nonspecific.Moreover, whereas ESP is heat-labile (data not shown), Co-EIF-1 is heat-stable. It may also be noted that we found eIF-3[Mr t700,000 (23)] to have no effect on ternary complex for-mation (data not shown). The fact that phosphorylation of theeIF-2 # subunit inhibits neither translation in lysates nor eIF-2-ESP interaction whereas phosphorylation of the a subunitinhibits both is consistent with the view that translational in-hibition by HCI is due to blocking of the eIF-2-ESP interaction.This view is further supported by the fact that, at the low levelsof eIF-2 in lysates, the factor is virtually inactive in the absenceof ESP. For a molecular weight of 150,000, 75% purity of PCeIF-2, and 41% yield, it can be calculated from Table 1 that theconcentration of eIF-2 in lysates is about 15 nM, and the samevalue has been determined by Safer et al. (24) by an isotopedilution method (cf. also ref. 5). It is clear from earlier (5, 6) andthe present work (Table 4, experiment 2) that essentially noternary complex is formed at these levels of eIF-2 unless ESPis present. Interestingly, if the purity of step 8 ESP is taken as50% and its molecular weight as 350,000, it can be calculatedfrom Table 2 that the lysate contains approximately equimolaramounts of eIF-2 and ESP.

We thank Christa Melcharick for excellent technical assistance. Wealso thank Dr. N. K. Gupta for a gift of homogeneous Co-EIF-l andDr. W. C. Merrick for a gift of approximately 80% pure eIF-3.

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