COMMUNICATION
Translational Repression by Human 4E-BP1 in Yeast Specifically Requires Human eIF4E as Target*

John M. X. HughesDagger , Marina Ptushkina, Md. Manjurul Karim, Nadejda Koloteva, Tobias von der Haar, and John E. G. McCarthy

From the Posttranscriptional Control Group, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, P. O. Box 88, Manchester M60 1QD, United Kingdom

    ABSTRACT
Top
Abstract
Introduction
References

4E-binding proteins (4E-BPs) are believed to have important regulatory functions in controlling the rate of translation initiation in mammalian cells. They do so by binding to the mRNA cap-binding protein, eIF4E, thereby inhibiting formation of the cap-binding complex, a process essential for cap-dependent translation initiation. We have reproduced the translation-repressive function of human 4E-BP1 in yeast and find its activity to be dependent on substitution of human eIF4E for its yeast counterpart. Translation initiation and growth are inhibited when human 4E-BP1 is expressed in a strain with the human eIF4E substitution, but not in an unmodified strain. We have compared the relative affinities of human 4E-BP1 for human and yeast eIF4E, both in vitro using an m7GTP cap-binding assay and in vivo using a yeast two-hybrid assay, and find that the affinity of human 4E-BP1 for human eIF4E is markedly greater than for yeast eIF4E. Thus yeast eIF4E lacks structural features required for binding to human 4E-BP1. These results therefore demonstrate that the features of eIF4E required for binding to 4E-BP1 are distinct from those required for cap-complex assembly.

    INTRODUCTION
Top
Abstract
Introduction
References

The initiation of translation requires recognition by small ribosomal subunits of the 5' end of mRNA. In eukaryotes, this is mediated by the assembly of a complex of proteins, known as eukaryotic initiation factor (eIF)14F, which recognizes the mRNA 5' cap structure. Modulation of eIF4F assembly is an important mechanism by which the rate of translation initiation is regulated according to the general demand for protein synthesis. The two most conserved components of eIF4F, found in all eukaryotes, are eIF4E, which binds to the cap structure directly, and eIF4G, a multifunctional polypeptide that binds to eIF4E and to other essential translation initiation factors. The binding of eIF4E to eIF4G is crucial for cap-dependent translation initiation and is believed to be subject to an important regulatory mechanism in mammalian cells involving 4E-binding proteins (4E-BPs), the subject of this study (reviewed in Refs. 1-6). 4E-BPs constitute a family of small polypeptides, of which one, 4E-BP1, has been the most extensively studied. 4E-BP1 shares with eIF4G a binding site for eIF4E and competes with eIF4G for binding to eIF4E, thus inhibiting the assembly of eIF4F (7, 8). The competition appears to be inversely phosphorylation-dependent. 4E-BP1 is known to be phosphorylated in response to stimuli that promote increases in the rate of translation (9), but only binds to eIF4E in its hypophosphorylated state (10, 11). Phosphorylation has been shown in vitro to cause dissociation of 4E-BP1 from eIF4E (10), thus relieving the competition for eIF4G binding (7). How important a regulatory mechanism is this in vivo? And, if regulation of translation initiation through 4E-BPs is important in mammals, to what extent has this regulatory mechanism been conserved throughout evolution? In order to start to answer these questions and to establish a system with which to dissect the details of 4E-BP function, we have reproduced the activity of human 4E-BP1 in the yeast Saccharomyces cerevisiae.

In S. cerevisiae, a small polypeptide, p20, co-purifies with the cap-binding complex and, like mammalian 4E-BPs, contains an amino acid sequence that resembles the 4E-binding domain of eIF4G (12). It is not clear to what extent p20 and mammalian 4E-BPs are related; however, they do share some functional similarities. p20 can also be phosphorylated (although, unlike mammalian 4E-BP1, p20 is phosphorylated following heat-shock, Ref. 13). Furthermore, a recent study shows that p20 interacts with some, but not all, of the same evolutionarily conserved residues on eIF4E that are recognized by eIF4G (14). The binding affinity of p20 for yeast eIF4E, however, appears to be significantly lower than that of eIF4G, suggesting that p20 may not be an efficient competitor of eIF4G binding (14).

Here we show that when human 4E-BP1 is expressed in an unmodified yeast strain, no effects on growth are observed. However, when the same protein is expressed in a strain in which human eIF4E is expressed instead of the endogenous yeast eIF4E (mammalian eIF4E functions in S. cerevisiae, Ref. 15), growth is strongly impaired, and this correlates with an apparent inhibition of translation initiation, as indicated by the intracellular accumulation of 80 S ribosomal subunit dimers relative to translating ribosomes. These results indicate that the translation regulatory function of human 4E-BP1 can be reproduced in yeast, but that its activity is specifically dependent on the co-expression of human eIF4E. Using both a cap-binding assay in vitro and two-hybrid analysis in yeast, we show that the binding affinity of human 4E-BP1 for human eIF4E is greater than that for yeast eIF4E. Thus, the specificity of 4E-BP function in yeast is a direct reflection of the difference between its binding affinities for the two eIF4E proteins. We conclude that, whereas the determinants on eIF4E for functional eIF4G binding are conserved between man and yeast, the determinants for 4E-BP1 binding are not, which suggests that the binding sites on human eIF4E for eIF4G and for 4E-BP1 are not identical.

    EXPERIMENTAL PROCEDURES

Yeast Strain and Plasmid Construction-- Plasmid DNA containing the human 4E-BP1 coding sequence was kindly provided by Dr. A.-C. Gingras of the Department of Biochemistry, McGill University, Montreal, Canada. This DNA was used as template in a polymerase chain reaction to generate DNA encoding 4E-BP1 with an N-terminal "Flag" (16) and flanked by NdeI and BamHI restriction sites, in which the ATG of the NdeI site forms the initiating methionine codon of the Flag sequence. The sequence of the Flag tag, inferred from the DNA, was MDYKDDDDKTM, the second M being the initial amino acid of 4E-BP1. The NdeI-BamHI restricted DNA was inserted at the NdeI and BglII sites of the yeast expression plasmid YCpSupex2, which contains a strong, galactose-inducible promoter (composed of the fused upstream regions of the yeast GAL1 and PGK1 genes) upstream of the insertion site and a PGK1 gene transcription terminator downstream of the insertion site (17). A ClaI restriction fragment containing the complete recombinant gene, including the promoter and terminator elements, was then excised and inserted at the ClaI site of pRS313, a yeast centromeric plasmid containing the HIS3-selectable marker gene (18). The resulting plasmid is named YCp313-fBP1.

Human cDNA encoding eIF4E was kindly provided by Prof. C. Proud, Department of Anatomy and Physiology, University of Dundee, Scotland. After amplification with suitable primers in a polymerase chain reaction, it was inserted between the NdeI and XbaI sites of YCp22-FL (19), the ATG of the NdeI site forming the initiator codon of the human eIF4E coding sequence. In this plasmid the eIF4E sequence is flanked upstream by the yeast TEF1 gene promoter and downstream by the PGK1 gene transcription terminator. This plasmid supports constitutive expression of human eIF4E in yeast and contains the TRP1 marker gene for selection. It is named YCpTrp-hu4E.

A haploid S. cerevisiae strain expressing human instead of yeast eIF4E, Jo56: cdc33::LEU2 his3 ura3 ade2 [YCpTrp-hu4E TRP1], was derived from strain T93C (20) after crossing to introduce the his3 auxotrophic mutation and transforming with the plasmid YCpTrp-hu4E. In order to test the effects of 4E-BP1 expression, plasmid YCp313-fBP1 was introduced into this and into strain Jo8: CDC33 his3-Delta ura3-52 leu2-3,112 lys1-1 ade2-1 can1-100, which carries an intact chromosomal eIF4E gene. Cultures were grown in appropriate selective nutrient "drop-out" media containing either 2% glucose or galactose.

Sucrose Density Gradient Centrifugation-- Cultures for the analysis of polysome profiles were grown to exponential phase initially in selective media containing glucose and then diluted into selective media containing galactose and cultured for a further 18 h at 30 °C. Cells were harvested and fractionated on sucrose density gradients as described previously (14).

Synthesis and Purification of Recombinant Proteins in Escherichia coli-- For expression in E. coli, the yeast and human eIF4E and 4E-BP1 DNA coding sequences were inserted between the NdeI and BamHI sites of the heat-inducible expression vector pCYTEXP3 (21). Six histidine codons were inserted between the first and second codons of the 4E-BP1 sequence. Expression was induced in the protease-deficient E. coli strain CAG629. Human and yeast eIF4E were purified from inclusion bodies and by affinity for m7GTP Sepharose (Amersham Pharmacia Biotech) as described previously (22, 23), but with the addition of 2 mM dithiothreitol to all buffer solutions following renaturation. His-tagged 4E-BP1 was purified denatured on nickel-nitrilotriacetic acid-agarose (Qiagen) following the supplier's recommended methods.

Analytical m7GTP-Sepharose Chromatography-- This was performed as described previously, using 10 µg of each purified protein per sample (14).

Yeast Two-hybrid Analysis-- The method of Fields and Song (24) in the form of the Matchmaker Gal4 Two-Hybrid System (CLONTECH) was used according to supplier's instructions (http://www.clontech.com) to investigate interactions between eIF4E and 4E-BP1 in yeast. The complete human or yeast eIF4E coding sequence was inserted between the SmaI and BamHI sites of plasmid pGBT9 to create a fusion in-frame with the Gal4p DNA-binding domain (BD, Fig. 4A). Meanwhile, the human 4E-BP1 coding sequence was inserted between the SmaI and BamHI sites of the plasmid pGAD424 to create a fusion in-frame with the Gal4p transcription activation domain (AD, Fig. 4A). The two recombinant plasmids encoding the hybrid proteins were introduced into the yeast strain HF7c, in which interaction between the two hybrid proteins is detected by expression of separate Gal4-responsive reporter genes, lacZ and HIS3.

    RESULTS

Expression of Human 4E-BP1 in Yeast Represses Growth in a Manner Specifically Dependent on Co-expression of Human eIF4E-- The human 4E-BP1 cDNA (kindly provided by Ann-Claude Gingras, McGill University, Montreal), including a sequence encoding a 10-amino acid N-terminal Flag polypeptide (Fig. 1A), was inserted downstream of a galactose-inducible promoter in a yeast centromeric plasmid expression vector. The recombinant plasmid was introduced into two different yeast strains: one carrying an unmodified yeast eIF4E gene (CDC33) and the second carrying a disrupted eIF4E gene (cdc33) but containing a constitutively expressed human eIF4E gene on a plasmid. CDC33 is an essential yeast gene, but eIF4E is sufficiently conserved so that the mammalian gene complements cdc33 (15). Transformants containing the 4E-BP1 expression plasmid were cultured initially in glucose-containing medium, in which the 4E-BP1 gene is repressed, and then transferred to galactose-containing medium in order to induce 4E-BP1 expression.


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Fig. 1.   Effects on yeast growth of expressing human 4E-BP1 in combination with either yeast or human eIF4E. A, structure of the recombinant 4E-BP1 gene expressed in yeast. The human 4E-BP1 coding sequence, incorporating a Flag tag at the 5' end, was placed downstream of a strong yeast galactose-inducible promoter in a yeast centromeric plasmid. B, each of two strains of S. cerevisiae, expressing either endogenous eIF4E or human eIF4E, were transformed with either the centromeric plasmid containing the galactose-inducible 4E-BP1 gene (+BP1) or the plasmid vector alone (Control) and allowed to grow on either galactose (4E-BP1-induced) or glucose-containing selective medium (4E-BP1-repressed). Independent, duplicate colonies were inoculated for each combination. C, the same yeast strains were cultured in selective liquid medium at 30 °C and transferred from glucose to galactose-containing medium at time 0 to induce 4E-BP1 expression. Growth rates were measured over a period of 2 days. The growth rate of the strain expressing 4E-BP1 in combination with human eIF4E decreased severely over this period, in which the cell doubling time increased to 15 h, compared with the controls, in which the cell doubling times remained constant at about 3 h. Filled circles, human eIF4E + BP1; open circles, human eIF4E control; filled triangles, yeast eIF4E + BP1; open triangles, yeast eIF4E control.

When the N-terminally flagged 4E-BP1 gene is expressed in the strain with yeast eIF4E, no effect on growth is observed (Fig. 1B, compare Yeast eIF4E + BP1 Induced and Repressed and the Control strain, which carries the appropriate plasmid expression vector without the 4E-BP1 gene.) However, when the flagged 4E-BP1 gene is expressed in the strain in which human eIF4E has been substituted for yeast eIF4E, a strong inhibition of growth is observed (Fig. 1B, compare Human eIF4E + BP1 Induced and Repressed and the Control strain.) When the growth rates of these strains are measured in liquid culture, 4E-BP1 expression with human eIF4E results in a cell doubling time of 15 h, compared with about 3 h when 4E-BP1 is expressed with yeast eIF4E and for the controls (Fig. 1C). Thus, the expression of human 4E-BP1 in yeast inhibits growth in a manner that is specifically dependent on co-expression of human eIF4E instead of endogenous yeast eIF4E.

Co-expression of Human 4E-BP1 and Human eIF4E in Yeast Impairs Translation Initiation-- Yeast strains expressing either yeast or human eIF4E and containing either the plasmid with the galactose-inducible 4E-BP1 gene, or the plasmid vector alone as control, were first cultured in glucose-containing medium and then transferred to galactose-containing medium in order to induce 4E-BP1 expression. After 18 h of induction, the cells were harvested, and polyribosomes were fractionated by sucrose density gradient centrifugation.

No changes in the polysome profiles due to expression of 4E-BP1 with yeast eIF4E are observed (Fig. 2, A and B), indicating that expression of 4E-BP1 does not affect translation in this strain. However, expression of 4E-BP1 with human eIF4E causes a marked relative accumulation of 80 S particles and of 40 S subunits (Fig. 2C) compared with the control (Fig. 2D). When 7 M NaCl is included in the fractionation buffers (Fig. 2, E and F), the large 80 S peak (Fig. 2C) is mainly replaced by 40 and 60 S peaks (Fig. 2E), suggesting that the 80 S particles accumulating as a result of 4E-BP1 expression are composed largely of subunit dimers, which are known to be less resistant to dissociation at high concentrations of salt, rather than translating ribosomes (25). The accumulation of subunit dimers has been observed in a number of conditions in which translation initiation is impaired (26, 27). It seems likely, therefore, that the growth inhibition caused by co-expression of human 4E-BP1 and human eIF4E in yeast is due to impaired translation initiation.


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Fig. 2.   Polysome profiles of yeast strains expressing 4E-BP1. A-D, yeast strains expressing either yeast (A, B) or human eIF4E (C, D) and containing either the plasmid with the galactose-inducible 4E-BP1 gene (A, C), or the plasmid vector alone as control (B, D), were harvested from selective medium after induction for 18 h with galactose. Cells were disrupted, and polysomes were fractionated by sucrose density gradient centrifugation. E and F, same as C and D except that fractionation buffers included 7 M NaCl to cause dissociation of ribosome subunit dimers. Peaks corresponding to ribosomes and subunits are indicated.

The Affinity of Human 4E-BP1 for Human eIF4E Is Greater than That for Yeast eIF4E in Vitro-- Given what is known of the function of mammalian 4E-BP1, we assume that the impaired growth and translation initiation observed when human 4E-BP1 and human eIF4E are co-expressed in yeast is due to direct interaction between these two proteins. Correspondingly, the lack of any effects due to the expression of 4E-BP1 in combination with yeast eIF4E could be due to a lack of binding. In order to test this hypothesis, we investigated the relative binding of 4E-BP1 to yeast and human eIF4E by observing the association of the purified proteins bound to m7GTP-Sepharose, for which eIF4E has affinity.

Human eIF4E and 4E-BP1 and yeast eIF4E were expressed and purified from E. coli. Equal amounts of either yeast or human eIF4E were mixed with 4E-BP1 and then adsorbed to m7GTP-Sepharose. After removing unbound material, protein complexes specifically bound to the m7GTP ligand were eluted by the addition of excess m7GDP and analyzed by SDS-polyacrylamide gel electrophoresis. The amount of human 4E-BP1 associated with m7GTP-bound human eIF4E was found to be significantly greater than that associated with m7GTP-bound yeast eIF4E (Fig. 3). This indicates that, under these conditions, the binding affinity of 4E-BP1 for human eIF4E is greater than that for yeast eIF4E.


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Fig. 3.   The affinity of human 4E-BP1 for human eIF4E is greater than that for yeast eIF4E in vitro. Purified proteins human eIF4E plus 4E-BP1 (Hu), yeast eIF4E plus 4E-BP1 (Ye), or 4E-BP1 alone (-) were mixed in equal quantities and incubated with m7GTP-Sepharose. Unbound material was removed by washing, and material remaining specifically bound to the m7GTP ligand was eluted with excess m7GDP. Bound and unbound material was analyzed by SDS-polyacrylamide gel electrophoresis and silver staining. Individual protein species in the gel are indicated.

The Affinity of Human 4E-BP1 for Human eIF4E Is Greater than That for Yeast eIF4E in Vivo-- We investigated the relative difference in the affinities between human and yeast eIF4E and 4E-BP1 in vivo using a yeast two-hybrid interaction assay. In this assay, evidence of interaction is provided by transcriptional activation of Gal4p-responsive reporter genes, due to reconstitution of Gal4p activity. Gal4p activity can be reconstituted when a test protein fused to the Gal4p DNA binding domain (BD) interacts with a second test protein fused to the Gal4p transcription activation domain (AD).

We fused either the human or the yeast eIF4E coding sequence in-frame with the Gal4p BD coding sequence and the human 4E-BP1 coding sequence with the Gal4p AD coding sequence (Fig. 4A). These two hybrid genes, in their appropriate plasmid expression vectors, were introduced into a yeast reporter strain in which both the yeast HIS3 and the bacterial lacZ genes can be expressed in response to Gal4p activity.


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Fig. 4.   The affinity of human 4E-BP1 for human eIF4E is greater than that for yeast eIF4E in vivo. A yeast two-hybrid assay was used to assess the relative strengths of the interactions between human or yeast eIF4E and human 4E-BP1. A, the assay tests the ability of hybrid proteins, consisting of either human or yeast eIF4E fused to the DNA binding domain (BD) of the Gal4p, and human 4E-BP1 fused to the transcriptional activation domain (AD) of the Gal4p, to interact and thereby activate transcription of the yeast HIS3 reporter gene, thus allowing the test strain to grow on medium lacking histidine. B, growth of the test strain, expressing various combinations of the hybrid proteins, on medium with and without histidine. The combinations of hybrid proteins are indicated. As controls (Con), the plasmids expressing hybrid proteins were combined with the respective plasmids expressing either the Gal4p BD or AD alone.

Fig. 4B illustrates the growth of the reporter strain expressing combinations of the hybrid proteins on selective media with or without histidine. Growth in the absence of added histidine reflects expression of the HIS3 reporter gene and is indicative of an interaction between the hybrid proteins. As controls, each of the hybrid proteins is also expressed in combination with either the Gal4p BD or AD alone. Only the combination of human eIF4E with 4E-BP1 supports growth on medium lacking histidine (Fig. 4B). Neither the combination of yeast eIF4E with 4E-BP1 nor the controls support growth without histidine. The same pattern of expression was observed for the lacZ reporter gene (data not shown). These data therefore provide evidence that human eIF4E and 4E-BP1, but not yeast eIF4E and 4E-BP1, are able to interact under physiological conditions in vivo.

    DISCUSSION

We have reproduced the translation-repressive function of human 4E-BP1 in yeast and find that it targets human eIF4E specifically. Co-expression of these two proteins in the absence of endogenous yeast eIF4E impairs growth (Fig. 1, B and C), probably as a direct result of inhibition of translation initiation (Fig. 2). Furthermore, we demonstrate using two independent techniques that the affinity of 4E-BP1 for human eIF4E is greater than that for yeast eIF4E (Figs. 3 and 4). Thus, the specificity of 4E-BP1 function in yeast is a direct reflection of the difference between its affinities for the two eIF4E proteins.

4E-BPs are believed to function by competing with eIF4G for binding to eIF4E, thus inhibiting cap-complex formation (7). Binding to eIF4E requires a conserved sequence of about 12 amino acids within the N-terminal domain of eIF4G and a similar sequence found in 4E-BPs (6 out of 12 residues in this sequence are identical in human and yeast eIF4G and human 4E-BP1, Ref. 8). The similarity of these sequences suggests that eIF4G and 4E-BPs share at least part of the same binding site on eIF4E. It is not known exactly which structural features of eIF4E are recognized by eIF4G and 4E-BPs. However, it is clear from our results that, whereas human eIF4E is sufficiently similar to yeast eIF4E to be recognized by yeast eIF4G and participate in yeast translation initiation, yeast eIF4E does not share all the structural features required for efficient recognition of human 4E-BP1. We conclude, therefore, that the binding sites on human eIF4E for 4E-BP1 and eIF4G are probably not identical. This situation may be similar to that of yeast p20 and eIF4G, for which it appears that p20 requires only a subset of those residues on yeast eIF4E that are required for binding by yeast eIF4G (14). Future work will be able to use the yeast model system described here to probe the detailed molecular basis of the interactions underlying this repressor-target specificity.

    ACKNOWLEDGEMENTS

We are indebted to Dr. A.-C. Gingras and Prof. C. Proud for the provision of plasmid DNA.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 44-161-200-8913; Fax: 44-161-200-8918; E-mail john.hughes{at}umist.ac.uk.

The abbreviations used are: eIF, eukaryotic initiation factor; 4E-BP, 4E-binding protein; BD, binding domain; AD, activation domain.
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Abstract
Introduction
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