Proc. Nati. Acad. Sci. USA
Vol. 76, No. 4, pp. 1741-1745, April 1979 Biochemistry
Further studies on the mode of action of the heme-controlled translational inhibitor (polypeptide chain initiation/protein phosphorylation/eukaryotic initiation factor 2 kinase/eukaryotic initiation factor 2 stimulating protein/ternary complex formation)
CESAR DE HARO* AND SEVERO OCHOAt Roche 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 formation of ternary and 40S initiation complexes by the eukaryotic polypeptide chain initiation factor 2 (eIF-2) at the low concentrations of eIF-2 present in reticulocyte lysates. The fact that stimulation of complex formation by ESP is virtually abolished when the small (38,000 daltons) subunit of eIF-2 is phosphorylated by ATP in the presence of eIF-2 kinase (heme-controlled inhibitor, HCI) is consistent with the notion that HCI inhibits translation in lysates by blocking the interaction of eIF-2 with ESP. Our present work, with highly purified eIF-2 and ESP, has additionally established that, unlike phosphorylation of the small- subunit, phosphorylation of the middle (52,000 daltons) subunit of eIF-2, which does not lead to translational inhibition in lysates, does not affect eIF-2-ESP interaction. This provides further support for our model of translational inhibition by HC1.
Incubation of the eukaryotic polypeptide chain initiation factor 2 (eIF-2) with heme-controlled inhibitor (HCI) and ATP phosphorylates its a subunit and inhibits translation (1-4). We have isolated (5, 6) a protein factor eIF-2 stimulating protein (ESP), that, at the low concentrations of eIF-2 in lysates, is essential for eIF-2 function-i.e., for formation of the ternary complex eIF-2-GTP-Met-tRNAj (Met-tRNAi is the initiator species of eukaryotic methionyl tRNA) that precedes the assembly of the 40S initiation complex. ESP has no activity in the absence of eIF-2. Phosphorylation of the eIF-2 a subunit does not prevent formation of ternary or 40S initiation complexes (7, 8) but abolishes the stimulatory effect of ESP (5, 6). It would then appear that, at physiological concentrations of eIF-2, phosphorylation of its a subunit inhibits translation by blocking the interaction of eIF-2 with ESP. Our earlier observations have now been confirmed with highly purified eIF-2 and ESP. Moreover, phosphorylation of the 18 subunit of eIF-2, which does not inhibit translation in lysates (9-11), does not affect the eIF-2-ESP interaction. Further experiments (12) indicate that formation of the ternary complex is preceded by formation of the binary complex eIF-2-GTP. ESP stimulates binary complex formation with intact eIF-2 but not with eIF-2 whose a subunit has been phosphorylated. MATERIALS AND METHODS Assays. eIF-2 and ESP were assayed by ternary complex formation. Assay A. The samples (50 Al) contained the following components added at 0°C in the order listed: 20 mM Hepes at pH 7.6, 100 mM KCI, 0.5 mM Mg(OAc)2, 1 mM dithiothreitol, and eIF-2, without or with ESP, as specified in the legends. 35S or 3H-labeled Met-tRNAj (as specified) and 22uM
GTP (GDP-free) were added last. After incubation for 6 min at 30'C, ternary complex formation was measured by Millipore filtration (6). Assay B. Samples (30 iil) containing 33.3 mM Hepes at pH 7.6,20 mM KCI, 0.83 mM Mg(OAc)2, 83gM ATP, 1.67 mM dithiothreitol eIF-2, and, when present, ESP and eIF-2 kinase as specified, were preincubated for 2 min at 30°C. They were then supplemented with KCI, GTP (final concentrations as in assay A) and labeled Met-tRNAi, as specified, made up to 50 Sl, and incubated and processed as in assay A. The final concentration of ATP was 50 AM. The effect of ESP was the same, and was similarly abolished by eIF-2 kinase, when ESP was added after the preincubation step in assay B. eIF-2 kinase was assayed routinely by inhibition of ternary complex formation (assay B); DE200 eIF-2 (see Table 1, step 2) served as a source of both eIF-2 and ESP. The # subunit of eIF-2 is phosphorylated by enzyme fractions that phosphorylate casein (10, 11); these enzyme(s) will be referred to throughout this paper as casein kinase. The activity was assayed with casein as substrate and ['y-32P]ATP as phosphate donor, essentially as described for histone phosphorylation (13), without addition of cyclic AMP (see Table 3). Other Methods. GDP-free GTP was used throughout. GDP has higher affinity for eIF-2 than does GTP and is a potent inhibitor of ternary complex formation (14-16). GTP was purified by chromatography on DEAE-Sepharose, essentially as described by Moffat (17). The presence of 7% GDP in commercial GTP inhibited ternary complex formation by 30%. Calf liver tRNA (Boehringer Mannheim) was charged with [35S]methionine (New England Nuclear) or [3H]methionine (Amersham/Searle) by using an Escherichia coli extract (the kind gift of J. Ofengand) as a source of tRNA ligases. This procedure charges only the initiator species of eukaryotic tRNAMet (18). [35S]Met-tRNAi of high specific radioactivity (t70,000 cpm/pmol) was used in experiments with small amounts of eIF-2 (2 pmol or less). Protein was determined spectrophotometrically or by the procedure of Lowry et al. (19) with bovine serum albumin as the standard. Preparation of eIF-2 and ESP. These were prepared from the ribosomal salt wash of rabbit reticulocyte lysate. All operations were conducted at 0-20C. Ribosomal wash from 450 ml of lysate (Pel-Freez), prepared as described (6), was concentrated by precipitation with ammonium sulfate at 80% saturation. The precipitate was dissolved in 25 ml of buffer A [20 Abbreviations: Met-tRNAj, initiator species of eukaryotic methionyl tRNA; 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,I and y, respectivelyl; ESP, eIF-2 stimulating protein; HCI, hemecontrolled inhibitor (eIF-2 kinase). * Present address: Centro de Biologia Molecular, Universidad Autonoma de Madrid, Madrid 34, Spain. t To whom correspondence and reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1741
1742
Biochemistry: de Haro and OchoaPProc. Natl. Acad. Sci. USA 76 (1979) Table 1. Purification of eIF-2
Step 1. High-salt wash, 80% sat. (NH4)2SO4 ppt. (120,gg) 2. DEAE-Cellulose, DE200 (24, g) CM-Sephadex, CM200 (30,ug) 3. CM-Sephadex, CM350 (12,ug) CM350 (1.2,Mg) + CM200 (30 Mg) 4. Phosphocellulose, PC (2,ug) PC (0.2 Mg) + CM200 (30 Mg)
Vol, ml 25 14 15 3.75 0.25
Protein, mg
Units
Specific activity*
1395 100 90 4.5 4.5t 0.6 0.6§
6188 7000 135 1485 5400 900 2520
4.4 70 1.5 330 1200 1500 4200
Yield, % No With ESPt ESPt 100 114
100
24
88 15 41
Assay A. Values in parentheses give the amount of protein actually used for assay. The following amounts of Met-tRNAi (in pmol) were used per 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 the latter 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 overnight against the same buffer (Table 1, step 1). The dialyzed solution was applied to a DEAE-cellulose (Whatman DE-52) column (1.5 X 16 cm) equilibrated with buffer A containing 80 mM KCl and washed with the same buffer until A280 nm decreased to
Vol. 76, No. 4, pp. 1741-1745, April 1979 Biochemistry
Further studies on the mode of action of the heme-controlled translational inhibitor (polypeptide chain initiation/protein phosphorylation/eukaryotic initiation factor 2 kinase/eukaryotic initiation factor 2 stimulating protein/ternary complex formation)
CESAR DE HARO* AND SEVERO OCHOAt Roche 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 formation of ternary and 40S initiation complexes by the eukaryotic polypeptide chain initiation factor 2 (eIF-2) at the low concentrations of eIF-2 present in reticulocyte lysates. The fact that stimulation of complex formation by ESP is virtually abolished when the small (38,000 daltons) subunit of eIF-2 is phosphorylated by ATP in the presence of eIF-2 kinase (heme-controlled inhibitor, HCI) is consistent with the notion that HCI inhibits translation in lysates by blocking the interaction of eIF-2 with ESP. Our present work, with highly purified eIF-2 and ESP, has additionally established that, unlike phosphorylation of the small- subunit, phosphorylation of the middle (52,000 daltons) subunit of eIF-2, which does not lead to translational inhibition in lysates, does not affect eIF-2-ESP interaction. This provides further support for our model of translational inhibition by HC1.
Incubation of the eukaryotic polypeptide chain initiation factor 2 (eIF-2) with heme-controlled inhibitor (HCI) and ATP phosphorylates its a subunit and inhibits translation (1-4). We have isolated (5, 6) a protein factor eIF-2 stimulating protein (ESP), that, at the low concentrations of eIF-2 in lysates, is essential for eIF-2 function-i.e., for formation of the ternary complex eIF-2-GTP-Met-tRNAj (Met-tRNAi is the initiator species of eukaryotic methionyl tRNA) that precedes the assembly of the 40S initiation complex. ESP has no activity in the absence of eIF-2. Phosphorylation of the eIF-2 a subunit does not prevent formation of ternary or 40S initiation complexes (7, 8) but abolishes the stimulatory effect of ESP (5, 6). It would then appear that, at physiological concentrations of eIF-2, phosphorylation of its a subunit inhibits translation by blocking the interaction of eIF-2 with ESP. Our earlier observations have now been confirmed with highly purified eIF-2 and ESP. Moreover, phosphorylation of the 18 subunit of eIF-2, which does not inhibit translation in lysates (9-11), does not affect the eIF-2-ESP interaction. Further experiments (12) indicate that formation of the ternary complex is preceded by formation of the binary complex eIF-2-GTP. ESP stimulates binary complex formation with intact eIF-2 but not with eIF-2 whose a subunit has been phosphorylated. MATERIALS AND METHODS Assays. eIF-2 and ESP were assayed by ternary complex formation. Assay A. The samples (50 Al) contained the following components added at 0°C in the order listed: 20 mM Hepes at pH 7.6, 100 mM KCI, 0.5 mM Mg(OAc)2, 1 mM dithiothreitol, and eIF-2, without or with ESP, as specified in the legends. 35S or 3H-labeled Met-tRNAj (as specified) and 22uM
GTP (GDP-free) were added last. After incubation for 6 min at 30'C, ternary complex formation was measured by Millipore filtration (6). Assay B. Samples (30 iil) containing 33.3 mM Hepes at pH 7.6,20 mM KCI, 0.83 mM Mg(OAc)2, 83gM ATP, 1.67 mM dithiothreitol eIF-2, and, when present, ESP and eIF-2 kinase as specified, were preincubated for 2 min at 30°C. They were then supplemented with KCI, GTP (final concentrations as in assay A) and labeled Met-tRNAi, as specified, made up to 50 Sl, and incubated and processed as in assay A. The final concentration of ATP was 50 AM. The effect of ESP was the same, and was similarly abolished by eIF-2 kinase, when ESP was added after the preincubation step in assay B. eIF-2 kinase was assayed routinely by inhibition of ternary complex formation (assay B); DE200 eIF-2 (see Table 1, step 2) served as a source of both eIF-2 and ESP. The # subunit of eIF-2 is phosphorylated by enzyme fractions that phosphorylate casein (10, 11); these enzyme(s) will be referred to throughout this paper as casein kinase. The activity was assayed with casein as substrate and ['y-32P]ATP as phosphate donor, essentially as described for histone phosphorylation (13), without addition of cyclic AMP (see Table 3). Other Methods. GDP-free GTP was used throughout. GDP has higher affinity for eIF-2 than does GTP and is a potent inhibitor of ternary complex formation (14-16). GTP was purified by chromatography on DEAE-Sepharose, essentially as described by Moffat (17). The presence of 7% GDP in commercial GTP inhibited ternary complex formation by 30%. Calf liver tRNA (Boehringer Mannheim) was charged with [35S]methionine (New England Nuclear) or [3H]methionine (Amersham/Searle) by using an Escherichia coli extract (the kind gift of J. Ofengand) as a source of tRNA ligases. This procedure charges only the initiator species of eukaryotic tRNAMet (18). [35S]Met-tRNAi of high specific radioactivity (t70,000 cpm/pmol) was used in experiments with small amounts of eIF-2 (2 pmol or less). Protein was determined spectrophotometrically or by the procedure of Lowry et al. (19) with bovine serum albumin as the standard. Preparation of eIF-2 and ESP. These were prepared from the ribosomal salt wash of rabbit reticulocyte lysate. All operations were conducted at 0-20C. Ribosomal wash from 450 ml of lysate (Pel-Freez), prepared as described (6), was concentrated by precipitation with ammonium sulfate at 80% saturation. The precipitate was dissolved in 25 ml of buffer A [20 Abbreviations: Met-tRNAj, initiator species of eukaryotic methionyl tRNA; 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,I and y, respectivelyl; ESP, eIF-2 stimulating protein; HCI, hemecontrolled inhibitor (eIF-2 kinase). * Present address: Centro de Biologia Molecular, Universidad Autonoma de Madrid, Madrid 34, Spain. t To whom correspondence and reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1741
1742
Biochemistry: de Haro and OchoaPProc. Natl. Acad. Sci. USA 76 (1979) Table 1. Purification of eIF-2
Step 1. High-salt wash, 80% sat. (NH4)2SO4 ppt. (120,gg) 2. DEAE-Cellulose, DE200 (24, g) CM-Sephadex, CM200 (30,ug) 3. CM-Sephadex, CM350 (12,ug) CM350 (1.2,Mg) + CM200 (30 Mg) 4. Phosphocellulose, PC (2,ug) PC (0.2 Mg) + CM200 (30 Mg)
Vol, ml 25 14 15 3.75 0.25
Protein, mg
Units
Specific activity*
1395 100 90 4.5 4.5t 0.6 0.6§
6188 7000 135 1485 5400 900 2520
4.4 70 1.5 330 1200 1500 4200
Yield, % No With ESPt ESPt 100 114
100
24
88 15 41
Assay A. Values in parentheses give the amount of protein actually used for assay. The following amounts of Met-tRNAi (in pmol) were used per 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 the latter 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 overnight against the same buffer (Table 1, step 1). The dialyzed solution was applied to a DEAE-cellulose (Whatman DE-52) column (1.5 X 16 cm) equilibrated with buffer A containing 80 mM KCl and washed with the same buffer until A280 nm decreased to
