Regulation of Protein Synthesis in Rabbit Reticulocyte Lysates

Transcription

1 Eur. J. Biochem. 104, (1980) Regulation of Protein Synthesis in Rabbit Reticulocyte Lysates Phosphorylation of eif2 Does Not Inhibit Its Capacity to Recycle Rob BENNE, Marcel SALIMANS, Hans COUMANS, Hans AMESZ, and Harry 0. VOORMA Department of Molecular Cell Biology, University of Utrecht (Received June 5 /December 24, 1979) eif2, phosphorylated in the a subunit (M, = ), was isolated from rabbit reticulocyte lysates in which protein synthesis was blocked by incubation in the presence of heminregulated translational inhibitor. Such eif2 was compared to nonphosphorylated eif2 from control lysates in three assay systems: (a) ternary complex formation with GTP and MettRNAY"', (b) methionylpuromycin synthesis in a purified system and (c) rescue of a hemindeficient rabbit reticulocyte lysate. No difference between the eif2 preparations could be detected in any of these assays. Addition of 1 eif2 resulted in (a) the binding of 0.65 [3H]MettRNAYe' into a ternary complex in the first assay system, (b) the synthesis of 0.6 methionylpuromycin in the second reaction and (c) the synthesis of globin in the hemindeficient lysate, with all eif2 preparations tested. In the first two assay systems no phosphorylation or dephosphorylation of added eif2 could be detected. However, in a hemindeficient lysate added nonphosphorylated eif2 was readily phosphorylated (t1p E 2.5 min), whereas the phosphate group of added phosphorylated eif2 was rapidly exchanged (tip z 2.5 min), without significantly affecting the overall level of phosphorylation of the factor. A protein factor, antagonistic in its mode of action to the inhibitor (therefore called antiinhibitor) was isolated from the postribosomal supernatant of rabbit reticulocyte lysate [Amesz, H., Goumans, H., HaubrichMorree, T., Voorma, H. 0. and Benne, R. (1 979) Eur. J. Biochem. 98, This factor induced the synthesis of 20 mol globin/mol eif2 present in the lysate, although no reduction of the level of phosphorylation of eif2 was observed. On the contrary, addition of the factor to the lysate system resulted in an almost twofold increase of phosphate incorporation into eif2a (from 0.7 mol to about 1.2 mol phosphate/mol eif2). These observations support the idea that phosphorylation of eif2a is not the sole cause (if any) of cessation of protein synthesis in rabbit reticulocyte lysates. In recent years evidence has accumulated linking the inhibition of protein synthesis observed in hemindeficient lysates to the phosphorylation of the M, (ci) subunit of initiation factor eif2 by a CAMPindependent kinase [l61 (for a review see [7]). The kinase activity, which has been isolated in a number of laboratories [8 lo], copurifies with the protein responsible for the impairment of protein synthesis (heminregulated translational inhibitor), hence is seemed feasible that the inhibitory and phosphorylating activities are both mediated by this protein. Abbreviations. eif20(, small subunit of eukaryotic initiation factor 2; PeIF20(, phosphorylated eif2cc; inhibitor, hemeregulated translational inhibitor; antiinhibitor, a protein factor that reverses inhibition of protein synthesis in hemedeficient lysates; Hepes, 4(2hydroxyethyl)lpiperazineethanesulphonic acid. However, several observations argue against a direct causal relation between cessation of protein synthesis in a hemindeprived lysate and the phosphorylation of eif2a : (a) a decrease in eif2 activity due to the phosphorylation of the a subunit has not been demonstrated unambiguously [2, ; (b) protein synthesis in a hemindeprived lysate can be restored to its maximum level by the addition of hemin [14] or a protein factor (antiinhibitor [15]) without any decrease in the level of phosphorylation of eif2cc; (c) a substantial proportion (20 %) of eif2a remains nonphosphorylated in fully inhibited lysates [5]. These results indicate that other components besides eif2 may be involved in the blockade of protein synthesis in hemindeficient lysates. Such a suggestion, based on a large variety of evidence, has been made

2 502 Phosphorylation of eif2 by many others [2,5,7,11, (see Discussion). Therefore, it seemed worthwhile to determine whether the inability of a hemindeficient lysate to synthesize globin is, in any way, reflected in the activity of the (predominantly phosphorylated) eif2 that can be isolated from it. This paper describes experiments in which such eif2 is compared to nonphosphorylated eif2 [5,16] from control lysates in a number of reactions such as ternary complex formation with MettRNApt and GTP, methionylpuromycin synthesis and rescue of a hemindeficient lysate. No difference between these eif2 preparations could be detected, supporting the hypothesis that phosphorylation of eif2 per se does not inactivate the factor. EXPERIMENTAL PROCEDURES Chemicals [Y~~P]ATP (3000 Ci/mmol), [14C]leucine(354 Ci/ mol) and [3H]methionine (8.8 Ci/mmol) were purchased from Radiochemical Centre Amersham, England; spermine, creatine phosphate, ATP, GTP, creatine kinase, AUG and puromycin from Sigma and phosphocellulose (P11) from Whatman. Buffers Buffer A: 20 mm potassium phosphate ph 7.2, 7 mm 2mercaptoethanol and 10 (v/v) glycerol. Buffer B: 20 mm TrisHC1, ph 7.6, 7 mm 2mercaptoethanol, 100 mm KC1 and 10% (v/v) glycerol. Buffer C (sample buffer according to Laemmli [17]): M TrisHC1 ph 6.8, 5 "/, sodium dodecylsulphate, 10 (v/v) glycerol and 10 (v/v) 2mercaptoethanol. Biological Components Rabbit reticulocyte lysate and ribosomal subunits were prepared from rat liver according to Schreier and Staehelin [18]; purified initiation factors were obtained from rabbit reticulocytes as described previously [ ; [3H]methionyltRNA~e' (5000 counts min' ') was prepared according to Bose et al. [23], as modified by Stanley [24]. Inhibitor (20000 units/mg) was isolated as described by Ranu and London [8] with some minor modifications [15]. Antiinhibitor was isolated as described previously [ 151. Ternary Complex Formation This assay was performed as described by Gupta et al. [25] with some minor modifications [26]. A typical mixture of 25 p1 contained: amounts of eif2 as indicated, 5 [3H]MettRNAp and 0.5 unit crea tine kinase in 1 mm magnesium acetate, 70 mm KCl, 7 mm 2mercaptoethanol, 1 mm ATP. Mg2 +, 0.4 mm GTP. Mg2+ and 5 mm creatine phosphate. Methionylpuromycin Formation This assay was performed essentially as described previously [15]. A volume of 25 p1 contained: amounts of eif2 as indicated, 0.5 unit creatine kinase, 1 pg AUG, 10 eif3, 10 eif4c, 10 eif 4D, 3 eif5, 0.15 A260 unit 40S ribosomal subunits, 0.35 A260 unit 60S subunits and 5 [3H] MettRNA?" in 20 mm TrisHC1, ph 7.6, 0.6 mm magnesium acetate, 70 mm KCl, 7 mm 2mercaptoethanol, 100 pm spermine, 1 mm ATP. Mg2+, 0.4 mm GTP. Mg2+, 5 mm creatine phosphate and 1 mm puromycin. Protein Synthesis in a Crude Lysate [14C]Leucine incorporation in a reticulocyte lysate was performed essentially as described previously [15]. Incubation mixtures contained per 10 pl: 2.5 pl crude lysate, 0.2 unit creatine kinase and varying amounts of eif2, hemin or inhibitor as indicated in 20 mm Hepes/KOH ph 7.6, 100 mm KCl, 1 mm magnesium acetate, 1 mm dithiothreitol, 1 mm ATP, 0.4 mm GTP, 5 mm creatine phosphate, 50 pm each of 19 unlabeled amino acids and 7 pm [14C]leucine. Incubation time and mixture volume varied as indicated. Phosphorylation of Purified eif2 100 pg highly purified eif2 [20] was phosphorylated in the presence of 10 pg inhibitor, 200 pm [Y~~P]ATP (7000 counts min' '), 5 mm magnesium acetate, 20 mm TrisHC1 ph 7.6 and 100 mm KC1 essentially as described by Safer and Jagus [27]. [32P]PeIF2 was repurified by chromatography on a 2ml phosphocellulose column equilibrated in buffer A containing 300 mm KC1. After extensive washing with the same buffer, the [32P]PeIF2 was stepeluted with buffer A containing 1200 mm KCl, pooled and dialyzed against buffer B. A typical preparation yielded 50 pg [32P]PeIF2 containing approximately 0.8 mol 32P04/mol eif2a as determined on slab gels according to Laemmli [17]. The gels were dried, autoradiographed, the regions containing the phosphorylated eif2cc cut out; the radioactivity was determined after digestion for 16 h at 37 "C in 0.5 ml Luma Solve and 5 ml Lip0 Luma. Specific Activity of [y32p]atp in the Lysate System Incubation of [Y~'P]ATP in the crude lysate assay mixture results in its hydrolysis yielding ADP and 32P04. The energyregenerating system used, consisting

3 R. Benne, M. Salimans, H. Goumdns, H. Amesz, and H. 0. Voorma 503 of creatine phosphate and creatine phosphokinase, is capable of converting all generated ADP into (nonradioactive) ATP, maintaining the ATP concentration at the 1 mm level for a prolonged period of incubation (at least 90 min, not shown). Therefore, the specific activity of [y32p]atp will gradually decrease during the incubaton period, which will affect any calculation of phosphorylation levels of eif2a, provided that exchange between eif2 phosphate and ATP yphosphate groups occurs (see next section). For this reason the rate of hydrolysis of [Y~'P]ATP in the lysate system was determined by analysis of the assay mixtures on ionexchange thinlayer chromatographs. The results are shown in Table 1. It is clear that, particularly during the early stages of incubation, rapid hydrolysis of [p3'pjatp occurred, resulting in decrease of its specific activity (tip z 20 min). These results were taken into account when phosphorylation levels of eif2a were calculated (see next section). Phosphorylation of eif2cr in a Lysate 10pl mixtures containing 2.5 p1 lysate and all of the components required for protein synthesis, as described above, were incubated at 25 "C in the presence of varying amounts of [p3'p]atp, hemin, eif2 or inhibitor as indicated. The mixtures were analyzed on slab gels and the radioactivity in eif2cr was determined as described above. The level of phosphorylation of eif2a was calculated taking the decrease of the specific activity of [Y~'P]ATP(see Table 1) during the incubation period into account. Two assumptions were made. (a) The concentration of eif2 in the lysate is 150 nm (see below and [7,12,30]). (b) The specific activity of the [32P]phosphate of ATP approaches that of ["PJPeIF2a at any point during the incubation, which will be the case if the rate of phosphate exchange betweeen PeIF2cr and ATP is high compared to the rate of ATP hydrolysis. Indeed, the measured rate of exchange (see Fig. 4, tlp z 2.5 min) was sufficiently high to keep the error introduced well within the experimental accuracy. Partial Purfication of eif2 from Rabbit Reticulocyte Lysate Three different types of lysate were prepared: lysate A, no incubation, no addition; lysate B, incubated for 60 min at 25 "C under conditions optimal for protein synthesis as described above in the presence of 10 pm hemin; lysate C, as B but without hemin in the presence of 40 pg purified inhibitor. After the incubation period the salt concentration of the 1ml lysates was raised to 300 mm KC1 and 0.5 pg (10000 Table 1. Hydrolysis of [Y~~PIATP during incubation in thc c.urtkc lysate [Y~'P]ATP(1 pci) was added to the lysate system composed as described in the text. The mixtures were incubated for varying periods of time as indicated, after which proteins and nucleic acids were removed by precipitation with 5 % HC pl of each supernatant fraction was analyzed by ionexchange thinlayer chromatography as described by Sy and Lipmann [28] with 3.3 M ammonium formate, 4.2% boric acid ph 7.0 as developing solution. The positions of [Y~~PIATP and free [32P]phosphate(s) were determined by autoradiography. The spots were cut out and counted. The extent of ATP hydrolysis at any point during incubation was calculated as the amount of 3zP present in ATP as a percentage of total 32P (i.e. in ATP and phosphate). Additions: in expt I, none; expt 11, 0.2 pg inhibitor; expt 111, 2.4 pg antiinhibitor Time min % Hydrolysis of [Y~~PIATP in expt I I counts/min) [Me'4C]eIF2 was added, which was prepared according to Ottesen and Svensson [29] as described previously [30]. The mixtures were applied onto 2ml columns of phosphocellulose equilibrated in buffer A containing 300 mm KCl and the eif2 was isolated as described under the section 'Phosphorylation of Purified eif2'. Under these conditions no inhibitor or antiinhibitor, but all of the eif2, was adsorbed to the resin. A typical preparation yielded approximately 150 pg protein containing 17 pg eif2/ ml lysate, as judged from gel analysis of the phosphocellulose fractions with known amounts of purified eif2 as standard. 75% of the [Me14CJeIF2 was recovered under these conditions, suggesting a total amount of 23 pg eif2/ml lysate, which results in a concentration in the lysate of about 150 nm, a value in good agreement with literature values [7,12,30] and with the [3HJMettRNAp binding capacity of these fractions, since 1 of eif2 from the phosphocellulose fractions is capable of binding approximately 0.7 [3H]MettRNAye' (see Table 2 and [26]). RESULTS Phosphorylation of eif2a and Inhibition of Protein Synthesis by HeminRegulated Translational Inhibitor eif2 occurs predominantly in the nonphosphorylated form in lysates of rabbit reticulocytes (not shown) [5,16] and remains nonphosphorylated

4 504 Phosphorylation of eif2 during incubation of such lysates in the presence of hemin [5,9,15]. However, when purified heminregulated translational inhibitor is added instead of hemin, the M, subunit of eif2 (eif2a) becomes readily phosphorylated, an event coupled with a complete inhibition of protein synthesis [5,8,15]. At present, little information is available on the rate of phosphorylation as related to the onset of the inhibition, although some indication exists that a direct correlation of the two processes is lacking [7,14]. Since one of our objectives was to isolate maximally phosphorylated eif2 from fully inhibited lysates, we first performed an analysis of the kinetics of both phosphorylation and inhibition. The results of such an experiment are shown in Fig. 1. A small, but consistent, rate difference between the two processes existed : the inhibition of protein synthesis by the inhibitor became apparent only after 5 min of incubation and reached a maximum value (= 100% inhibition) after 10 min, indicating that the blockade of protein synthesis is a rather abrupt process. Phosphorylation of eif24 however, occurred more gradually and did not result in stoichiometric incorporation of phosphate into eif2a: % of the eif2 present in the lysate was phosphorylated under these conditions, which is in good agreement with literature values [5]. Addition of more inhibitor did not increase further either the extent or the rate of the phosphorylation, suggesting that the eif2 present in the lysate was maximally phosphorylated under these conditions. Activity of eif2 from Incubated and Nonincubated Lysates In order to correlate the activity of eif2 and the phosphorylation level of its a subunit to the ability of the reticulocyte lysate to synthesize globin, a partial purification was performed of eif2 from three lysates : (A) nonincubated, (B) incubated in the presence of hemin and (C) incubated in the presence of saturating amounts of inhibitor. Lysates A and B are active in globin synthesis and contain (predominantly) nonphosphorylated eif2 (not shown), whereas in lysate C protein synthesis has been completely blocked and eif2a is phosphorylated for % [5,9,14,16] (see previous section and Fig. 1). The purification procedure, which caused no decrease in the level of 32P attached to eif24 is described under Experimental Procedures. The different eif2 preparations were assayed in several reactions : (a) ternary complex formation with GTP and MettRNAp and methionylpuromycin synthesis in a purified system; (b) rescue of a hemindeficient rabbit reticulocyte lysate. Ternary Complex Formation and Methionylpuromycin Synthesis. The eif2 preparations from the ; 100.$.6 60 : :. t 40.._ 0._ c i." H 6 H o a ' I 0 r l " " I Fig. 1. Phosphorylation ojelf2a in the lysate (11) and inhibition of' protein synthesis by the heminregulated inhibitor (I). (I) Incubation mixtures of 10 p1 containing all of the components required for protein synthesis as described under Experimental Procedures were incubated at 25 C and the rate of incorporation of [14C]leucine was determined either in the presence or absence of 0.2 pg (4 units) of purified inhibitor. Both curves were corrected for the incorporation obtained in the presence of 1 pm edeine (which completely blockes initiation of protein synthesis but does not affect elongation [31]) and the percentage of inhibition of the incorporation rate by the inhibitor was calculated (00).(11) Experiment I was repeated replacing ['4C]leucine by unlabeled leucine and adding 20 pci [Y~'P]ATP to the mixtures. The reaction was stopped by the addition of 25 p1 buffer C and the mixtures were applied onto a 10 "/, polyacrylamide slab gel. Gel analysis was performed according to Laemmli [I71 as described previously [15]. After staining and destaining, the gels were dried and exposed to Kodak XR1 films for 2 days. The autoradiograms are shown on the right, the arrow indicates the position of eif2cc. The region of the gels containing elf2cc was cut out, digested in LipoLuma and LumaSolve as described previously [I31 and counted for 32P. Phosphorylation levels of eif2cc were calculated as described under Experimental Procedures and given as the molar ratio of phosphate per eif ( three different lysates were tested for their ability to form ternary complexes with MettRNAp' and GTP. The results are shown in Table 2. It is clear that the capacity of eif2 to form ternary complexes is not affected by a prolonged incubation of the lysate with inhibitor : approximately 0.65 mol MettRNA?'/mol eif2 added was retained on the filters with all eif2 samples tested. The same activity is displayed by routinely prepared, highly purified factor [26]. The activity of these preparations in the formation of methionylpuromycin was also tested (Table 3). The synthesis of methionylpuromycin also did not depend on the treatment of the lysate from which the eif2 was isolated : approximately 0.6 mol methionylpuromycin was formed/mol eif2 added. Rescue of a HeminDeficient Lysate. The outcome of the experiments of the previous section are in line with the observations of Trachsel and Staehelin who obtained similar results with highly purified eif2 phosphorylated in vitro [ll]. In order to explain these results, it was postulated that phosphorylation of

5 R. Benne, M. Salimans, H. Goumans, H. Amesz, and H. 0. Voorma 505 Table 2. Activity of eif2 from different lysates in ternary complex formation eif2 from different lysates was assayed for activity in ternary complex formation with [3H]MettRNAr" and GTP as described under Experimental Procedures Lysate eif2 [3H]MettRNA!''e' MettRNA? bound/ added bound eif2 added A B C Table 3. Activity of eif2 from different lysates in methionylpuromycin synthesis eif2 from different lysates was assayed for activity in methionylpuromycin (MetPur) synthesis as described under Experimental Procedures. A background of 0.1 methionylpuromycin, obtained in the absence of eif2, has been subtracted Lysate eif2 added MetPur formed MetPur formed/ eif2 added A B C eif2cc impairs the capability of the factor to recycle, i.e. to participiate in another round of initiation [11,30]. The results of Fig.1 seem to support this assumption. Unfortunately, however, no assay system is available in which recycling of eif2 can be tested directly, since stimulation of hemindeficient lysates by eif2 does not seem to result in the synthesis of more than 1 mol globin/mol eif2 added [15,30,32]. The dependence of protein synthesis in a hemindeficient lysate on the addition of highly purified eif2 is shown in Fig.2. Indeed, large amounts of eif2 (> 5 ) induced the synthesis of approximately 1 mol globin/mol eif2 added. At low concentrations (< 1 ), however, the added eif2 was capable of initiating at least three chains of globin. Under these conditions a limited recycling of factor seems to occur. Therefore, small amounts of the different eif2 preparations were tested in this assay. The results are shown in Table 4. Obviously, no difference in stimulatory activity existed between the various eif2 preparations (experiment A), not even during the first few minutes of incubation (experiment B). Irrespective of its origin, the eif2 induced the synthesis of approximately 3 mol globin/mol eif2 added, in good agreement with the stimulatory activity of highly purified factor (Fig. 2). Apparently, treatment of eif2 in the lysate with inhibitor does not inactivate the factor per se, since eif2 isolated from an inactive lysate stimulates protein synthesis in a hemindeficient lysate to the same extent as eif2 isolated from active lysates. Fate of the Phosphate from Phosphoryluted eif2u in the Various Assay Systems A valid comparison of the activity of phosphorylated and nonphosphorylated eif2 requires a check on the fate of the phosphate group of PeIF2% in the various assay systems. Therefore, the reactions were performed with [32PfPeIF2, prepared as described Table 4. Activity of eif2 preparations in a hemedeficient lysate Experiment A. eif2 (1.8 from lysate A, 1.4 from lysate B and 1.3 from lysate C) was added to 5Opl incubation mixtures containing 12.5 pl lysate and other components needed for protein synthesis, in the absence of hemin (see Experimental Procedures). Incorporation of ['4C]leucine into protein was determined in 5pl aliquots of these mixtures and of a control mixture to which no eif2 was added. The values given in the table are those obtained after 60 min of incubation after which no significant further increase of the incorporation leyel occurred. The stimulation (S) of globin synthesis induced by added eif2 was calculated as described in the legend to Fig.2. The ratio S/eIF2 is given in the table Experiment B. 2.1 eif2 from lysate A and 2.6 eif2 from lysate C was added to 100pl incubation mixtures, which were preincubated for 30 min at 25 "C. At the times indicated 15pI aliquots were taken and ['4C]leucine incorporation into globin was determined as described for experiment A. Values obtained in a control lysate (3.3 ) were subtracted and the stimulation (S) of globin synthesis induced by added eif2 determined as described for experiment A Experiment A : Nonpreincuhated lysate Lysate Globin Stimulation (S) S/eIF2 formed by added eif A B C 1.oo Experiment B: Preincuhated lysate Incubation eif2 from lysate A eif2 from lysate C time S S/eIF2 S S/eIF2 min

6 506 Phosphorylation of eif2 under Experimental Procedures and the amount of 32P present in PeIF2a was determined during the incubation period normally used in these assays. The results are shown in Fig. 3. It is clear that during the methionylpuromycin synthesis (Fig. 3 A) and ternary complex formation (not shown), no dephosphorylation of the PeIF2a took place. However, in the lysate system (Fig.3B) a rapid loss of 32P from [3zP]PeIF2a occurred (t~p z 2.5 min) in good agreement with the results of Safer and Jagus [27] who reported an even faster rate of 'dephosphorylation' (tljz = 20 s) elf2 added () Fig. 2. Rescue by eif2 uf a hemindeji'cienf lysate. 20.~1 incubation mixtures (see Experimental Procedures) were incubated for 90 min at 25 "C in the absence of hemin with varying amounts of highly purified eif2 (prepared as described in [20]). The reaction was stopped with 5 trichloroacetic acid and [14C]leucine incorporation determined as described under Experimental Procedures (00). 1 globin (= 15 leucine) contains approximately 2400 counts I4C/min, as calculated from isotope dilution experiments (not shown). The value obtained without added eif2 was subtracted from those obtained in its presence and the ratio of globin synthesis induced/eif2 added was calculated (OO) The Level of Phosphorylution of eif2a in the Lysate System The loss of [32P]phosphate from [32P]PeIF2a does not necessarily imply a decrease in phosphorylation level of the factor but might simply be explained by an exchange reaction of labeled and unlabeled phosphate groups. In order to test this possibility, lysates were preincubated with nonradioactive ATP in the presence of inhibitor under the conditions described in Fig. 1, which resulted in rapid phosphorylation of eif2a. Following the preincubation, [y"pi ATP was added either with or without nonradioactive PeIF2 and analysis of the kinetics of the appearance of phosphate on eif2a was performed. The results are shown in Fig.4. It is clear from Fig.4(11) that the incorporation of phosphate into eif2a reached the same level as was observed in Fig. 1, which showed phosphate incorporation into nonphosphorylated eif2a in a lysate without any preincubation. Therefore, a rapid exchange must have occurred between the unlabeled phosphate group incorporated into A B 34 " Fig. 3. Dephosphorylation of [32P] PeIF2u. (A) Methionylpuromycin synthesis. 5 [3ZP]PeIF2c( counts/min), prepared as described under Experimental Procedures, was incubated in the assay for methionylpuromycin synthesis as described under Experimental Procedures. After 20 min the reaction was stopped by the addition of 25 pl buffer C and "P in the eif2cc subunit was determined both on autoradiogram (shown on the right, A) a din scintillation liquid (shown on the left, A) as described in the legend to Fig. I. In the control experiment ('0 min') buffer C was added prior to the [32P]PeIF2. (B) Hemedeficient preincubated lysate. 10pl incubation mixtures, containing 2.5 pl each of crude lysate and all of the other components as described under Experimental Procedures except for the replacement of [14C]leucine by unlabele&@cine, were incubated in the absence of hemin for 30 min at 25 "C. At t = 30 min, 0.7 [3ZP]PeIF2 (5000 counts/min) was added and the incubation was continued for the time indicated. The reaction was stopped by the addition of 25 pi buffer C and gel analysis performed as described in the legend to Fig. 1. The radioactivity is shown on the left (B), the autoradiogram on the right (B). The arrow indicates the position on the gel of eif2u

7 R. Benne, M. Salimans, H. Goumans, H. Amesz, and H. 0. Voorma 507 elf2a during the preincubation period and the [p32p]p group of the ATP, added after 30min of preincubation. The exchange was complete within 5 min, approximately the time needed for the disappearance of [32P]phosphate from [32P]PeIF2 shown in Fig.3. Apparently the kinase and phosphatase activities act in a closely coupled reaction. lo 2o I II Fig. 4. E.diange on PrIF2a of unlabeled phosphate by [3ZP]phosphatr incubation mixtures containing 5 pl lysate were preincubated for 30 min in the absence of hemin as described in the legend to Fig.3. Then, 10 pci [Y~'P]ATP was added with (I) or without (11) 1 nonradioactive PeIF2 prepared essentially as described for [32P]PeIF2 under Experimental Procedures. The reaction was continued for the times indicated in the figure, stopped with 25 pl buffer C and analyzed on gels. The gels were processed as described in the legends to Fig. 1 and 3. Phosphate incorporation in eif2a is shown on the left, the autoradiograms on the right. The arrow indicates the position of eif2ct The addition of 1 nonradioactive PeIF2 (Fig. 4,I) or nonphosphorylated eif2 (not shown) resulted in an approximate 1.7fold increase of the amount of 32P in eif2a whereas the amount of eif2 present was raised 2.3fold, if one assumes a concentration for eif2 in the lysate of 150 /ml (see Experimental Procedures). These values resulted in the curves of Fig. 4, which suggest a comparable level of phosphorylation of endogenous and added eif2. The results of Table 4 (experiment B) indicate that eif2, when added to the preincubated hemindeprived lysate system, is capable of initiating the synthesis of three chains globin/mol. The phosphorylation (or phosphate exchange) occurring after the addition of eif2 (Fig. 4) reaches maximum levels before the first globin chain is completed, suggesting that phosphorylated eif2 is able to recycle at least twice. Eflect of Antiinhibitor on Protein Synthesis and Phosphorylution of eif2a The results presented so far indicate that phosphorylated eif2 is capable of reinitiating at least once (Table 2 and 3) or twice (Table 4). Experiments in which hemin 1141 or a protein factor called antiinhibitor [15] are added to a hemindeficient preincubated lysate show no decrease in the phosphorylation level of eif2a, although globin synthesis is restored to its normal rate. In such systems the (phosphorylated) factor is able to initiate a much larger number of globin chains. Fig. 5 depicts the analysis of the kinetics of globin synthesis and eif2a phosphorylation in the lysate both in the absence and presence of antiinhibitor. It is obvious that addition of antiinhibitor to the A B E ti 0.9 a, c. 1.2 p 0.6 E A m b a c n " Time (rnin) II Time (rnin) Fig. 5. Stimulation of protein synthesis in the lysate by antiinhibitor and phosphorylation of eif2ct. (A) 10 p1 incubation mixtures containing 2.5 PI crude lysate were incubated at 25 "C for the times indicated without hemin in the presence of all of the other components required for protein synthesis (see Experimental Procedures) either without (I, w0) or with (11, 0) 2.4 pg purified antiinhibitor [15]. The incubation was stopped with 5 % trichloroacetic acid and incorporation determined as described (Experimental Procedures [15]).(B) The experiment described under A was repeated in the presence of 20 pci [Y~*P]ATP, replacing [14C]leucine by unlabeled leucine. Phosphorylation of the eif2a subunit was determined as described in the legend to Fig. 1. Phosphate incorporation is shown on the left, the autoradiogram on the right. The arrow indicates the position of eif2a. (I) Without antiinhibitor (M); (11) with antiinhibitor (0) I

8 508 Phosphorylation of eif2 lysate results in a pronounced stimulation of globin synthesis, particularly at later stages of the incubation (Fig. 5A). The stimulation of protein synthesis was not coupled, however, with a decrease of eif2% phosphorylation : phosphorylation of eif2a proceeded at a higher rate and reached higher levels upon the addition of antiinhibitor : the molar ratio of phosphate/eif2 was raised from 0.7 to 1.2 (Fig. 5 B). These results, although puzzling (see Discussion), suggest that phosphorylation per se does not impair the ability of the factor to participate in more than one round of initiation, since in the presence of antiinhibitor, eif2 recycles at least 19 times (if one assumes an eif2 concentration in the lysate of 150 nm, see Experimental Procedures). DISCUSSION eif2a in a hemedeficient, inhibitorsupplemented lysate is readily phosphorylated (til2 = 2.5 min), whereas the inhibitory effect of added inhibitor on protein synthesis does not become apparent before 7 8 min of incubation (Fig. 1). This observation, also reported by others [14,31], could be explained by the assumption of Cherbas and London [31] that eif2 in a hemindeficient lysate is used stoichiometrically and is inactivated by the inhibitor after one round of initiation. However, several lines of evidence argue against this explanation, as follows. a) The transit time for the completion of a globin chain has been estimated to be less than 1 min [31], suggesting that several rounds of initiation might occur with phosphorylated eif2 (see also [7]). b) No difference could be detected between phosphorylated and nonphosphorylated eif2 when tested in model reactions with eif2 isolated from inhibitorsupplemented lysates and from control lysates (Tables 2 and 3), suggesting that lysates inactivated by the inhibitor still contain active (though phosphorylated) eif2. c) In hemindeficient lysates, in which all added eif2 is rapidly phosphorylated (Fig. 4), the addition of eif2 results in the initiation of at least 3 mol globin/mol eif2 added (Table 4). d) When protein synthesis is measured in the presence of antiinhibitor a large number of globin chains is initiated by phosphorylated eif2 (Fig. 5), approximately the same number of chains as is initiated by eif2 present in a normal heminsupplemented lysate in which the factor remains predominantly in the nonphosphorylated form [5,9,14,15]. Apparently, phosphorylation of initiation factor 2 is not the only prerequisite (if at all) for the cessation of protein synthesis. Presumably, other components are modified by the inhibitor during the period of time preceding the appearance of its inhibitory effect on protein synthesis in a lysate. Such components, obviously, did not copurify with eif2 on phosphocellulose (see Experimental Procedures) since the resulting preparations were active in all assay systems tested. At present, the nature and/or mode of action of these components remains unclear. It has been postulated that, besides eif2, the 40S ribosomal subunit [2], other (protein) factors [14,15,34] or MettRNA? deacylase [35] are involved in the inhibitormediated inhibition of protein synthesis. The hypothesis that a difference between phosphorylated and nonphosphorylated eif2 can only be measured in the presence of cofactors for ternary complex formation such as eif2stimulatory protein [36] is not in line with the results of Table 2, in which both forms of eif2 are highly (> 60%) active in this reaction. We feel, however, that extreme care should be taken in interpreting any stimulation of ternary complex formation by other proteins, when the reaction is performed with minute quantities of highly purified eif2. The observed stimulation may, in great part, be due to an enhanced recovery of the factor on the nitrocellulose filters and not to an increased affinity of eif2 for MettRNA (see [26]). The rate of phosphate exchange on phosphorylated eif2a is extremely rapid (Fig. 4); therefore the possibility still exists that a temporary dephosphorylation of the factor could take place before each initiation event. However, the stimulation of protein synthesis by antiinhibitor cannot be explained via such a mechanism, since no effect by this factor on the exchange reaction could be measured (not shown). The stimulation of eif2a phosphorylation by antiinhibitor is a puzzling result, since purified eif2 is not phosphorylated by this factor [15]. Antiinhibitor, though, can be isolated from the rabbit reticulocyte lysate complexed with eif2 [15]. It is feasable that complex formation between eif2 and antiinhibitor enhances the accessibility of the phosphorylation site or induces a second one. Indeed, incorporation of more than one phosphate per eif2 molecule has been reported [14]. The physiological significance of the (supposed) second site remains to be elucidated. We thank Thea HaubrichMorree and Marcelle Kasperaitis for skilful technical assistance. This work was supported by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) with financial support from the Netherlands Foundation for Chemical Research (SON). REFERENCES 1. Levin, D. H., Ranu, R. S., Ernst, V. & London, I. M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, Kramer, G., Cimadevilla, M. & Hardesty, B. (1976) Proc. Nut1 Acad. Sci. U.S.A. 73,

9 R. Benne, M. Salimans, H. Goumans, H. Amesz, and H. 0. Voorma Gross, M. & Mendelewski, J. (1977) Biochem. Biophys. Res. Commun. 74, Farrell, P. J., Balkow, J., Hunt, T., Jackson, R. J. & Trachsel, H. (1977) Cell, 11, Farrell, P. J., Hunt, T. & Jackson, R. J. (1978) Eur. J. Biochem. 89, Balkow, K., Mizuno, S., Fisher, J. M. & Rabinovitz, M. (1973) Biochim. Biophys. Acta, 324, Safer, B. &Anderson, W. F. (1978) CRC Crit. Rev. Biochem. 5, Ranu, R. S. & London, I. M. (1976) Proc. Nut1 Acad. Sci. U.S.A. 73, Traugh, J. A,, Tahara, S. M., Sharp, S. B., Lundak, T. S., Safer, B. & Merrick, W. C. (1978) Proc. Nutl Acad. Sci. U.S.A. 75, Trachsel, H., Ranu, R. S. & London, I. M. (1978) Proc. Nutl Acad. Sci. U.S.A. 75, Trachsel, H. & Staehelin, T. (1978) Proc. Natl Acud. Sci. U.S.A. 75, Safer, B., Lloyd, M., Jagus, R. & Kemper, W. (1978) Fed. Proc. 37, deharo, C., Datta, A. & Ochoa, S. (1978) Proc. Nutl Acad. Sci. U.S.A. 75, Lloyd, M. (1978) Dissertation, Bethesda, U.S.A. 15. Amesz, H., Goumans, H., HaubrichMorree, T., Voorma, H. 0. & Benne, R. (1979) Eur. J. Biochem. 98, Benne, R., Edman, J., Traut, R. R. & Hershey, J. W. B. (1978) Proc. Nutl Acad. Sci. U.S.A. 75, Laemmli, U. K. (1970) Nature (Lond.) 227, Schreier, M. H. & Staehelin, T. (1973) J. Mol. Biol. 73, Benne, R. & Hershey, J. W. B. (1976) Proc. Nut1 Acad. Sci. U.S.A. 73, Benne, R., Wong, C., Luedi, M. & Hershey, J. W. B. (1976) J. Biol. Chem. 251, Benne, R., Luedi, M. & Hershey, J. W. B. (1977) J. Bid. Chem. 252, Benne, R., BrownLuedi, M. L. & Hershey, J. W. B. (1978) J. Bid. Chem. 253, Bose, K. K., Chaterjee, N. K. & Gupta, N. K. (1974) Methods Enzymol. 29, Stanley, W. M., Jr (1974) Methods Enzymol. 29, Gupta, N. K., Woodley, C. L., Chen, Y. C. & Bose, K. K. (1973) J. Bid. Chem. 248, Benne, R., Amesz, H., Hershey, J. W. B. & Voorma, H. 0. (1979) J. Biol. Chem. 254, Safer, B. & Jagus, R. (1979) Proc. Nut1 Acad. Sci. U.S.A. 76, Sy, J. & Lipmann, F. (1973) Proc. Natl Acad. Sci. U.S.A. 70, Ottesen, M. & Svensson, B. J. (1974) C.R. Trav. Lab. Carlsberg, 38, Benne, R., BrownLuedi, M. & Hershey, J. W. B. (1979) Methods Enzymol. 60, Cherbas, L. & London, I. M. (1976) Proc. Natl Acad. Sci. U.S.A. 73, Safer, B., Kemper, W. & Jagus, R. (1978) J. Biol. Chem. 253, Safer, B., Peterson, D. & Merrick, W. C. (1977) in Translation of Syntheticu and Natural Polynucleotides (Legocki, A. B., ed.) pp , University of Agriculture, Poznan, Poland. 34. Ranu, R. S. & London, I. M. (1979) Proc. Nut1 Acad. Sci. U.S.A. 76, Gros, M. (1979) J. Biol. Chem. 254, Ochoa, S. (1979) Eur. J. Cell Bid. 19, R. Benne, M. Salimans, H. Goumans, H. Amesz, and H. 0. Voorma Vakgroep Moleculaire Celbiologie, Rijksuniversiteit Utrecht, Padualaan 8, NL3584CH Utrecht, The Netherlands