Translation of cellular mRNAs occurs by a cap-dependent
mechanism whereby the 40 S ribosomal subunit interacts with the
5`-terminal cap structure and subsequently migrates to the AUG start
codon(1, 2) . This process involves the activities of
a number of protein factors of the eIF4 (
)group(3, 4, 5) . The formation
of the complex termed eIF4F is thought to allow joining of the
ribosomal subunit to mRNA through its interaction with the mRNA cap
structure on the one hand and association with ribosomes on the
other(6) . eIF4F consists of three
polypeptides(7, 8, 9) : (i) eIF4E, which
specifically binds the cap; (ii) eIF4A, which possesses RNA helicase
activity; and (iii) eIF4G, a 220-kDa polypeptide on SDS gels. There is
a strong requirement for eIF4E and eIF4G in cap-dependent
initiation(1, 10, 11, 12, 13) .
Recent work has shed light on the potential role of eIF4G in
translation initiation(14, 15) . These studies have
identified an eIF4E-binding motif on eIF4G and have also identified
likely sites of interaction with eIF4A and eIF3. Thus eIF4G seems to
mediate joining of the mRNA and ribosomes by interaction with both the
cap-binding protein, eIF4E, and with eIF3 already bound to ribosomes.
eIF4A is believed to catalyze the unwinding of upstream mRNA secondary
structures. It is recycled through the eIF4F complex during successive
rounds of initiation(6, 16, 17) , possibly
explaining the presence of, and requirement for, abundant quantities of
uncomplexed eIF4A protein (6, 8, 18) . The
unwinding activity of eIF4A is stimulated in the presence of eIF4B
which may interact directly with
mRNA(19, 20, 21, 22) and/or 18 S
rRNA (23) through RNA recognition motifs.
eIF4E has been the
focus of intensive study in recent years. Current data suggest that
this factor exists in particularly low abundance both in the
reticulocyte lysate (24, 25) and in HeLa
cells(26) , indicating that it may quantitatively limit
translation rates. The biological importance of eIF4E levels is
illustrated by in vivo overexpression experiments, which
result in a transformed phenotype (27) or aberrant
growth(28) . Several observations stress the importance of
eIF4E phosphorylation in regulation of eIF4E activity during cell
growth and development (see Rhoads (5) and references
therein)(29, 30, 31) . Growth stimulation
correlates with increased de novo phosphorylation of eIF4E
(reviewed in Rhoads (5) and Morley(31) ) and eIF4F
complex
formation(30, 31, 32, 33, 34) ,
while the reverse applies when translation rates are down-regulated
during mitosis(35) , the heat-shock response (26, 36) and viral
infection(37, 38) . Recent work has determined that
the primary site of eIF4E phosphorylation in reticulocytes (39) and CHO cells (40) is serine 209. Interestingly, Saccharomyces cerevisiae and plant homologs of eIF4E lack a
serine at the equivalent position (41, 42) (for
comparison, see Hernández and
Sierra(43) ). The effect of phosphorylation on the affinity of
eIF4E for cap structures and components of the translation machinery
remains uncertain. While hyperphosphorylation of eIF4E does not affect
its recovery from HepG2 cell extracts on
m
GTP-Sepharose(32) , other experiments do suggest
an enhanced affinity for cap analogs and mRNA(44) . eIF4E
recovered with eIF4G from Ehrlich ascites tumor cells is highly
phosphorylated relative to uncomplexed eIF4E(45) ; however, the
phosphorylation of eIF4E alone may not alter its affinity for other
elements of the translational apparatus(15, 32) .
It is now becoming clear that eIF4E interacts specifically with both
eIF4G and at least one other binding protein, PHAS-I (also known as
eIF4E-BP1)(15, 46, 47, 48, 49) .
Current evidence suggests that PHAS-I acts to sequester eIF4E protein.
In hormone-responsive cells phosphorylation of PHAS-I in response to
insulin results in the release of sequestered eIF4E, thereby relieving
inhibition of translation(47) . Further work has shown that
PHAS-I and eIF4G possess a common eIF4E-binding motif (15) and
indeed compete for binding to eIF4E(50) . Thus PHAS-I
suppresses eIF4E activity by impairing its ability to form active
complexes in association with eIF4G. Whether translation regulation by
PHAS-I is important in reticulocytes is unknown at present.
In this
report we present data indicating that, contrary to previous
impressions, the reticulocyte lysate contains a functional excess of
eIF4E. The small proportion of the factor associated with ribosomes in
actively translating lysates shows enhanced phosphorylation. In
addition we provide evidence that levels of eIF4E in the reticulocyte
lysate are significantly higher than previously published and that the
molar relationship of eIF4E to PHAS-I in the postribosomal supernatant
is close to 1:1, supporting a regulatory role for PHAS-I in the
reticulocyte lysate.
EXPERIMENTAL PROCEDURES
Materials
All reagents were reagent grade and
purchased from Sigma unless indicated otherwise.
Buffers
Buffer A, 40 mM Tris/HCl, pH 7.4,
0.25 mM dithiothreitol, 0.1 mM EDTA, 50 mM sodium fluoride, 20 mM
-glycerophosphate, 2 mM benzamidine, 0.05% (v/v) Tween 20, 10% (v/v) glycerol; Buffer B,
40 mM Mops/KOH, pH 7.2, 125 mM NaCl, 2.5 mM EGTA, 40 mM
-glycerophosphate, 40 mM sodium
fluoride, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 7 mM
-mercaptoethanol; Resuspension
buffer (RB), 20 mM Mops/KOH, pH 7.2; 10 mM NaCl; 1.1
mM MgCl
; 0.1 mM EDTA; 75 mM KCl;
0.5 mM dithiothreitol; 5% (v/v) glycerol; RIPA, 50 mM Tris/HCl, pH 8.0, 1% (v/v) Triton X-100, 1% (v/v) Nonidet P-40,
0.1% (w/v) SDS, 125 mM NaCl, 10 mMp-nitrophenyl phosphate, 1 µM microcystin
(Calbiochem), 2 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride; Low salt buffer (LSB), 20 mM Mops/KOH, pH 7.2, 10 mM NaCl, 25 mM KCl, 1.1
mM MgCl
, 0.1 mM EDTA, 0.5 mM dithiothreitol; IEF sample buffer, 9 M urea, 7% (v/v)
Ampholine, pH 3.5-10 (Pharmacia Biotech Inc.), 2% (v/v)
-mercaptoethanol, 5% (w/v) CHAPS, 1 µM microcystin
(Calbiochem).
m
GTP-Sepharose Chromatography
Prior to
use, the m
GTP-Sepharose affinity resin (Pharmacia) was
equilibrated in RB, and the buffer was removed by aspiration using a
needle attached to a vacuum line. Depletion of eIF4E was achieved in
batch by addition of 4.5 volumes of undiluted reticulocyte lysate onto
1 volume of m
GTP-Sepharose, gentle agitation on ice for 15
min, and recovery of the unbound fraction by a brief centrifugation in
a microcentrifuge. In all cases, unbound material from a parallel
treatment using Sepharose 4B was used as the nondepleted control.
In Vitro Translation
Reticulocyte lysates were
prepared as described(51) . Translation reactions contained 50%
(v/v) reticulocyte lysate with the following added components (final
concentrations): 25 µM hemin, 25 µg/ml creatine
phosphokinase, 75 mM KCl, 0.8 mM magnesium acetate,
50 µM amino acids without methionine and leucine, 200
µM leucine, 3 mMD-glucose, 5 mg/ml
creatine phosphate. The translational activity of the system was
measured by inclusion of 0.1 mCi/ml
[
S]methionine and measurement of trichloroacetic
acid-precipitable radioactivity by scintillation counting.
Fractionation and Reconstitution of the Reticulocyte
Lysate
Ribosomal components of the intact reticulocyte lysate
were separated from the soluble fraction by centrifugation at 100,000
rpm (approximately 430,000
g) for 25 min at 4 °C
using the TL-100.2 rotor in a Beckman TL-100 ultracentrifuge,
essentially as described elsewhere(52) . When required for
subsequent translation assays, the undiluted reticulocyte lysate was
used as the starting material and the top two-thirds of the
postribosomal supernatant removed for use, while the bottom third of
the supernatant was discarded. The ribosomal pellet was resuspended in
RB buffer to 0.1 of the starting volume, yielding a 10-fold
concentrated (10
) stock of ribosomes. For reconstitution, 0.45
volume of postribosomal supernatant was combined with 0.05 volume of
10
ribosomes in a 50% translation reaction as above, with the
additional inclusion of 15 mM 2-aminopurine. For measurements
of the subcellular distribution of translation factors (see
``Results and Discussion'') the reticulocyte lysate was
preincubated under full translation conditions (as above) prior to
ultracentrifugation. In these cases the ribosomal pellet was
resuspended in RB buffer to 0.2 of the volume of the original
incubation.
Purification of eIF4E by Fast Protein Liquid
Chromatography
Chromatography on Mono Q (Pharmacia) to separate
the reticulocyte lysate into fractions enriched in eIF4F or eIF4E was
based on the method of Lamphear and Panniers(45) , with some
modifications. Briefly, 700 µl of reticulocyte lysate previously
incubated for 15 min under full translation conditions was loaded onto
a 1-ml Mono Q column equilibrated with 10 ml of Buffer A containing 50
mM KCl. The ``free'' and complexed forms of eIF4E
were step-eluted with Buffer A containing 130 mM KCl and 330
mM KCl, respectively. Pooled eluates were diluted to 100
mM KCl by addition of KCl-free Buffer A and eIF4E purified by
affinity chromatography using a 0.3 ml m
GTP-Sepharose
column, previously equilibrated in Buffer B. Bound eIF4E was eluted in
0.5 ml of Buffer B containing 150 µM m
GTP,
concentrated by acetone precipitation and the sample dissolved directly
into IEF sample buffer (see above).
Co-immunoprecipitation
Following preincubation
under full translational conditions, 40 µl of translation mix was
brought to 200 µl with RIPA prior to addition of affinity purified
polyclonal antiserum as indicted in the figures. The mixture was
incubated on ice for 90 min and IgG recovered by addition of 50 µl
of a 50% (v/v) Protein A-Sepharose 4B suspension (Pharmacia) in RIPA.
Following agitation at 4 °C for 30 min, the resin was recovered by
centrifugation, washed three times in 1 ml of RIPA and bound proteins
eluted directly into SDS or IEF sample buffer, as indicated in the
figure legends.
Expression and Purification of Recombinant eIF4E,
[His]
-tagged PHAS-I, and L
Protease
Recombinant proteins were expressed in, and purified
from, Escherichia coli (BL21(DE
) strain containing
the plasmid pLysS (Novagen)) as described for eIF4E(53) ,
PHAS-I(54) , and L protease (Lb)(55) . In the final
step of purification, recombinant protein was dialyzed overnight at 4
°C against LSB and the protein concentration measured by the
Bradford assay (Bio-Rad).
SDS-Polyacrylamide Gel Electrophoresis
(PAGE)
Samples were analyzed on 12% polyacrylamide mini-gels
(Protean II, Bio-Rad) as described elsewhere(33) .
Vertical Slab Isoelectric Focusing
(VSIEF)
One-dimensional VSIEF of eIF4E was carried out
essentially as described previously (53) using a Protean II
mini-gel apparatus (Bio-Rad). Briefly, samples were combined with at
least one volume of IEF sample buffer before focusing on a 5.6%
acrylamide gel for 3 h at 500-750 V (50 V increments, every 30
min) in the presence of 9 M urea, 7.5% (v/v) Ampholine, pH
3.5-10, and 2% (w/v) CHAPS. 0.01 M histidine was used at
the cathode (outer chamber) and 0.05 M glutamic acid at the
anode (inner chamber).
Western Blotting
After SDS-PAGE or VSIEF, proteins
were transferred to polyvinylidene difluoride membrane (Immobilon P,
Millipore) under semidry conditions (Semi-Phor, Hoefer Scientific
Instruments). Blots were incubated with primary antiserum; eIF4G,
polyclonal rabbit anti-peptide, raised against the N-terminal sequence
KKEAVGDLLDAFKEVN; eIF4E, polyclonal rabbit anti-peptide, raised against
the peptide WALWFFKNDKSKTWQANL; PHAS-I, polyclonal rabbit anti-peptide
raised against the peptide CSSPEDKRAGGEESQFE(46) ; eIF4B,
polyclonal goat anti-eIF4B protein antiserum, kindly provided by Dr. J.
Hershey; eIF4A, monoclonal antiserum, the kind gift of Dr. M. Altmann.
Bands were revealed by incubation with the appropriate alkaline
phosphatase-conjugated secondary antibody. Anti-peptide antibodies were
affinity purified on Affi-Gel 10 (Bio-Rad) prior to use, in accordance
with the manufacturers instructions.
RESULTS AND DISCUSSION
The availability of eIF4E is widely believed to limit
translation in eukaryotic cells(5, 49) . This belief
is based on measurements of initiation factor concentrations in HeLa
cells (26) and reticulocytes (24, 25) , which
indicated a very low ratio of eIF4E to ribosomes. However, another
common observation is that eIF4E is found in cells in both low and high
molecular weight forms(33, 45, 56) . The
latter, which is usually isolated from ribosomal salt-wash fractions,
is a complex of eIF4E with other initiation factors, notably eIF4A and
eIF4G, which together make up the eIF4F
complex(7, 9) . This is believed to be the functional
form of eIF4E, and good correlations between rates of protein synthesis
and the proportion of eIF4E associated with these complexes have been
observed in a number of physiological
states(30, 33, 34) . In contrast, isolation
of eIF4E from cytosolic (postribosomal supernatant) fractions of cells
by m
GTP-Sepharose affinity chromatography invariably yields
the low molecular weight form(9, 57, 58) ,
suggesting that cells contain substantial amounts of the factor not
involved in protein synthesis(45) .
Much of the Endogenous eIF4E Can Be Removed from the Intact
Reticulocyte Lysate with Only a Small Effect on Translational
Activity
To examine the effect of partial depletion of eIF4E on
protein synthesis we have employed the affinity matrix
m
GTP-Sepharose to specifically remove eIF4E from the rabbit
reticulocyte lysate (RRL) in vitro translation system. To
preclude concomitant removal of eIF4G, the post-ribosomal supernatant
was first separated from the ribosomes and then subjected to treatment
with the affinity resin, using a batch method. Separation of the
ribosomal and postribosomal supernatant (PRS) fractions from small
volumes of reticulocyte lysate can be achieved very rapidly using
conditions previously established (52) (see also
``Experimental Procedures''). Ribosomal and PRS fractions can
be reconstituted into a translation system that retains approximately
70% of the activity of the parental lysate and maintains polysome
integrity in vitro (data not shown). Using the initiation
inhibitor edeine, we find that the reconstituted translation system
used here is 90% initiation-dependent; this value is only slightly
below that for the intact reticulocyte lysate (data not shown). Fig. 1A shows Western blots demonstrating the degree of
depletion of the PRS as well as the quantities of eIF4E remaining after
reconstitution of the translation system with ribosomes
(``incubation''). In the control incubation, the PRS was
mock-depleted using Sepharose 4B resin (see ``Experimental
Procedures''). Lanes 1 and 2 show that all
detectable eIF4E was removed from the PRS following treatment with
m
GTP-Sepharose. The small proportion of eIF4E that
reappeared upon reconstitution (lane 4) is attributable to the
added ribosomes. The influence of this treatment on translation of
endogenous mRNA in the reconstituted lysate system is shown in Fig. 1B. Surprisingly, despite the extensive removal of
eIF4E (80-90%), only a 30% reduction in
[
S]methionine incorporation was observed after
60 min. Although the rate of translation is seen to fall relative to
the undepleted control during the late part of the time course depicted
in Fig. 1B, this was a characteristic of this
experiment and was not a reproducible finding. Experiments in which the
batch depletion was applied instead to the reconstituted system
following recombination of the PRS with ribosomes consistently show an
effect more in line with the extent of eIF4E depletion (data not
shown). These results raise two interesting questions. First, the
maintenance of approximately 70% of the protein synthesis activity in a
system depleted of 80-90% of its content of eIF4E is not
consistent with the concept that this factor is present in
rate-limiting
amounts(5, 25, 26, 49) ; rather it
indicates that much of the factor in the PRS fraction is
non-functional. This is consistent with the finding that eIF4E may be
withheld from participation in initiation complexes through
sequestration by PHAS-I in other cell
systems(47, 48) . The second point relates to the more
severe effect of depleting the reconstituted lysate, rather than the
PRS alone, prior to reconstitution (data not shown). This result may be
explained by the concomitant removal of eIF4G by the
m
GTP-Sepharose affinity matrix due to its interaction with
eIF4E. Alternatively, the ribosomal fraction may contain a highly
active (e.g. highly phosphorylated) form of
eIF4E(59) , which is removed by depleting the reconstituted
lysate. In order to address these points we have undertaken a more
rigorous study of the distribution of eIF4E between the
ribosome-associated and PRS fractions of actively translating
reticulocyte lysates, and, for each population of the factor, we have
analyzed its extent of phosphorylation and its association in complexes
with eIF4G and PHAS-I.
Figure 1:
Extensive depletion of eIF4E in the
reticulocyte lysate causes only a moderate inhibition of protein
synthesis. A, the PRS of a reticulocyte lysate was subjected
to batchwise treatment with either Sepharose 4B (Control) or
the affinity matrix m
GTP-Sepharose 4B (Depleted)
for 15 min on ice as described under ``Experimental
Procedures.'' Samples corresponding to 1 µl of the parent
reticulocyte lysate were analyzed by SDS-PAGE and the depletion of
eIF4E monitored by immunoblotting, as described (PRS, lanes 1 and 2). In addition, equivalent samples were
analyzed in a similar manner following reconstitution of the
reticulocyte lysate (see below) (Incubation, lanes 3 and 4). The immunoblots were quantified by scanning
densitometry and the data are presented as a histogram of optical
density expressed as arbitrary units (AU). B, control
and depleted postribosomal supernatant (0.45 volume) were recombined
with resuspended 10
ribosomes (0.05 volume) and translation mix
(0.5 volume) prior to incubation at 30 °C. Aliquots (2 µl) were
removed at the times shown and processed for incorporation of
[
S]methionine into acid-precipitable protein.
These data are representative of those obtained in three separate
experiments.
Subcellular Distribution of eIF4E and Associated Proteins
in the Reticulocyte Lysate
Fig. 2A shows the results
of an experiment where an intact RRL was incubated for 15 min at 30
°C under full translational conditions (see ``Experimental
Procedures'') and subsequently separated into soluble (PRS) and
ribosomal fractions. The complete removal of all ribosomal components
from the PRS was confirmed by sucrose density gradient analysis (data
not shown). Equivalent samples of PRS and resuspended ribosomes were
analyzed by gel electrophoresis followed by Western blotting, using
antibodies recognizing eIF4E, eIF4G, eIF4B, eIF4A, and PHAS-I (see
``Experimental Procedures''). The intensity of the resultant
bands was quantified by densitometry and these data are summarized in Table 1. In all cases the immunochemical signal of the blot was
within the linear range of antibody response (data not shown). The
distribution of the various proteins differed substantially;
approximately 80% of the total eIF4G co-sedimented with ribosomes,
while only about 25% of total eIF4E and no detectable PHAS-I were found
in this fraction. In contrast both eIF4B and eIF4A were fairly evenly
distributed between both the PRS and ribosomal fractions. The presence
of most of the eIF4E in the PRS, apparently not directly associated
with the translational machinery in the ribosomal fraction, may be
explained by sequestration into inactive complexes by specific binding
proteins such as
PHAS-I(47, 48, 60, 61) . Indeed our
unpublished results show that removal of eIF4E from the PRS using
m
GTP-Sepharose concomitantly removes PHAS-I, indicating a
stable interaction between eIF4E and PHAS-I in that fraction. The
presence of about 80% of the eIF4G in the ribosome fraction suggests
that this factor, rather than eIF4E, may be limiting for translation,
although it is possible that the association of eIF4G with ribosomes is
independent of active protein synthesis. Indeed, Joshi et al.(62) have demonstrated association of radioactively
labeled eIF4G with 43S preinitiation complexes in reticulocyte lysates,
suggesting that the factor binds to ribosomes in the absence of mRNA.
Figure 2:
Subcellular distribution of eIF4E and
associated proteins in the reticulocyte lysate; effects of cleavage of
eIF4G by L protease. A, following a 15-min preincubation at 30
°C under full translation conditions (see ``Experimental
Procedures'') reticulocyte lysate was rapidly fractionated by
centrifugation at 100,000 rpm (approximately 430,000
g) for 25 min in a Beckman TL-100 centrifuge. The S 100 was
removed (PRS), and the pellet was resuspended in RB buffer on ice to
0.2 of the original incubation volume (see ``Experimental
Procedures''). Samples corresponding to 1 µl of unfractionated
reticulocyte lysate were analyzed by SDS-PAGE and Western blotting
using the specific antibodies shown. B, the reticulocyte
lysate was further incubated for 10 min at 30 °C following the
addition of a partially purified form of recombinant FMDV L protease
prior to fractionation and immunoblot analysis as for A. Cp
= eIF4G cleavage product,
N-terminal domain; Cp
= eIF4G
cleavage product, C-terminal domain. These data are representative of
those obtained in three separate
experiments.
The Association of eIF4E with Ribosomes Is
Functional
In order to test the functional significance of eIF4E
associated with ribosomes (Fig. 2A), we have employed
the eIF4G-specific FMDV L protease as a tool to block the initiation
phase of translation. The use of L protease in preference to other
inhibitors of initiation allowed us to evaluate simultaneously the role
of eIF4G in mediating the association of eIF4E with the ribosomal
fraction. In the experiment shown in Fig. 2B, the
reticulocyte lysate translation system was briefly incubated under
protein synthesis conditions, followed by addition of L protease and a
further incubation. The lysate was subsequently fractionated into PRS
and ribosomal fractions as described and the distribution of initiation
factors determined by SDS-PAGE and immunoblotting. Separate
anti-peptide antibodies recognizing eIF4G were used to monitor the
N-terminal (termed Cp
) and the C-terminal (Cp
)
cleavage products (the latter a kind gift from Dr. R. Rhoads, Louisiana
State University). A summary of the densitometric quantification of
these Western blots is shown in Table 1. After L protease
treatment of the reticulocyte lysate the C-terminal domain of eIF4G
remains mostly ribosome-bound, while, in contrast, the N-terminal
domain of eIF4G is released into the PRS (compare Fig. 2, A with B). Fig. 2B also demonstrates that
the L protease treatment results in the complete loss of eIF4E from the
ribosomal fraction, while the distribution of eIF4B, eIF4A, and PHAS-I
is not affected. These observations suggest a functional role for the
ribosome-bound eIF4E in the intact reticulocyte lysate, mediated by the
N-terminal domain of eIF4G, rather than nonspecific binding or
contamination of the ribosomal pellet by supernatant material. The data
are therefore consistent with recent observations suggesting that the
C-terminal domain of eIF4G interacts with eIF4A, eIF3, and ribosomes,
while the N-terminal domain binds
eIF4E(14, 15, 49, 63) . Thus in the
presence of L protease the distribution of the C-terminal domain of
eIF4G and eIF4A between the PRS and ribosomes remains unchanged, while
there is a shift of the N-terminal domain and associated eIF4E into the
soluble fraction.
The Removal of eIF4E from the Ribosomal Fraction by
Treatment with L Protease Is Not Dependent on the Loss of Polysome
Integrity
An outstanding question from the work detailed above
is whether eIF4G cleavage directly causes the release of eIF4E from cap
structures, or whether the movement of eIF4E into the soluble fraction,
shown in Fig. 2, could be explained by a shift of mRNA itself
into the PRS after ribosome run-off, with eIF4E remaining bound to the
mRNA cap structure. In order to examine the latter possibility, we used
the elongation inhibitor emetine to prevent ribosome run-off in the
presence and absence of L protease. Lysates were incubated as described
in Fig. 2B but in the presence of emetine to maintain
polysomes (data not shown); lysates were then fractionated and the
level of eIF4E associated with the ribosomal fraction analyzed by
immunoblotting. Fig. 3shows that maintenance of polysome
integrity does not prevent the release of ribosome-associated eIF4E
into the postribosomal supernatant following L protease treatment. This
intriguing result can be explained by at least three distinct
mechanisms. First, if all ``ribosomal'' eIF4E is normally
cap-associated, the most probable explanation for these data is that L
protease-induced cleavage of eIF4G results in the dissociation of eIF4E
from the mRNA cap. A cleavage-induced conformational alteration in the
eIF4G domain containing the eIF4E-binding site may be transmitted to
eIF4E itself, reducing its affinity for the cap structure. Although
eIF4E alone does have a high affinity for mRNA cap structures in
vitro(44) , this is apparently stimulated in the presence
of eIF4G(4, 6, 19) ; hence, cleavage of eIF4G
might conversely reduce its affinity for the cap. However, work in
several laboratories, including our own, (
)has shown that
the Cp
eIF4E complex retains its ability to bind to
cap analogs(9, 14, 58) . A second alternative
is that the association between eIF4E and the cap is very transient,
with eIF4E being released from the cap as the 40S subunit begins to
scan toward the initiation codon. In this model the eIF4E present in
``48 S'' complexes(62, 64) , where the 40 S
subunit is presumably located at the initiation codon, would be
attached solely via its interaction with eIF4G. Finally, one might
argue that only a small proportion of ribosomal eIF4E is cap-associated
and that the majority is in fact involved in 43 S preinitiation
complexes formed in the absence of mRNA. eIF4E in such complexes,
presumably bound via the N-terminal domain of eIF4G, would be released
into the soluble fraction following cleavage of eIF4G. Prevailing
evidence is against the presence of eIF4E in 43 S preinitiation
complexes(62, 64) , but uncertainty on this point
remains (65) .
Figure 3:
Emetine does not prevent the loss of eIF4E
from the ribosomal fraction following cleavage of eIF4G by L protease.
Three parallel reactions of intact reticulocyte lysate under full
translation conditions (see ``Experimental Procedures'') were
incubated for 15 min at 30 °C. Emetine (25 µM final
concentration) was added for 2 min where indicated, prior to the
addition of recombinant L protease (or an equivalent volume of LSB) and
the incubation continued for a further 10 min at 30 °C. Reactions
were stopped by cooling on ice and lysates were fractionated by
ultracentrifugation, as described in Fig. 2. The resultant
ribosome pellets were resuspended as described and aliquots
corresponding to 3 µl of undiluted reticulocyte lysate were
analyzed by SDS-PAGE and the presence of eIF4E monitored by Western
blotting. These data were reproduced on two separate
occasions.
eIF4E in the Ribosomal Fraction of the Reticulocyte
Lysate Is Highly Phosphorylated
There are strong circumstantial
links between eIF4E phosphorylation and enhanced activity of this
factor(5, 29, 30, 49) . Fig. 4A shows typical measurements of eIF4E phosphorylation
obtained when samples of PRS and resuspended ribosomes are subjected to
VSIEF followed by Western blotting (see ``Experimental
Procedures''). Clearly, the small proportion of eIF4E that is
ribosome-bound is enriched for the phosphorylated form. This effect is
reproducible between lysates, although the actual degree of
phosphorylation shows some variation. Nonetheless, it is noteworthy
that first, we never observe 100% phosphorylation of ribosomal eIF4E
and, second, the soluble eIF4E pool is itself approximately 30% phosphorylated. By combining data from Fig. 2A and Fig. 4A we can estimate the distribution of the
phosphorylated form of eIF4E between subcellular fractions in the
reticulocyte lysate (Table 2) and find that, while the ribosomal
fraction is enriched for the phosphorylated form, the postribosomal
supernatant contains more than half of the total phosphorylated eIF4E
protein in the reticulocyte lysate. However, the PRS contains only
small amounts of eIF4G (Fig. 2A). Hence, while
phosphorylation of eIF4E may affect its ability to participate in
protein synthesis, it cannot alone be sufficient to promote an
association with eIF4G. Indeed, unpublished studies discussed by Mader et al.(15) have indicated that phosphorylation of
eIF4E does not alter its interaction with eIF4G in a far Western
blotting assay. Rather, concomitant phosphorylation of eIF4E and eIF4G
may be required to stimulate their interaction(32) .
Figure 4:
The phosphorylation status of eIF4E in
subcellular fractions of the reticulocyte lysate. A, the
intact reticulocyte lysate was incubated for 15 min at 30 °C prior
to centrifugal fractionation into PRS and ribosomes. The following
samples were then removed for VSIEF, and the proportion of total eIF4E
in the phosphorylated state was visualized by immunoblotting: 4 µl
of the original incubation, corresponding to 2 µl of reticulocyte
lysate; PRS (4 µl); ribosomes (10 µl), corresponding to 5
µl of reticulocyte lysate. The upper band, indicated as P,
corresponds to the phosphorylated form of eIF4E. In each case,
quantification of these data by scanning yielded the percent of total
eIF4E in the phosphorylated form. The data are representative of six
independent experiments. B, reticulocyte lysate was incubated
under full translation conditions as above prior to chromatography on
Mono Q as described under ``Experimental Procedures.''
Material corresponding to uncomplexed eIF4E (Light) was eluted
from the column at 130 mM KCl, while complexed eIF4E (Heavy) was eluted at 330 mM KCl. eIF4E was isolated
as described under ``Experimental Procedures.'' Precipitated
proteins were resuspended directly in IEF sample buffer and subjected
to VSIEF. A sample of the Mono Q load, corresponding to 2 µl of
reticulocyte lysate, was also analyzed (Starting material).
Quantification by scanning yielded values for the percentage of
phosphorylation. The data are representative of two separate
experiments. C, reticulocyte lysate was incubated as in B; immunoprecipitation of eIF4E was carried out using
affinity-purified antibodies recognizing eIF4G, eIF4E, and PHAS-I, as
described under ``Experimental Procedures.'' Immune complexes
were isolated with Protein A-Sepharose in batch, as described, and the
bound protein eluted directly into IEF sample buffer. Starting material
shows VSIEF analysis of 4 µl of the original translation
incubation. These data are representative of four separate
experiments.
eIF4E Involved in Complexes Is More Phosphorylated than
the ``Free'' Form When These Are Separated by Fast Protein
Liquid Chromatography
As an alternative approach to
differentiating between populations of eIF4E in the reticulocyte lysate
we used Mono Q chromatography to separate complexed from uncomplexed
eIF4E in samples of actively translating reticulocyte lysate (see
``Experimental Procedures''), using a procedure similar to
Lamphear and Panniers (45) . Pools corresponding to
eIF4E
eIF4G complexes (``heavy'') and uncomplexed
(``light'') fractions were analyzed by both SDS-PAGE (data
not shown) and VSIEF (Fig. 4B). In support of the data
obtained with fractions separated by ultracentrifugation (Fig. 2A), approximately 80% of the eIF4E was recovered
at a KCl concentration previously found to elute the uncomplexed form
of eIF4E protein(45) , while 20% showed elution properties
typical of the eIF4E
eIF4G complex (data not shown). This latter
fraction was, however, more highly phosphorylated (60%) (Fig. 4B). These data thus support those in Fig. 4A, indicating that the functional pool of eIF4E
is enriched for, but does not contain exclusively, the phosphorylated
form of the protein.
Anti-eIF4G Antibody Co-immunoprecipitates Mostly
Phosphorylated eIF4E, while Anti-PHAS-I Antibody Co-immunoprecipitates
eIF4E That Is Less Phosphorylated
To confirm data on the
phosphorylation status of eIF4E in complexed versus ``free'' form (Fig. 4, A and B), we examined the phosphorylation status of eIF4E in
immunoprecipitates obtained with antibodies against eIF4E, eIF4G, and
PHAS-I (see ``Experimental Procedures''). Fig. 4C shows that, relative to the starting material (31% phosphorylated)
or that recovered with anti-eIF4E antibody (29% phosphorylated), eIF4E
co-immunoprecipitating with anti-eIF4G antibody is more highly
phosphorylated (54%). However, the eIF4E co-immunoprecipitating with
anti-PHAS-I antibody appears, if anything, to be less phosphorylated
(20%) than the starting material.Thus we find that eIF4E, selected
by three distinct methods (Fig. 4, A-C), on the
basis of its association with active translational complexes, is
consistently enriched for the phosphorylated form. This observation
supports the large body of evidence (5, 29, 30) which correlates increased eIF4E
phosphorylation with enhanced eIF4E activity. While there is in
vitro evidence for a greater affinity of phosphorylated eIF4E for
mRNA cap structures(44) , it is not clear at this stage which
aspect(s) of eIF4E activity are affected by its phosphorylation in
vivo. Numerous studies linking increased eIF4E phosphorylation and
enhanced complex/eIF4F formation (30, 32, 33, 34) would indicate an
improved ability to bind eIF4G; however, no evidence has been presented
to refute the alternative possibility that eIF4E phosphorylation occurs
only as a consequence of recruitment into complexes.
Binding of eIF4G or PHAS-I to eIF4E Is Mutually
Exclusive
Recent work has provided evidence for a common
eIF4E-binding motif within both eIF4G and PHAS-I(15) . This
suggests a competitive scenario where PHAS-I and eIF4G are unable to
interact with eIF4E simultaneously. Recent studies with purified
proteins by Haghighat et al.(50) have provided
evidence in support of this hypothesis. Most work in favor of an
important regulatory role for PHAS-I in controlling protein synthesis
has involved insulin-responsive cells(48, 60) .
However, the potential role of PHAS-I in reticulocytes is not known at
this time. As seen in Fig. 2A, we have detected
significant quantities of PHAS-I in the reticulocyte lysate. Indeed the
preferential retention of this protein in the post-ribosomal
supernatant is consistent with current views for the role of PHAS-I
which would preclude an association with any ribosomal component.We
have therefore extended the use of co-immunoprecipitation to
investigate molecular interactions between eIF4G, eIF4E, and PHAS-I in
the intact reticulocyte lysate under full translational conditions. Fig. 5shows Western blots of co-immunoprecipitated material
following SDS-PAGE. While this procedure is not quantitative, each
employed antiserum predictably precipitated its specific antigen. Fig. 5shows that, in addition, anti-eIF4G antibody
co-immunoprecipitates eIF4E, while anti-eIF4E co-immunoprecipitates
eIF4G and PHAS-I, and anti-PHAS-I antibody co-immunoprecipitates eIF4E.
The doublet due to PHAS-I is likely to represent two differentially
phosphorylated forms, as seen in 3T3-L1
cells(48, 60) . These data are internally consistent
in their support for interactions between eIF4E and eIF4G, as well as
between eIF4E and PHAS-I. However, the failure of anti-eIF4G antibodies
to co-immunoprecipitate PHAS-I and, conversely, the absence of eIF4G in
anti-PHAS-I immunoprecipitates, in spite of the presence of eIF4E in
both cases, provides evidence for a mutually exclusive interaction of
PHAS-I and eIF4G with eIF4E in reticulocytes. It is likely, therefore,
that PHAS-I and eIF4G compete for available eIF4E, where the affinity
of PHAS-I(47, 48, 60) , and possibly that of
eIF4G, for eIF4E, is influenced by phosphorylation. These data are in
agreement with recent work by Mader et al.(15) and
Haghighat et al.(50) . While we have established that
eIF4E phosphorylation is insufficient to explain association with eIF4G
(see Fig. 2A and Fig. 4A, summarized in Table 2), it is not known whether the phosphorylation status of
eIF4E influences its association with PHAS-I. Fig. 4C shows that eIF4E co-immunoprecipitated with PHAS-I antibody is
only 20% phosphorylated, compared with a phosphorylation status of
approximately 30% in the starting material. The significance of this
small difference is not clear; whereas dephosphorylation of eIF4E might
favor binding of PHAS-I, it may equally occur as a consequence of
association with PHAS-I.
Figure 5:
Analysis of protein-protein interactions
by co-immunoprecipitation with affinity purified antibodies to eIF4G,
eIF4E, and PHAS-I. For immunoprecipitation studies, reticulocyte
lysate, under full translation conditions (40 µl), was incubated
for 15 min at 30 °C before dilution to 200 µl in RIPA.
Polyclonal affinity-purified antibodies, specific for eIF4G, eIF4E, and
PHAS-I, were then added and incubated for 90 min at 4 °C. Immune
complexes were purified with Protein A-Sepharose, and the bound protein
was eluted directly into sample buffer for SDS-PAGE. Proteins were
transferred to polyvinylidene difluoride, and eIF4G, eIF4E, and PHAS-I
in the sample were visualized by immunoblotting. These data are
representative of those obtained in four separate
experiments.
The Molar Ratio of eIF4E to PHAS-I in the Postribosomal
Supernatant of the Reticulocyte Lysate Is Approximately 1:1
To
further assess the potential role of PHAS-I in the regulation of
protein synthesis in reticulocyte lysates, we have estimated the molar
concentrations of eIF4E and PHAS-I proteins in this system. To date,
the only quoted estimate of the ratio of PHAS-I to eIF4E in the
reticulocyte lysate is 1:20(47) , although no data were
presented. We have used purified, recombinant eIF4E (Fig. 6) and
PHAS-I (Fig. 7) proteins as standards in Western immunoblotting
assays, using a procedure similar to that employed by Pause et
al.(17) . Fig. 6A shows >90% purity of the
recombinant eIF4E protein. Fig. 6B shows a Western blot
demonstrating a linear immunological response to increasing levels of
recombinant eIF4E. In the same experiment, three different volumes of
reticulocyte lysate were used to estimate the average endogenous
concentration of eIF4E (Fig. 6C). From these data we
calculate that 1 ml of reticulocyte lysate contains approximately 10.7
± 0.8 µg (S.D., n = 3) of eIF4E. Fig. 6D confirms this result by showing that 0.5 µl
of reticulocyte lysate (lane 1) yields an immunological
response similar to that observed with 5.5 ng of recombinant eIF4E (lane 3). A similar value was obtained in assays on three
separate lysates (data not shown). In molar terms this value is
equivalent to a concentration for eIF4E of
0.4 µM, as
compared to previous estimates of 8 nM(24) and 33
nM(25) in the reticulocyte lysate and a calculated
0.35 nM in HeLa cells(26) . Consistent with eIF4A
being one of the most abundant initiation
factors(18, 26) , the recent estimation of 3.4
µM (calculated from 17) reveals eIF4A levels approximately
10-fold greater than those calculated above for eIF4E. Initiation
factor concentrations are often best expressed relative to the
translational apparatus itself. We have measured the molar
concentration of ribosomes in different preparations of reticulocyte
lysate and obtain a value of 0.2 µM ± 0.04 (S.D., n = 4). Thus we observe a eIF4E to ribosome ratio of
2:1 in reticulocytes, compared with previous data of 0.02 in
reticulocytes (24) and 0.26 in HeLa cells(26) . Our
revised estimates of eIF4E abundance are therefore significant in that
they are at least an order of magnitude greater than those published to
date. Furthermore, these estimates are consistent with observations
published herein that quantities of eIF4E per se are not
limiting for translation in the reticulocyte lysate (see Fig. 1)
and that the majority of this protein is not engaged in translation
under optimal conditions (see Fig. 2A).
Figure 6:
Quantification of the levels of eIF4E
protein in the reticulocyte lysate. A, recombinant,
bacterially expressed eIF4E protein (2 µg), prepared as described
under ``Experimental Procedures,'' was analyzed by SDS-PAGE
and Coomassie staining; the resultant gel is presented. B, the
immunological response to increasing amounts of recombinant eIF4E was
tested by Western blotting (upper panel) and quantified by
densitometric scanning (lower panel; AU =
arbitrary units). C, the same experiment included a dose
response of eIF4E antibodies to three different volumes of reticulocyte
lysate. An estimation of eIF4E concentration in the reticulocyte lysate
(see ``Results'') was made by taking the average of the
calculated concentration, obtained from each of the three doses of
reticulocyte lysate. D, to ensure reliability of the Western
blotting method, 0.5 µl of reticulocyte lysate (lane 1)
and 5.5 ng of recombinant eIF4E (lane 3) were tested for their
ability to give an additive immunological response when combined (lane 2). These data are representative of those obtained in
five experiments using three different lysate
preparations.
Figure 7:
Quantification of the level of PHAS-I
protein in the reticulocyte lysate. A, recombinant
histidine-tagged PHAS-I ([His
]PHAS-I) was
prepared as described under ``Experimental Procedures.'' The
immunological response to increasing amounts of recombinant
[His
]PHAS-I was tested by Western blotting; the
data were quantified by scanning and are presented on the lower panel
as an optical density in arbitrary units (AU) expressed as a
function of increasing amounts of PHAS-I (ng). B, the same
experiment included a dose response of PHAS-I antibodies to three
different volumes of reticulocyte lysate. An estimation of PHAS-I
concentration in the reticulocyte lysate (see ``Results'')
was made by taking the average of the calculated concentration,
obtained from each of the three doses of reticulocyte lysate. Similar
data were reproduced in two further experiments using different lysate
preparations.
In Fig. 7, we show a similar method to quantify the concentration
of PHAS-I in reticulocyte lysates. The recombinant PHAS-I protein used
was electrophoretically distinct from endogenous PHAS-I, because of a
histidine tag used to facilitate its purification (data not shown, see
``Experimental Procedures''). Assuming that the histidine tag
had no influence on the immunoreactive properties of the recombinant
protein, we estimate the concentration of PHAS-I to be 3.2 ± 0.4
µg/ml of reticulocyte lysate (S.D., n = 3) (see Fig. 7B), representing a molar concentration of
0.3 µM. From these data the ratio of eIF4E to PHAS-I
in the reticulocyte lysate (and particularly in the PRS, containing
75% of total eIF4E and all endogenous PHAS-I) (see Fig. 2A) is close to 1:1. This close molar
stoichiometry between eIF4E and PHAS-I is strongly suggestive of a role
for PHAS-I in the regulation of translation in rabbit reticulocytes as
well as in insulin responsive cells.