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INTRODUCTION |
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.
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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-
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.
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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.
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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.
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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.
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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.
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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.
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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.