The proteolytic cleavage of eukaryotic initiation factor (eIF) 4G is prevented by eIF4E binding protein (PHAS-I; 4E-BP1) in the reticulocyte lysate
1997; Springer Nature; Volume: 16; Issue: 4 Linguagem: Inglês
10.1093/emboj/16.4.844
ISSN1460-2075
Autores Tópico(s)Plant Virus Research Studies
ResumoArticle15 February 1997free access The proteolytic cleavage of eukaryotic initiation factor (eIF) 4G is prevented by eIF4E binding protein (PHAS-I; 4E-BP1) in the reticulocyte lysate Theophile Ohlmann Theophile Ohlmann Department of Biochemistry, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG UK Search for more papers by this author Virginia M. Pain Virginia M. Pain Department of Biochemistry, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG UK Search for more papers by this author Wendy Wood Wendy Wood Department of Biochemistry, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG UK Search for more papers by this author Michael Rau Michael Rau Department of Biochemistry, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG UK Search for more papers by this author Simon J. Morley Corresponding Author Simon J. Morley Department of Biochemistry, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG UK Search for more papers by this author Theophile Ohlmann Theophile Ohlmann Department of Biochemistry, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG UK Search for more papers by this author Virginia M. Pain Virginia M. Pain Department of Biochemistry, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG UK Search for more papers by this author Wendy Wood Wendy Wood Department of Biochemistry, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG UK Search for more papers by this author Michael Rau Michael Rau Department of Biochemistry, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG UK Search for more papers by this author Simon J. Morley Corresponding Author Simon J. Morley Department of Biochemistry, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG UK Search for more papers by this author Author Information Theophile Ohlmann1, Virginia M. Pain1, Wendy Wood1, Michael Rau1 and Simon J. Morley 1 1Department of Biochemistry, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG UK The EMBO Journal (1997)16:844-855https://doi.org/10.1093/emboj/16.4.844 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A common feature of viral infection is the subversion of the host cell machinery towards the preferential translation of viral products. In some instances, this is partly mediated by the expression of virally encoded proteases which lead to the cleavage of initiation factor eIF4G. The foot-and-mouth disease virus encodes two forms of a cysteine proteinase (L protease) which bisects the eIF4G polypeptide into an N-terminal fragment containing the eIF4E binding site, and a C-terminal fragment which contains binding sites for eIF4A and eIF3 and which associates with the 40S ribosomal subunit. Previously, we have demonstrated that the cleavage of eIF4G by L protease stimulates the translation of uncapped transcripts encoding cellular proteins and supports internal initiation driven by picornavirus internal ribosome entry segment (IRES) elements. Use of reticulocyte lysates manipulated to deplete them of eIF4E and the N-terminal fragment suggests that the C-terminal fragment of eIF4G is responsible for these effects, and we have now confirmed this by purifying the C-terminal fragment and analysing its effects directly in the absence of L protease. Interestingly, we find that pre-incubation of reticulocyte lysates or ribosomal salt wash fractions with the specific eIF4E binding protein, PHAS-I (eIF4E-BP1), blocks the proteolytic cleavage of eIF4G by L protease. This effect can be reversed by addition of recombinant eIF4E. These data are consistent with a model whereby the L protease cleavage site in eIF4G is inaccessible until a change in conformation is induced by the binding of eIF4E. This may have implications for a role for eIF4E binding in triggering changes that expose other domains in the eIF4G molecule during initiation of translation. Introduction The control of polypeptide synthesis plays an important role in cell proliferation, with physiological regulation of protein synthesis almost always exerted at the level of polypeptide chain initiation. Cap-dependent initiation of translation involves the assembly of initiation factors at the 5′ end of mRNA, including the cap-binding protein, eIF4E, the ATP-dependent RNA helicase, eIF4A, and the eIF4G polypeptide to form the eIF4F complex (reviewed in Hershey, 1989; Merrick, 1992; Rhoads, 1993; Morley, 1994, 1996; Pain, 1996). There is a strong requirement for eIF4E and eIF4G in cap structure-dependent initiation, and the eIF4F complex is believed to promote the unwinding of mRNA secondary structure to facilitate the binding of the 40S ribosomal subunit (reviewed by Morley, 1994, 1996; Pain 1996). Consistent with its proposed regulatory role, eIF4E exists in both phosphorylated and non-phosphorylated forms, and is believed to be regulated both by phosphorylation and the availability of the factor to enter the eIF4F complex. It is now becoming clear that eIF4E can interact specifically with either eIF4G or a new family of regulatory binding proteins (such as PHAS-I, also known as 4E-BP1; Lin et al., 1994; Pause et al., 1994a; Haghighat et al., 1995; Mader et al., 1995; Morley and Pain, 1995; Rau et al., 1996). The amino acid sequence in eIF4G which binds to eIF4E is similar to the sequence in PHAS-I which binds to eIF4E (Haghighat et al., 1995; Mader et al., 1995). Hence, PHAS-I acts to sequester eIF4E and prevent its interaction with eIF4G and the formation of the eIF4F complex (Lin et al., 1994; Pause et al., 1994a; Haghighat et al., 1995; Mader et al., 1995; Azpiazu et al., 1996; Beretta et al., 1996; Kimball et al., 1996; Mendez et al., 1996; Whalen et al., 1996). Recent work has shed light on the potential role of eIF4G in translation initiation (Lamphear et al., 1995; Mader et al., 1995). These studies have identified an eIF4E binding motif on eIF4G in the N-terminal domain, and likely sites of interaction with eIF4A and eIF3 in the C-terminal domain. Thus, eIF4G seems to mediate joining of the mRNA and ribosomes by interaction with both the cap-binding protein, eIF4E, and with eIF3 aready associated with the ribosome. eIF4A is believed to catalyse the unwinding of upstream secondary structures and is recycled through the eIF4F complex during successive rounds of initiation (Pause et al., 1994b). When eIF4G is cleaved by viral proteases such as 2A of poliovirus, coxsackie virus and human rhinovirus (Etchison et al., 1982; Liebig et al., 1993) or the leader (L) protease of foot-and-mouth disease virus (FMDV) (Devaney et al., 1988; Belsham and Brangwyn, 1990; Ohlmann et al., 1995), translation of capped mRNAs is disrupted, but that of picornavirus RNA is maintained (Etchison et al., 1982; Devaney et al., 1988). Picornavirus RNAs are naturally uncapped and possess highly structured sequences [called internal ribosome entry segments (IRESes)] within their 5′ untranslated regions (5′ UTRs) which direct 40S ribosomal subunits to bind to internal elements rather than at the extreme 5′ end (reviewed by Jackson et al., 1990, 1994, 1995). mRNAs possessing an IRES element in the 5′ UTR can still be translated when the eIF4G subunit is proteolytically cleaved (Liebig et al., 1993; Ohlmann et al., 1995, 1996; Ziegler et al., 1995) and several reports have suggested that proteolytic cleavage of eIF4G, rather than merely discouraging translation of competing cellular mRNAs, may exert a positive role in promoting internal initiation on picornavirus RNAs in infected cells (Buckley and Ehrenfeld, 1987; Hambidge and Sarnow, 1992; Scheper et al., 1992; Macadam et al., 1994; Pause et al., 1994b; Lamphear et al., 1995). More direct studies with in vitro systems have demonstrated that cleavage of eIF4G enhances translation driven by enterovirus or rhinovirus IRES elements (Liebig et al., 1993; Ziegler et al., 1995), while translation of coding sequences downstream of a cardiovirus IRES element was not affected (Thomas et al., 1992; Ohlmann et al., 1995; Ziegler et al., 1995). Recently, we (Ohlmann et al., 1996) and others (Ziegler et al., 1995) have demonstrated that in in vitro translation systems the cleavage of eIF4G by FMDV L protease stimulates the translation of uncapped transcripts encoding cellular proteins, as well as supporting internal initiation driven by picornavirus IRES elements. Use of reticulocyte lysates manipulated to deplete them of eIF4E and the N-terminal domain of eIF4G suggests that the C-terminal domain of eIF4G is responsible for these effects (Ohlmann et al., 1996). Curiously, we also found that translation of uncapped cellular mRNAs was inhibited by addition of PHAS-I, an effect prevented by prior treatment of the translation system with L protease (Ohlmann et al., 1996). In order to confirm that these effects of L protease were due to generation of the C-terminal fragment, we have now purified this fragment of eIF4G and demonstrated that it stimulates the translation of uncapped mRNA when added directly to the reticulocyte lysate. In addition, we show that the action of L protease in cleaving eIF4G requires the interaction of eIF4E with eIF4G. Cleavage of eIF4G by L protease is prevented by pre-incubation of reticulocyte lysate or ribosomal salt wash with exogenously added PHAS-I, an effect reversed upon addition of excess recombinant eIF4E protein. Results The C-terminal domain of eIF4G stimulates the translation of uncapped cyclin mRNA We showed previously that cleavage of eIF4G in reticulocyte lysates by the FMDV Lb protease exerted little effect on initiation driven by a TMEV IRES and was actually stimulatory for translation of an uncapped transcript encoding a cellular mRNA (Ohlmann et al., 1995, 1996). Moreover, data from experiments employing modified lysate translation systems strongly suggested that the C-terminal cleavage product of eIF4G could support translation of uncapped or IRES-driven mRNAs under conditions where eIF4E and the N-terminal domain of eIF4G were severely depleted (Ohlmann et al., 1996). The first aim of the present work, therefore, was to demonstrate a direct effect of the C-terminal fragment on translation and, accordingly, we attempted to isolate this product. In the native reticulocyte lysate (not treated with micrococcal nuclease), most of the eIF4G is ribosome associated (Rau et al., 1996). We therefore incubated a 0.5 M KCl wash from reticulocyte ribosomes (see Materials and methods) with L protease for 15 min, then added 300 μM elastatinal to inhibit further protease action (Ohlmann et al., 1995, 1996; Ziegler et al., 1995) and subjected the material to m7GTP–Sepharose affinity chromatography. The affinity resin absorbs virtually all the eIF4E, together with the N-terminal cleavage product of eIF4G, which includes the eIF4E binding site (Lamphear et al., 1995; Mader et al., 1995), whereas the C-terminal cleavage product is recovered in the run-through (Ohlmann et al., 1996). The run-through fraction was then applied to an FPLC Mono-Q column, which was developed with a 50–600 mM KCl gradient in Buffer B. Figure 1A shows the protein elution profile from Mono-Q. Fractions were examined directly by SDS–PAGE and Western immunoblotting (Figure 1B), using antibodies recognizing eIF4E, eIF4A and the C-terminal half of eIF4G, as described previously (Ohlmann et al., 1996). The elution profile of the C-terminal fragment was quite distinct from that of the residual intact eIF4G which eluted at higher salt. Although there was considerable overlap in the elution profiles of the C-terminal fragment and eIF4A, the bulk of eIF4A eluted in fractions 9 and 10. There is an eIF4A binding site in the C-terminal domain of eIF4G (Lamphear et al., 1995), but it seems unlikely that the elution similarity was simply due to association between these two proteins since the peaks do not superimpose completely (Figure 1B). Each fraction, following concentration, was tested for its ability to influence the translation of capped and uncapped transcripts encoding Xenopus cyclin A. As shown in Figure 1C, the peak of stimulatory activity coincided exactly with that of the C-terminal fragment and eluted later than the peak of eIF4A. However, it should be noted that the peak fractions concentrated for use in the experiments to be described, and denoted ‘Ct’, contained significant amounts of eIF4A as well as the C-terminal domain of eIF4G. They were, however, free of eIF4E and the N-terminal domain of eIF4G. This preparation of the C-terminal fragment is referred to as ‘Ct’ in the remainder of this paper. Figure 1.Partial purification of the C-terminal domain of eIF4G. (A) Reticulocyte lysate ribosomal HSW was prepared as described in Materials and methods. An aliquot (3.5 ml) was treated with L protease and subjected to m7GTP–Sepharose as described and the unbound material applied to a FPLC Mono Q column equilibrated in Buffer B. Bound proteins were resolved with a 50–600 mM NaCl gradient in the same buffer and 1 ml fractions collected. The resulting A254 profile is presented. (B) Samples (10 μl of 1 ml fractions) were resolved by SDS–PAGE (7.5% acrylamide), proteins transferred to PVDF and visualized by immunoblotting with antisera recognizing: upper panel, the C-terminal part of eIF4G; middle panel, eIF4A; lower panel, eIF4E. HSW refers to the starting material prior to cleavage with L protease and HSW+L that following cleavage. RT shows the material which was not retained by the m7GTP–Sepharose column and was the fraction applied to the Mono Q column. (C) Following concentration and re-equilibration in Buffer A, samples (1 μl) were assayed for their ability to influence the translation of capped (hatched bars) or uncapped cyclin mRNA (filled bars) in the MDL translation system (10 μl), as described. Incubations were carried out in the presence of 300 μM elastatinal and were for 45 min at 30°C. Samples (2 μl) were then processed as described to determine the level of [35S]methionine incorporation into protein (Ohlmann et al., 1996). With the uncapped transcript, incorporation by the same lysate in the absence of L protease treatment was 27 698 c.p.m./2 μl, and following L protease treatment was 97 322 c.p.m./2 μl. These data are representative of those obtained in at least three separate experiments. Download figure Download PowerPoint Figure 2 shows the effect of adding different amounts of Ct prepared in this way to translation systems (in the presence of 300 μM elastatinal) based on native (RRL) and messenger-dependent (MDL) reticulocyte lysates. The addition of Ct did not affect the translation of endogenous globin mRNA in the RRL (Figure 2A) and only slightly stimulated translation of the influenza virus NS′ coding sequence driven by the TMEV IRES (JODA 1099; Hunt et al., 1993) in the MDL (Figure 2B). However, translation of uncapped cyclin A mRNA was enhanced in the presence of additional Ct in a dose-dependent manner (Figure 2C). This provides evidence for a direct stimulatory effect of Ct on the translation of uncapped mRNA, observable in the presence of intact eIF4G and in the absence of L protease. The result shown in Figure 2C was obtained with three separate preparations of Ct; in each case the stimulatory activity, though evident following one freeze–thaw cycle, was most pronounced when the fresh preparation was used (see the legend to Figure 1). In the remaining experiments shown in this paper, Ct was added to translation systems at 1 μl/10 μl assay. The immunoblots shown in Figure 2D (and Figure 4B) illustrate that this amount of added Ct is similar to the levels of endogenous eIF4G in the system. The samples analysed in Figure 2D were removed from the incubation mixtures at the end of the experiment, and the results (lanes 3 and 6) indicate that addition of the Ct preparation did not result in proteolysis of the endogenous eIF4G. Figure 2.Fractions enriched for the C-terminal domain of eIF4G stimulate the translation of uncapped mRNA in the reticulocyte lysate in the absence of L protease. Translation assays (10 μl) based on the reticulocyte lysate (A) or MDL (B and C) were set up as described in Materials and methods, in the presence of 300 μM elastatinal and in the absence of (A) or presence of 25 μg/ml uncapped influenza virus NS′ driven by the TMEV IRES sequence [JODA 1099; (B)] or uncapped cyclin A (pXLcycA1) mRNA (C) and supplemented with Buffer A or Ct as indicated. Aliquots (2 μl) were removed after incubation for 45 min at 30°C and processed to measure incorporation of [35S]methionine into protein. These data are representative of those obtained on at least three separate occasions using different reticulocyte lysates and preparations of Ct. (D) Translation mixes either in the absence or presence of 1 μl Ct were resolved by SDS–PAGE (7.5% acrylamide), proteins transferred to PVDF and visualized by immunoblotting with antiserum recognizing the C-terminal domain of eIF4G. Lanes 1–3 and 4–6 represent analysis of two separate preparations of Ct in two different lysates; the migration of the intact eIF4G and the C-terminal product is indicated. These data are representative of those obtained in at least three separate experiments. Download figure Download PowerPoint Previously, we found that the ability of MDL systems to translate either capped or uncapped cellular mRNAs and, to a lesser extent, IRES-driven cistrons, was impaired if the systems were pre-treated with m7GTP–Sepharose to deplete them of most of the endogenous eIF4E (Ohlmann et al., 1996). A possible explanation for this effect is that translation of these mRNAs is sensitive to partial depletion of eIF4G, some of which is removed concomitantly with eIF4E on the affinity resin. In support of this possibility, we found that the inhibitory effect on translation of uncapped or IRES-bearing mRNAs was prevented by inducing cleavage of eIF4G in the lysate with L protease prior to the treatment with m7GTP–Sepharose. In the doubly treated lysate, eIF4E and the N-terminal fragment of eIF4G were both reduced to very low levels, but virtually all the C-terminal fragment generated by eIF4G cleavage was retained. The maintenance of full translational activity for uncapped and IRES-bearing mRNAs in such a lysate thus suggested that the requirement for eIF4G could be met by an equivalent molar concentration of the free C-terminal domain (Ohlmann et al., 1996). We have now tested this directly by addition of partially purified Ct to m7GTP–Sepharose-treated lysates. Figure 3A shows an immunoblot analysis of the lysates used in such an experiment. Treatment of an MDL with m7GTP–Sepharose affinity resin resulted in the loss of nearly all the eIF4E, and concomitantly removed ∼40% of the eIF4G (compare lanes 1 and 2), which was recovered following elution of the resin with m7GTP (lanes 3 and 4). The ability of the depleted and control lysates to translate uncapped cyclin mRNA and NS′ driven by the TMEV IRES in the presence and absence of 1 μl additional free Ct is shown in Figure 3B and C. In each case, prior treatment of the lysate with m7GTP–Sepharose decreased translation, as observed previously (compare 1 and 3, and 5 and 7). Addition of Ct stimulated that of uncapped cyclin mRNA (Figure 3B) and restored the translation of the IRES-driven mRNA to the level seen in lysates treated with uncoupled Sepharose resin (Figure 3C). Similar data (not shown) have been obtained with at least two different lysates and three preparations of Ct, although there were minor differences between them as to whether or not the addition of Ct completely equalized the translation in m7GTP–Sepharose and control Sepharose-treated samples (2 versus 4, and 6 versus 8). Figure 3.Fractions enriched for the C-terminal domain of eIF4G are sufficient to promote translation of uncapped and IRES-driven translation in an eIF4E-depleted MDL. (A) Reticulocyte MDL (100 μl) was subjected to batch absorption with m7GTP–Sepharose (1 and 3) or Sepharose 4B (2 and 4) as described and the unbound lysate fraction retained. The resins were washed with 4 vols of Buffer A and bound proteins eluted with 150 μl SDS–PAGE sample buffer. Aliquots (1.5 μl) of the unbound fraction or eluted fractions (10 μl) were resolved by SDS–PAGE, proteins transferred to PVDF and visualized by immunoblotting with antisera recognizing eIF4G (upper panel) or eIF4E (lower panel). (B) and (C) Uncapped versions of cyclin A (B) or TMEV IRES-containing mRNA (C) were translated in assays based on the reticulocyte lysate (10 μl) containing 300 μM elastatinal and subjected to the pre-treatments described above, in the presence of 1 μl Buffer A (1 and 3) or 1 μl Ct (2 and 4) as indicated. Following incubation for 45 min at 30°C, aliquots (2 μl) were removed to measure the incorporation of [35S]methionine into protein. These data are representative of those obtained in at least three separate experiments. Download figure Download PowerPoint Figure 4.Pre-incubation of MDL with PHAS-I prevents the stimulation of translation uncapped cyclin A mRNA by L protease. (A) Translation assays (10 μl) based on the reticulocyte lysate (MDL) were carried out for 45 min at 30°C in the presence of Buffer A (1, 4, 7 and 10), 1 μl L protease (2, 5, 8, 11) or 1 μl Ct (3, 6, 9 and 12) with addition of 300 μM elastatinal. Incubations were conducted as follows: condition (i), in the absence of PHAS-I (1–3); condition (ii), with PHAS-I added at the same time as the other components (4–6); condition (iii), PHAS-I added 10 min after the start of the translation assay (7–9); condition (iv), PHAS-I added during a 10 min pre-incubation at 30°C prior to the addition of L protease or Ct (10–12). Aliquots (2 μl) were removed after 45 min further incubation at 30°C and processed to measure incorporation of [35S]methionine into protein. These data are representative of those obtained on at least three separate occasions using different preparations of Ct. (B) At the end of the translation reaction, samples (2 μl) from assays 4–6 and 10–12 were resolved by SDS–PAGE (7.5% acrylamide), proteins transferred to PVDF and visualized by immunoblotting with antisera recognizing the C-terminal domain of eIF4G; the migration of the intact eIF4G and C-terminal product is indicated. These data are representative of those obtained in at least three separate experiments. Download figure Download PowerPoint Addition of PHAS-I prevents the action of L protease in the reticulocyte lysate An additional way of depleting translation systems of functional eIF4E, without the complication of concomitant removal of variable amounts of eIF4G, is to add the specific eIF4E binding protein, PHAS-I or 4E-BP1 (Lin et al., 1994; Pause et al., 1994a). Previously, we used this approach to demonstrate that translation of uncapped transcripts encoding cyclin A or cyclin B2 was very sensitive to eIF4E depletion, whereas internal initiation directed by an IRES element was unaffected (Ohlmann et al., 1996). Interestingly, however, the inhibitory effect of PHAS-I was prevented by prior treatment of the lysate with L protease. To examine more directly the role of the C-terminal domain of eIF4G in this response, we tested the effect of adding PHAS-I, Ct and L protease to lysates in different combinations on the translation of uncapped cyclin A mRNA (Figure 4A). Low levels of L protease were employed in these studies to limit proteolysis of eIF4G to one primary cleavage event, separating the N- and C-terminal domains. The first set of data [condition (i)] shows control levels of translation of this mRNA and the stimulation resulting from the addition of either L protease or Ct at zero time. The apparently greater effectiveness of Ct relative to L protease treatment in stimulating translation probably reflects the fact that these components were added at the beginning of, rather than prior to, the period of translation measurement. There would, therefore, have been a lag period before C-terminal fragments generated by the low levels of L protease employed in these experiments would have accumulated and exerted their full effect. In condition (ii), PHAS-I was added at zero time in the absence or presence of these components. Under these conditions, PHAS-I alone inhibited the translation of uncapped cyclin A mRNA by 70%; these data confirmed the inhibitory effect of the binding protein, and demonstrated that Ct added directly was able to prevent it. Essentially similar results were obtained [condition (iii)] if PHAS-I was added after a 10 min incubation with L protease or Ct. However, the most interesting result was obtained under condition (iv), where PHAS-I was added at zero time, followed by either L protease or Ct at 10 min. In this case, complete rescue was still achieved by addition of Ct, whereas the stimulatory effect of L protease was completely abrogated. To investigate the basis of this result, we compared the effectiveness of L protease in cleaving eIF4G when added to the lysate either at the same time as [condition (ii)] or 10 min after [condition (iv)] the addition of PHAS-I. The immunoblot in Figure 4B clearly demonstrates that cleavage of eIF4G occurred when L protease was added at the same time as PHAS-I (lane 5), but was severely inhibited when the lysate was pre-incubated for 10 min with the binding protein (lane 11). Since PHAS-I is generally regarded as acting specifically to sequester eIF4E and withhold it from interacting with eIF4G, this raised the interesting possibility that the susceptibility of eIF4G to cleavage by L protease was in some way dependent on the integrity of the eIF4F complex. Inhibition of L protease-induced cleavage of eIF4G by PHAS-I is reversed by eIF4E The simplest explanation of our findings is that eIF4G is only vulnerable to cleavage by L protease when eIF4E is bound to it. A possible mechanism for this would invoke a model where the binding of eIF4E induced a change in conformation of eIF4G that led to the exposure of the cleavage site (see Discussion). Sequestration of eIF4E by the binding protein would therefore shift most of the eIF4G into the protected conformation. To test this model, we examined the ability of exogenously added eIF4E to facilitate cleavage of eIF4G in MDL pre-incubated with PHAS-I. In the experiment shown in Figure 5, samples of lysate were incubated with different amounts of PHAS-I (0.03–1 μg) for 10 min, followed by the addition of L protease. Cleavage of eIF4G was monitored by immunoblotting after a further 20 min of incubation. A protective effect of PHAS-I was seen at all concentrations used (lanes 3–7). In a duplicate set of incubations, recombinant wild-type eIF4E (0.25 μg) was added 10 min after L protease and the state of eIF4G examined 10 min later (lanes 8–12). It is clear that the subsequent addition of eIF4E overcame the protective effect of the lowest concentrations of PHAS-I (compare lanes 8 and 9 with 3 and 4), but was ineffective when amounts of PHAS-I were in excess. These data are consistent with the possibility that the binding of eIF4E to eIF4G facilitates the cleavage of the latter by L protease in the untreated reticulocyte lysate. Figure 5.The inhibition of cleavage of eIF4G by PHAS-I in the presence of L protease is reversed by the addition of wild-type eIF4E. Samples of unsupplemented MDL (7 μl) were pre-incubated with 1 μl Buffer A (lane 1) or increasing concentrations of PHAS-I (1 μl containing 0.03, 0.06, 0.2, 0.4 or 1 μg; lanes 3–7 and 8–12, respectively) for 10 min at 30°C. L protease was added (1 μl) where indicated and the incubation continued for another 10 min. At this stage, either Buffer A (1 μl; lanes 1–7) or recombinant wild-type eIF4E (1 μl, 0.25 μg; lanes 8–12) was added. Lane 10 contains approximately a 1:1 ratio of added PHAS-I:eIF4E. Following a further 10 min incubation, samples (1 μl) were resolved by SDS–PAGE, proteins transferred to PVDF and visualized by immunoblotting with antisera recognizing the C-terminal domain of eIF4G. The position of the intact eIF4G and C-terminal product is indicated. These data are representative of those obtained in at least three separate experiments. Download figure Download PowerPoint Next we tested whether this effect of eIF4E could be exerted by two different mutant forms of the factor, where Ser-53 was mutated to Ala-53 and Ser-209 was mutated to Ala-209. The Ser-53 to Ala-53 variant has been found previously to be non-functional either in the formation of 48S initiation complexes (Joshi-Barve et al., 1990) or in inducing a transformed phenotype when overexpressed in mammalian cells (Lazaris-Karatzas et al., 1990). The other variant used had the serine residue, now identified as the major physiological phosphorylation site (Ser-209), mutated to Ala-209 (Flynn and Proud, 1995; Joshi et al., 1995; Makkinje et al., 1995). Recombinant eIF4E proteins were expressed and purified by m7GTP–Sepharose chromatography as described in Materials and methods; a Coomassie-stained SDS–PAGE analysis of the preparations of eIF4E and PHAS-I used in these experiments is shown in Figure 6A. As shown in Figure 6B, addition of PHAS-I prevented the action of L protease (compare lanes 2 and 3). Addition of the Ala-53 mutant eIF4E (lanes 7–9) gave results similar to the wild type (lanes 4–6), in that it was able to restore the proteolysis of eIF4G in the presence of PHAS-I. Ho
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