THE JOURNAL OF BIOLOGICAL CHEMISTRY

Prrnted in U.S.A. vol. 258, No. 9, Issue of May 10, pp. 5804-5810, 1983

New Initiation Factor Activity Required for Globin mRNA Translation*

(Received for publication, July 30, 1982)

Jamie A. Gri€o$, Stanley M. Taharag, Maureen A. Morgan& Aaron J. Shatkintj, and William C. Merrick From the Department of Biochemistry, Case Western Reserve Uniuersity, School of Medicine, Cleueland, Ohio 44106 and the §Roche Institute of Molecular Biology, Nutley, New Jersey 07110

A reconstituted reticulocyte translation system orig- inally designed to be deficient in eukaryotic initiation factor 4B (eIF-4B) was used to identify a new activity required for maximal synthesis of rabbit globin. This new activity purifies as a stable, high molecular weight complex by a variety of chromatographic procedures and is termed eIF-4F. The purified globin stimulatory activity also restores translation of capped mRNAs in extracts of poliovirus-infected HeLa cells. Like restor- ing activity that was obtained as a protein complex by different procedures (Tahara, S. M., Morgan, M. A. and Shatkin, A. J. (1981) J. Biol. Chem. 256, 791-794), eIF- 4F includes the 24,000-dalton cap binding protein and major polypeptides of M, -200,000 and -46,000. The latter component comigrates with eIF-4A by two-di- mensional gel electrophoresis and, like eIF-4A, chemi- cally cross-links to the 5’-end of capped mRNA by an ATP-dependent, m‘GDP-sensitive reaction. Unlike eIF- 4F, cap binding protein of M, -24,000 isolated by affin- ity chromatography on m’GDP-Sepharose does not stimulate globin synthesis in the reconstituted system.

eIF1-4B is a protein involved in binding mRNA to the 40 S ribosomal subunit. It has been partially purified as an 80,000- dalton polypeptide by several investigators (1-3). Various activities ascribed to it include cap recognition (41, mRNA discrimination ( 5 ) , a role in the poliovirus-mediated shut-off of host protein synthesis (6), and ATP-dependent binding of mRNA ( 7 ) . However, preparations of eIF-4B also contain the 24,000-dalton cap binding protein (8), and it is unclear which of the above processes are mediated by these two polypep- tides. Recently we observed that some partially purified prep- arations of eIF-4B were able to restore capped mRNA trans- lation in cell extracts made from poliovirus-infected HeLa cells. Polyacrylamide gel analyses of these eIF-4B prepara- tions indicated the presence of polypeptides similar in molec- ular weight to those comprising the large (7 S) form of CBP I1 described previously (9-11). This form of the cap binding

* This work was supported in part by Grant GM 26796 from the National Institute of General Medical Sciences. The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- rnent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. + Supported in part by Grant T32-GM-07250 from the National Institute of General Medical Sciences. This work was done in partial fulfillment of the requirements for the Ph.D. degree. .

The abbreviations used are: eIF, eukaryotic initiation factor; &pes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Na- DodSO,, sodium dodecyl sulfate; CBP I, cap binding protein of -24,@3? CBP 11, cap binding protein complex containing CBP I and other polypeptides as described.

protein possesses restoring activity in the infected cell extract while the 24,000-dalton cap binding protein (CBP I) does not (9, 10).

As part of ongoing studies of the mRNA binding step of initiation of protein synthesis, it was necessary to obtain a well-defined preparation of eIF-4B for functional characteri- zation. Procedures developed for the purification and assay of eIF-4B enabled us to resolve and identify a new initiation factor activity, termed eIF-4F, which co-purified with eIF-4B by previous isolation methods. This activity increases globin synthesis in a reconstituted system, apparently by enhancing the binding of mRNA to ribosomes. The new factor has a composition similar to CBP I1 (10) and also restores capped mRNA translation in extracts of poliovirus-infected HeLa cells, suggesting that both activities are associated with a single, multi-subunit factor.

EXPERIMENTAL PROCEDURES

Materials-Sources of most of the reagents have been described (7). In addition, Ultrogel AcA-34 was purchased from LKB Instru- ments, and agarose-hexane-adenosine-5’-triphosphate type 4 and m7GDP were from P-L Biochemicals. All other chemicals were re- agent grade.

Polyacrylamide Gel Electrophoresis-Polypeptides were analyzed in 12.5% polyacrylamide gels (ratio of acrylamide to bisacrylamide = 58.5) in the presence of NaDodSO, (12). For two-dimensional com- parative analysis of eIF-4A and eIF-4F, proteins were separated by electrophoresis in 7.5% polyacrylamide containing 6 M urea at pH 4.5, followed by 10% polyacrylamide in 0.1% NaDodSOd (13).

Globin Synthesis Assays-The basic, 50-p1 reaction mixture con- sisted of 20 mM Tris-HC1 (pH 7.5); 5 mM magnesium acetate; 100 mM KCl; 40 PM each of 18 unlabeled amino acids; 20 p~ [“Clleucine (specific activity, 312 mCi/mmol); 20 p~ [I4C]valine (specific activity, 342 mCi/mmol); 1 m~ ATP; 0.2 mM GTP; 4 mM phosphoenolpyru- vate; 0.3 IU of rabbit muscle pyruvate kinase; I mM dithiothreitol; and 0.2 Aaso units of “sucrose cushion” ribosomes (1, 14). Assay mixtures deficient in eIF-4A included the indicated amounts of phos- phocellulose fractions B and C (see Fig. 1). Assays deficient in eIF-4B and eIF-4F contained fractions A and C and 9 pg of sucrose gradient purified eIF-3. To examine globin synthesis dependence on purified factors eIF-4A, eIF-3, and eIF-4F, assays were done in the presence of fraction C. Reactions incubated a t 37 “C were terminated after 20 min by addition of 2 ml of cold 10% trichloroacetic acid, and after hydrolyzing the unused aminoacyl-tRNA, the newly synthesized pro- tein was collected on nitrocellulose filters and counted (7).

Translation of Capped mRNA in Extracts of Poliouirus-infected HeLa Cells (Restoring Actiuityl-Protein synthesis in extracts pre- pared from infected HeLa cells has been described (6, 10). Reactions of 25 gl containing -0.1 pg of Sindbis virus mRNA and 10 pCi of [35s] methionine were incubated for 60 min at 37 “C. Samples of 5 p1 were spotted on Whatman 3” filters which were boiled in 10% trichlo- roacetic acid, washed, and counted. Acetone (1 ml) was added to the remaining incubation volumes to terminate the reactions, and protein precipitates were analyzed by polyacrylamide gel electrophoresis and autoradiography.

Chemical Cross-lhking of Factors to mRNA-Reovirus mRNA containing [3H]methyl-labeled, 5”terminal m7GpppC“ (specific activ- ity, 1.5 x lo” cpm/gg) was synthesized in vitro, periodate oxidized

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and cross-linked to protein fractions by NaBH&N reduction. The resulting radiolabeled proteins were analyzed by polyacrylamide gel electrophoresis and fluorography (8, 15).

Binding of [3H]"t-tRNA,'ue' and [32P]mRNA to 40 S Subunits- Reactions were done in 100-pl mixtures containing initiation factors as indicated, 15 pmol of [%]Met-tRNA?, approximately 2 pmol of 3'-end-labeled globin r3'P]mRNA, 100 pmol of 40 S ribosomal sub- units, 0.5 nm guanosyl-5'-yl imidodiphosphate and 1 m ATP. Sam- ples incubated for 15 min at 37 "C were processed as described (14).

3'-End Labeling of Globin mRNA-Purified globin mRNA was radiolabeled with [32P]pCp as described (7) except that incubation was for 30 min at 37 "C followed by overnight incubation at 4 "C. The radiolabeled mRNA was shown to be intact by agarose gel electro- phoresis under denaturing conditions with marker ribosomal and transfer RNAs (16).

mRNA Filter Binding Assay-Incubations were carried out at 37 "C for 2 min in a total volume of 50 pl, containing 20 mM Tris-HC1, pH 7.5, 90 mM KCl, 3 mM magnesium acetate, 1 mM dithiothreitol, 3 mM phosphoenolpyruvate, 0.2 IU of pyruvate kinase and 33,000 cpm of "'P-labeled globin mRNA (7). Additions of protein factors, Mg"/ ATP, and m7GDP are indicated in the figure legends. Reactions were stopped by addition of 2 ml of ice-cold Buffer A (20 ~ l l ~ Tris-HC1, pH 7.5, 1 mM dithiothreitol, 0.1 mM EDTA, 10% glycerol) containing 2.5 mM magnesium acetate and 100 mM KC1 and immediately applied to nitrocellulose filters which were then dried and counted.

Purification of Initiation Factors-Rabbit reticulocyte polysomes (170,000 A260 units, -300 A , ~ / m l ) were incubated in the presence of 0.5 M KC1 for 30 min with gentle mechanical stirring. This procedure and all subsequent ones were carried out at 4 "C (1). The sample was centrifuged in a Spinco Ti 70 rotor a t 50,000 rpm for 90 min. Super- natants (0.5 M KC1 ribosomal wash, see Fig. 1) were pooled, dialyzed for 10 h against Buffer A-150 (Buffer A made 150 mM KCl), and divided into four aliquots of 4.3 g of protein which were fractionated separately. Protein was applied at a flow rate of 50 ml/h to a phosphocellulose column (2.5 X 38 cm) equilibrated in Buffer A-150. The unadsorbed material (containing eIF-4A) was designated fraction A (16,000 A260 units; see Fig. 1). After eluting unadsorbed material with Buffer A-150, the column was washed with Buffer A-450 (Buffer A made 450 mM KC1) to obtain fraction B (1.45 g of protein) which included eIF-3, eIF-4B, and eIF-4F. Finally, the column was washed with Buffer A-1000 (Buffer A made 1 M KC1) to elute protein con- taining eIF-2, eIF-4C, and eIF-5 (fraction C; 636 mg). Fractions A, B and C were used as starting material to obtain individual factors.

Purification of eZF-M-Fraction A (16,000 A ~ M ) units) was purified by Sephadex G-100 gel filtration followed by DEAE-cellulose chro- matography according to procedures described previously (7). The resulting protein (27.4 mg, >90% pure by polyacrylamide gel analysis), was stored at the vapor temperature of liquid nitrogen.

Separation of eZF-3, eZF-4B, and eZF-4F"Fraction B (1.45 g) was precipitated by 70% saturated ammonium sulfate, and the pellet was resuspended in Buffer A-100 without glycerol. Aliquots of 60 mg were

Tris-HCI, pH 7.5, 0.5 M KCl, 0.1 nm EDTA, and 1 mM dithiothreitol. applied to linear 5-20s sucrose gradients which contained 20 mM

Gradients were centrifuged in an SW27 rotor a t 4 "C for 22 h at 22,000 rpm. Samples of the resulting 17 fractions from each gradient were precipitated with cold 10% trichloroacetic acid and analysed by NaDodS04-polyacrylamide gel electrophoresis. Samples were pooled according to the stained gel patterns, i.e. crude eIF-3 in fractions 2-6 and crude eIF-4B containing eIF-4F in fractions 10-16 from the gradients.

The crude eIF-4B (691 mg) was fractionated in four batches by application to a Ultrogel AcA-34 column (0.8 X 99 cm) equilibrated in Buffer A-100. Fractions 15-18 contained both globin stimulatory and restoring activities and were pooled. The combined protein (132 mg) was applied to a DEAE-cellulose column (0.5 X 37.5 cm) equilibrated in Buffer A-100 and eluted with a gradient of 100-400 nm KC1 (90 ml each) at a flow rate of 6 ml/h. Fractions of 4.6 ml were collected; globin stimulatory and restoring activities eluted between 160 and 220 mM KCl. The pooled material was precipitated by 70% saturated ammonium sulfate, resuspended in Buffer A-500 and the total protein (52 mg) was re-applied to the Ultrogel AcA-34 column equilibrated in Buffer A-500. Fractions of 5 ml collected at a flow rate of 8 ml/h yielded restoring activity slightly behind the void volume (fractions 15-19, Fig. 2B) while globin synthesis stimulatory activity measured in the eIF-4B-deficient assay was also present in fractions that eluted later (fractions 20-23, Fig. 2B). Vo was determined by elution of blue dextran.

Fractions containing restoring activitv were Dooled (29.9 me) and

applied to m7GDP-Sepharose (17) equilibrated with Buffer A-100 minus glycerol but with Hepes in place of Tris-HC1 (Buffer B) in this and subsequent steps. After collecting the unbound protein, the column was eluted with 70 p~ m7GDP in the same buffer followed by Buffer B-1000. The bulk of the protein (18 mg) including the globin synthesis and restoring activities eluted in Buffer B-1000 under these conditions and was applied to an ATP-agarose column (0.5 X 19 cm) equilibrated in Buffer B-150. The column was washed with Buffer B- 150 containing 1 mM ATP before applying a gradient of 150-700 mM KC1 (50 ml each) in Buffer B. The peak of globin synthesis stimulatory and restoring activities co-eluted at 265-340 m KC1. The active fractions were pooled, concentrated with Aquacide 11, and dialysed against Buffer B-100. The yield of eIF-4F was 5.5 mg.

The eIF-4B activity in the fractions pooled from the AcA-34 column (11.8 mg) was applied to m7GDP-Sepharose. The fractions eluted in Buffer B-1000 (8.7 mg) contained eIF-4B activity but no restoring activity. After passage through ATP-agarose in Buffer B- 100, the eIF-4B (2.94 mg) was dialyzed against Buffer B-100 and stored at the vapor temperature of liquid nitrogen.

Affinity purified CBP I1 was prepared as previously described (10).

RESULTS

In order to try to define more precisely the translational function(s) of eIF-4B, we devised a new scheme for its purifi-

0.5 M KC1 WASH OF POLYSOMES

I Phosphocellulose

A B C e I F - 4 A e I F-3 e l F - 2 e l F - 4 D e l F - 4 B e l F - 4 C

e l F - 4 F e l F - 5

A Sucrose Gradient 0 . 5 M KC1

e l F - 3 e l F - 4 B (<losi (13-18s) e lF-4F

I I Ultrogel ACA-34

0.1 M KC1

DEAE -Cellulose

e l F - 4 F e l F - 4 B

1 m 7GDP- Sepharose I 1 ATP-Agarose I FIG. 1. Flow scheme for purification of eIF-4B and eIF-4F.

Ribosomal wash was batch-eluted from phosphocellulose as described under "Experimental Procedures." Fractions A. B and C remesent crude initiation factor preparations which were ais0 used in the-factor-

- Y deficient globin synthesis assay.

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FIG. 2. Gel filtration of partially purified eIF-4B. A, crude eIF-4B (173 mg) obtained after sucrose gradient cen- trifugation of fraction B (see Fig. 1) was chromatographed on a column of Ultro- gel AcA-34 in buffer containing 0.1 M KC1 (A-100). The column was eluted at a flow rate of 8 ml/h and fractions of 5 ml were collected. Column fractions were tested for stimulation of globin synthesis (10-pl samples) in assay mixtures con- taining 325 and 50 pg, respectively of phosphocellulose fractions A and C and 9 pg of eIF-3 as described under “Ex- perimental Procedures.” For restoration of Sindbis virus capped mRNA transla- tion, samples of 5 pl were assayed in extracts of poliovirus-infected HeLa cells (10). B, fractions 15 to 19 from the activ- ity profile shown in A were pooled, ap- plied to DEAE-cellulose and eluted with a KC1 gradient as described under “Experimental Procedures.” Fractions were assayed as in A above, and peak fractions were pooled and re-applied to the AcA-34 column equilibrated with 0.5 M KCI. The eluted fractions were as- sayed as in A but with 3pl samples for restoring activity. Inset, samples of the fractions obtained from the profile in B were analyzed by NaDodS04-polyacryl- amide gel electrophoresis. Gels were stained with Coomassie brilliant blue (7). Molecular weight standards in lane 23 were: phosphorylase a (94,000). bovine serum albumin (67,000). ovalbumin (43,- 0 0 0 ) . carbonic anhydrase (30,000), soy- bean trypsin inhibitor (20,100), and a- lactalbumin (14,100). Gel lanes are num- bered according to the column fraction numbers. V, void volume.

FRACTION NUMBER

cation (Fig. 1). In addition, this purification protocol resulted in the identification of a new translational activity (eIF-4F) that also restores capped mRNA function in extracts of polio virus-infected cells and appears to correspond to CBP I1 (10). Purification of these proteins involved phosphocellulose col- umn chromatography and sucrose gradient centrifugation in 0.5 M KC1 to minimize aggregation. Protein obtained from sucrose gradients was applied to Ultrogel AcA-34 in the pres- ence of 0.1 M KCl, and column fractions were tested for stimulatory activity in the eIF-4B-deficient globin synthesis assay (Fig. 2 4 ) . A major peak of eIF-4B activity eluted slightly behind the void volume at the same elution position as most

FRACTION NUMBER

of the restoring activity (measured by Sindbis virus capped mRNA translation in extracts of poliovirus-infected HeLa cells; Fig. 2 A ) . Polyacrylamide gel analysis indicated that a prominent protein with the mobility expected for eIF-4B (M, -80,000) was also present in these fractions (data not shown). Consequently, a t this stage of purification, it was not clear if the globin stimulatory and restoring activities resided in the same or in co-purifying protein(s).

Separation of eIF-4B and Restoring Activity-To resolve this question fractions 15-19 in Fig. 2A were pooled, and the proteins were purified further by DEAE-cellulose column chromatography. Both eIF-4B (as measured by globin syn-

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thesis), and restoring activity co-eluted a t 0.16-0.22 M KC1 (data not shown). These fractions were pooled and re-applied to Ultrogel AcA-34 in buffer containing 0.5 M KCl. Restoring activity again eluted slightly behind the void volume (Fig. 2B). By contrast, globin stimulatory activity eluted broadly and apparently as two peaks. Polyacrylamide gel electropho- resis indicated that eIF-4B was the main component in frac- tions 20-23 but was not detectable in fractions 15-18 (Fig. 2B, arrow). Fractions containing restoring activity and eIF-4B were separately pooled and purified further by affinity chro- matography on m’GDP-Sepharose followed by ATP-agarose. Stained polyacrylamide gel electrophoresis profiles of the purified proteins indicate that the eIF-4B preparation con- tains mainly a polypeptide of M, -80,000 (Fig. 3, lune 3). By contrast, eIF-4F contains major polypeptides with M, -24,000, 46,000, 73,000 and -200,000 (Fig. 3, lane 4). Three of these polypeptides correspond to the main components of CBP I1 when compared by electrophoretic mobility under denaturing conditions in a polyacrylamide gel; the fourth, a 73,000-dalton polypeptide (M, -55,000 a t acrylamide/bis = 37, Ref. lo), is not present in CBP 11, suggesting that it is not required for restoring activity (10). Furthermore, the 24,000-dalton poly- peptide in eIF-4F and CBP I1 cross-linked specifically to the capped 5’-end of oxidized mRNA, c o n f i i n g t h e presence of cap binding protein in both preparations (1 1, and Fig. 5 below). In addition to restoring activity, eIF-4F that was purified free of eIF-4B markedly stimulated globin synthesis in the eIF-4B deficient assay (fractions 15-18, Fig. 2B). This result prompted US to test eIF-4F for initiation factor activity in the reconsti- tuted system.

eIF-4F Requirement for Globin Synthesis-The reconsti- tuted globin synthesis assay was used to test for translational dependence on eIF-4F in the presence of an excess of eIF-4A and eIF-4B. Maximum stimulation of endogenous globin RNA translation required addition of eIF-4F under these conditions (Table I, Experiment 1). Omission of any one of the three protein preparations resulted in a 3-5-fold decrease in globin translation. Affinity purified CBP I (17) could not substitute

1 2 3 4 5 6 Mr

( x

94

67

43

30

20.1 ”

FIG. 3. Polyacrylamide gel analysis of purified initiation fac- tors. Electrophoresis was in gels containing an acrylamide to bisac- rylamide ratio of 58.5 as described under “Experimental Procedures.” Lane I , molecular weight standards as in Fig. 2; lane 2, eIF-4A (2 pg); lane 3, eIF-4B (1.3 pg); lane 4, eIF-4F (1.7 p g ) ; lane 5, eIF-3 (5.2 pg); and lane 6, eIF-2 (2 pg).

TABLE I Initiation fartor requirements for globin synthesis

Translation assays were performed as described under “Experimental Procedures” with eIF-3 (9 pg) , eIF-4A (3 pg) , eIF-4B (2.3 pg), eIF-4F (2.8 p g ) , CBP I (2 pg) or CBP I1 (2 pg) added as indicated. For Experiment 1, eIF-3 (9 pg) and phosphocellulose frac- tion C ( 5 0 pg; see Fig. 1) were included in all incubation mixtures; for Experiment 2, phosphocellulose fractions A (325 p g ) and C (50 pg) were present in each assay. Background values of 4500 cpm and 5600 cpm for Experiments 1 and 2, respectively have been subtracted.

Additions

eIF-4A eIF-4B eIF-4F CBP 1 Incorporated Maximum

cpm c.; Experiment 1

+ + + - 20,200 100

- 4,132 20 + + - 5,477 27 + + + 6,058 30

+ + +

- - 7,416 3 i - + - -

eIF-3 eIF-4B eIF-4F CBP I1

Experiment 2 + + + - 40,215 100 + + + 36,953 92

+ + - 23,092 57 11,292 28

- 1 1,455 28

- - + + +

- - - +

for eIF-4F. Furthermore, eIF-4A, eIF-4B, and eIF-4F added singly resulted in less than 4% of the maximum incorporation (data not shown). The same requirements were also observed in a similar assay system consisting of ribonuclease treated ribosomes (18) and exogenous globin mRNA (data not shown). In addition, a variation of Experiment 1 was performed with incubation mixtures containing saturating amounts of phos- phocellulose fractions A and C. Utilization of these crude fractions allowed reconstitution of an assay system that was complete with respect to eIF-2, eIF-4A, eIF-4C, eIF-4D, and eIF-5 but required the components purified from phosphocel- lulose fraction B, i.e. eIF-3, eIF-4F, and eIF-4B. In the pres- ence of eIF-3, omission of either eIF-4F or eIF-4B decreased globin synthesis by almost 4-fold (Table I, Experiment 2). The findings are indicative of independent requirements and pre- sumably different functional roles for eIF-3, eIF-4B, and eIF- 4F in cell-free globin synthesis. The ability of CBP I1 (10) to substitute for eIF-4F in this assay (Table I) provides addi- tional evidence that the two represent the same biological entity.

Initiation factor dependencies were also determined for the translation of Satellite Tobacco Necrosis Virus RNA, a natu- rally uncapped message. They were generally the same as shown in Table I for globin mRNA, i.e. incorporation of [“S] methionine into hot trichloroacetic acid-precipitable products was. diminished by 55 to 67% upon single omissions of eIF-4F, eIF-4A or eIF-4B (data not shown). Analyses by polyacryl- amide gel electrophoresis in NaDodS04 verified that these decreases reflected the relative yields of M, -22,000 putative coat protein of Satellite Tobacco Necrosis Virus. However, additional studies will be necessary to establish whether eIF- 4F stimulates the translation of other uncapped viral messages as well as capped mRNAs in the reconstituted system.

Presence of eIF-4A in eIF-4F-The polypeptide pattern and translational properties of eIF-4F isolated by the scheme shown in Fig. 1 correspond to those described previously for CBP I1 (10, 11). The predominant polypeptides of eIF-4F migrated differently from those in eIF-3 (Fig. 3, lane 5) and eIF-2 (lune 6). However, the M, 46,000 component of eIF-4F co-migrated with eIF-4A (Fig. 3, lanes 2 and 4). To examine further the possible identity of eIF-4A and the 46,000-dalton

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FIG. 4. Comparison of eIF-4F and eIF-4A by two-dimensional gel analysis. eIF-4F (11 pg) and eIF-4A (5.1 pg) were analyzed separately and as a mixture by electrophoresis for 11.5 h at 100 V in a 7.5% polyacrylamide gel containing 6 M urea, pH 4.5 (first dimension). Gel lanes were excised and cast separately into 10% polyacrylamide gels containing 0.1% NaDodS04. After electrophoresis in the second dimension for 4 h at 180 V, proteins were visualized by staining with Coomassie brilliant blue. Left, eIF-4F middle, eIF-4F + eIF-4A right, eIF-4A. The second-dimension migrations of eIF-4F and eIF-4A are also shown on the left and right sides, respectively of the middle panel.

polypeptide in eIF-4F, studies were initiated to compare di- rectly the chemical and functional properties of these proteins. By two-dimensional gel electrophoresis, the 46,000-dalton component of eIF-4F has the same apparent molecular weight and net charge as eIF-4A as determined from analyses of the individual proteins (Fig. 4, left and right) or a mixture of eIF- 4F and eIF-4A (Fig. 4, middle).

Functional similarity of the two M, 46,000 polypeptides was indicated by the ability of eIF-4F to replace eIF-4A in two model assays: ATP-dependent binding of mRNA and ATP- dependent cross-linking to the 5'-terminal m'G cap of oxidized reovirus mRNA. ATP-dependent retention of globin mRNA on nitrocellulose fiters required the presence of both eIF-4A and eIF-4B, and binding was inhibited by m'GDP, an analog of the mRNA 5'-terminal cap structure (7,9; Table 11, lines 1- 5). Purified eIF-4F also binds mRNA, but this binding, al- though inhibited by m7GDP, was not ATP-dependent (Table 11, lines 6-8). However, in the presence of eIF-4B, there was a large ATP-dependent increase in the amount of mRNA retained on fiters by eIF-4F, and this increase was sensitive to m7GDP inhibition (Table 11, lines 9-11). Thus, although eIF-4F binds mRNA in the absence of other factors, it can replace eIF-4A in the ATP-stimulated, eIF-4B-dependent binding of mRNA.

To determine if the M, 46,000 polypeptide in eIF-4F also has cross-linking characteristics similar to eIF-4A, eIF-4F was cross-linked to oxidized 'H-labeled reovirus mRNA in several different conditions. Previously it was shown that the 24,000- dalton polypeptide of CBP I1 cross-links to capped mRNA by an ATP-independent, m'GDP-inhibited reaction (1 l) , while purified eIF-4A cross-links by an ATP-dependent reaction in the presence of eIF-4B and the M , 24,000 CBP I (7,9). Only the 24,000-dalton component of eIF-4F was cross-linked to mRNA and radiolabeled in the presence of adenyl-5'-yl imi- dodiphosphate (Fig. 5, lune I ) . The same result was obtained in the absence of ATP with either eIF-4F or a mixture of CBP I, eIF-4A and eIF-4B (data not shown, Ref. 7). Cross-linking of eIF-4F in the presence of ATP yielded in addition a labeled band in the position of eIF-4A (lune 2). It was decreased 41% by 1 mM m7GDP, a cap analog concentration which com- pletely inhibited labeling of the M , 24,000 component (lune 3). Addition of eIF-4A to the ATP-dependent cross-linking of eIF-4F resulted in an augmented intensity of the 46,000-dalton band (lune 4) which was m7GDP-sensitive (73% inhibition,

TABLE I1 Protein-dependent retention of globin mRNA on nitrocellulose

filters Assay mixtures containing 33,000 cpm of ["'PImRNA and eIF-4A

(3 pg), eIF-4B (1.4 pg) or eIF-4F (0.55 pg), were analyzed as described under "Experimental Procedures." The concentrations of Mg"/ATP and m'GDP were 2 m. A background of 1,400 cpm obtained in the absence of added protein was subtracted from each value.

Additions

eIF-4A eIF-4B eIF-4F m'GDP MApT'i' mRNA Bound

c m

1. + 525 2. - + 743 3. + -I- - - - 2,157 4. + + + 7,184 5. + + + + 3,362

6. - + - - 3,315 7. - + + 3,100 8. - + + - 87 1

9. - + + - - 6,445 10. - + + + 12,306 11. - + + + + 1,558

lune 5). Radiolabeling of the 46,000-dalton component of eIF- 4F, like eIF-4A, was also increased by the addition of eIF-4B, consistent with a stimulatory effect of this factor (7) (lune 6). It was reported previously that the M , 80,000 eIF-4B cross- links to mRNA in the presence of ATP, eIF-4A, and CBP I (7). In agreement with this observation, eIF-4B cross-linked to mRNA in the presence of eIF-4F, and m'GDP inhibited the reaction (lunes 6 and 7). The similarity between these findings obtained with eIF-4F and those reported previously for eIF-4A cross-linking (7) provide further evidence that the 46,000-dalton component of eIF-4F corresponds to eIF-4A.

Other Functional Properties of eIF-4F-eIF-4F comprises a separate activity that is required for maximal hemoglobin synthesis (and Satellite Tobacco Necrosis Virus RNA trans- lation) in the reconstituted system. It was therefore of interest to determine whether eIF-4F was necessary for partial reac- tions which were previously determined to be dependent on eIF-3, eIF-4A, and/or eIF-4B. Since eIF-4F binds mRNA (Table 11), it may promote association of mRNA and ribo- somes during initiation of protein synthesis. We tested the

- - - - - - -

- - -

- - - -

-

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1 2 3 4 5 6 7 "W v 1 ---

-48

- 4A

- CBP

M$~/ATP: - + + + + + t m7GDP: - - + - + - +

FIG. 5. Cross-linking of eIF-4F to oxidized, [3H]methyl-la- beled reovirus mRNA. Purified eIF-4F (3.3 pg, lanes 1-7), eIF-4A (1.2 pg, lanes 4 and 5), and eIF-4B (2.7 pg, lanes 6 and 7) were incubated with 0.7 pg of mRNA in the presence of 3 mM magnesium acetate, 1 mM ATP (or 1 mM adenyl-5"yl imidodiphosphate in lane I ) and 1 mM m'GDP as indicated. Gel analyses and fluorography were as described in Experimental Procedures. The relative amounts of cross-linking were determined from densitometric tracings of the fluorogram.

TABLE 111 Protein requirements for binding of ["PJmRNA and ["HIMet-

tRNA?' to 40 S ribosomal subunits Assays were performed as described under "Experimental Proce-

dures" in incubation mixtures containing eIF-4A (5.0 pg), eIF-4B (4.6 pg), eIF-3 (18.4 pg), eIF-4F (5.5 pg). eIF-2 (8.0 pg) and 2 mM m'GDP as indicated. Binding was determined by sucrose gradient centrifu- gation and summing the radioactivity in the 40 S region of the gradient. Values have been corrected by subtracting 8,387 cpm of '"P and 765 cpm of "H that were obtained by gradient analyses of mixtures incubated in the absence of added factors.

Addition.. I'"PlrnRNA I"HIMet-tRNA;w"'

cpm Complete 60,746 -eIF-4A 24,680 -eIF-4B 26,448 -eIF-3 7,491 -eIF-4F 39,355 -eIF-2 23,957 +m'GDP 18,831

% binding 100 41 44 12 65 56 31

cpm % binding 26,172 100 23,932 91 21,872 84 20,435 78 29,930 114

1,268 5 23,095 88

effect of eIF-4F on 40 S initiation complex formation with 3'- end-labeled globin mRNA. Maximum mRNA binding re- quired the presence of eIF-4F in addition to the previously reported (2,3) dependence on eIF-2, eIF-3, eIF-4A, and eIF- 4B (Table 111). Binding to 40 S subunits was inhibited by the cap analog m'GDP (Table III), consistent with the functional association of protein factor(s) to the 5'-capped end of mRNA (7, Table 11). There were fewer factor requirements for 40 S- Met-tRNA? complex formation as compared to mRNA bind- ing; addition of eIF-2 was necessary but eIF-4F was not required (Table 111). Furthermore, there was little effect of

TARLE IV Effect of purified proteins on the translation of Sindbis cirus

capped mRNA in extract ofpoliovirus-infected HeLa cells Restoring activity was measured as described under "Experimental

Procedures" with eIF-2 (1.0 pg), eIF-3 (1.0 pg), eIF-4A (1.1 pg), eIF- 4B (0.9 pg), CBP I (0.9 pg), and eIF-4F (1.0 pg) added as indicated. CBP I1 and eIF-4F (0.5 pg each) were compared in a separate experiment. ['%]Methionine-labeled products were analyzed by poly- acrylamide gel electrophoresis and autoradiography. Sindbis virus capsid protein formation quantitated by densitometry is expressed in arbitrary units as described (9).

Protein added Sindbis virus Relative activ- capsid ity

arhitrary units Experiment 1

None 0 0 eIF-2 0 0 eIF-3 0 0 eIF-4A 0 0 eIF-4B 1 .o CBP I

0.04 1.3 0.05

CBP I + eIF-4A 5.2 0.2 1 CBP I + eIF-4A + eIF-4B 6.2 0.25 eIF-4F 24.6 1 .00

eIF-4F 7.5 CBP I1 10.5

Experiment 2

deleting eIF-4A, eIF-4B, or eIF-3. The Met-tRNA?" step was resistant to m'GDP inhibition, also suggesting that proteins with cap binding activity (e.g. CBP 11, eIF-4A and eIF-4B as measured by chemical cross-linking) are not required for assembly of 40 S-Met-tRNA"' complexes.

In addition to its structural similarity to CBP 11, a functional characteristic of eIF-4F is its ability to restore the translation of capped mRNAs in extracts of poliovirus-infected HeLa cells. Because eIF-4F contains eIF-4A as one of its compo- nents, attempts were made to reconstitute the "restoring activity" of eIF-4F by combining purified factors. As indicated in Table IV, only eIF-4F was highly active in the restoration assay. A mixture of CBP I and eIF-4A with or without eIF-4B yielded significant restoring activity, but on a microgram of protein basis, the level of activity was 10-fold lower than that obtained with eIF-4F. In a separate experiment, CBP I1 and eIF-4F were tested for restoring activity and found to be comparable (Table IV).

DISCUSSION

The complex process of initiation of eukaryotic protein synthesis has been partially elucidated by studies of cell-free translation systems reconstituted from purified rabbit reticu- locyte initiation factors and salt-washed ribosomes. The work reported here describes the purification of a new initiation factor that has stimulatory activity for globin synthesis and is functionally distinct from previously identified factors. I t ap- parently increases mRNA binding to ribosomes. In addition, the new activity has the capacity to restore capped mRNA translation in cell extracts prepared from poliovirus-infected HeLa cells. This property was previously shown to be associ- ated with CBP 11, a protein complex that, like eIF-4F, includes the 24,000 molecular weight cap binding protein (10). An immediate question is what are the similarities between CBP I1 and eIF-4F? Although neither factor preparation is reported as homogeneous, each contains three polypeptides of the same apparent molecular weights (Fig. 3, and Ref. lo), and both factors are similarly required for optimal translation of globin mRNA (Table I). Furthermore, both eIF-4F and CBP I1 have similar capacities to restore capped mRNA translation in poliovirus-infected HeLa cell extracts (Table IV, Ref. lo), and the 24,000-dalton peptide of each preparation can be specifi-

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5810 New Initiation Factor for Globin Synthesis

c d y cross-linked to the 5’-cap of oxidized mRNA (Fig. 5 , Ref. 11). On the basis of these considerations, it would appear that the active protein components in eIF-4F are the same as in CBP 11.

A slightly different question is whether or not the major bands of eIF-4F constitute the biologically active protein which is responsible for the number of activities that are associated with eIF-4F. Although homogeneous eIF-4F is not available, based upon several criteria, it would appear that one, or a combination of the main polypeptides of eIF-4F is responsible for the biological properties observed. This inter- pretation is based upon the following observations: 1) the microgram amounts of eIF-4F required for stimulation of globin synthesis (Table I), ATP-dependent mRNA binding (Table II), binding of mRNA to 40 S subunits (Table 111), and cross-linking to oxidized mRNA (Fig. 5, Ref. 9) are similar to the quantities of other factors (eIF-3, eIF-4A, and eIF-4B) required for comparable activity; 2) based upon the extent of cross-linking of eIF-4F to oxidized mRNA and the specific requirements for cross-linking to the 24,000- and 46,000-dalton bands of eIF-4F, these polypeptides are as active or more active than their apparent single polypeptide counterparts, CBP I and eIF-4A (9); 3) they have similar specific activities (ie. activity/pg of protein) for those assays in which eIF-4A and eIF-4F are both functional [mRNA binding (Table II), cross-linking to oxidized mRNA (9), RNA-dependent ATP hydrolysis (data not shown)]; 4) since eIF-4A and eIF-4F behave quite differently during purification by ion exchange or gel filtration chromatography, it is very unlikely that these factor preparations would share common minor contaminants which would account for the observed activities. The above arguments, while indirect, suggest strongly that the 24,000- and 46,000-dalton polypeptides are subunits of eIF-4F. The inclusion of the M , 200,000 polypeptide as part of eIF-4F is based mainly upon its co-purification with the 24,000- and 46,000-dalton peptides by different procedure (Figs. 2B, 3 and Ref. 10); correspondingly, the absence of a 73,000-dalton poly- peptide from CBP I1 (10) suggests that it is not an essential part of the active complex. The apparent high molecular weight of eIF-4F (Fig. 2B) is also consistent with a multi- component protein complex. Clearly, however, the final an- swer to the questions of assignment of biological activity to specific proteins or polypeptides will require studies using homogeneous preparations of eIF-4F.

As noted above, the subunit composition of eIF-4F is of interest with respect to the presence of eIF-4A and the 24,000 molecular weight cap binding protein (CBP I) in this complex. It is not possible to conclude on the basis of their purification properties that these two polypeptides exist in cells as mono- meric entities as well as components of eIF-4F. However, there is a recently described precedent for functional cycling of components of multimeric complexes of eukaryotic initia- tion factors. Factor eIF-2 purifies as a three subunit protein from ribosomal salt wash and is also found as a complex with five other polypeptides that together comprise the eIF-2-SP complex (19). It accounts for 10% of the total eIF-2 extractable from rabbit reticulocytes (19) and has been shown to facilitate recycling of eIF-2 during initiation of protein synthesis. Al- though the analogy to eIF-2-SP is clear, there is at present, no evidence for cycling of CBP I or eIF-4A through eIF-4F. In this regard, eIF-4A, CBP I, and a combination of both were incapable of substituting for the eIF-4F-mediated stimulation of globin translation in the factor-deficient reconstituted SYS-

tern or for restoration of capped mRNA translation in extracts of poliovirus-infected cells. Similarly, eIF-4F could not replace eIF-4A for globin mRNA translation.

The step of initiation which is least well characterized is

the factor-mediated binding of mRNA to the 40 S-Met- tRNA,Me‘ complex. This step of initiation requires eIF-4A and eIF-4B in a reaction resulting in ATP hydrolysis (2, 3 ) . Inhi- bition of the reaction by the cap analog, m7GDP suggests that it involves event(s) near or at the mRNA capped 5’-terminal sequence (7, 9). Stimulation of mRNA entry into 40 S initia- tion complexes by eIF-4F may also indicate an interaction of mRNA with the 24,000 molecular weight component (CBP I) and 46,000 molecular weight polypeptide (eIF-4A) in eIF-4F. Both polypeptides cross-link to the 5’-cap of oxidized mRNA in the presence of ATP, but the exact nature of initiation factor-mRNA interactions remains to be clarified.

Although there is uncertainty as to the differential functions of eIF-3, eIF-4A, eIF-4B, and eIF-4F, a complex process of proiein-mRNA interaction as judged by cross-linking to oxi- dized capped mRNA has been reported by Sonenberg (20). Using crude factor preparations, a number of polypeptides were identified as requiring ATP to effect specific cross-linking to mRNA. The molecular weights of the polypeptides identi- fied by gel electrophoresis in NaDodS04 were approximately the same as eIF-4A and eIF-4B. What is not clear is whether the 5’-proximal region of mRNA during translation interacts sequentially or in a concerted manner with the several initia- tion factors that bind to mRNA.

Acknowledgments-We wish to thank Dr. John Clark, Jr. and Nick Tripoulas for helpful discussion, J. S. Butler and J. Clark, Jr. for providing the Satellite Tobacco Necrosis Virus RNA, Janet Hansen for help in the preparation of the manuscript, and Carol Satler for assistance in the purification of reticulocyte initiation factors.

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