From the School of Life Sciences, Medical Sciences
Institute/Wellcome Trust Biocentre Complex, University of Dundee, Dow
Street, Dundee DD1 5EH, United Kingdom, the ¶ Department of
Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD,
United Kingdom, and the ** Research School of Biosciences, University of
Kent at Canterbury, Canterbury CT2 7NJ, United Kingdom
Received for publication, December 29, 2001, and in revised form, April 24, 2001
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ABSTRACT |
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Initiation factor eIF2B mediates a key regulatory
step in the initiation of mRNA translation, i.e. the
regeneration of active eIF2·GTP complexes. It is composed of five
subunits, Eukaryotic initiation factor
(eIF)1 2B is a
heteropentameric protein, which catalyzes a key step in translation
initiation, i.e. the exchange of guanine nucleotides on
initiation factor eIF2. eIF2 binds GTP and in this form can also
interact with the initiator methionyl-tRNA (Met-tRNAi) to
form the ternary complex eIF2·GTP·Met-tRNAi (1, 2).
Such complexes can then bind to the 40 S ribosomal subunit to form
pre-initiation complexes, bringing the Met-tRNAi into
position to recognize the AUG start codon of the mRNA following
ribosomal scanning. The eIF2-bound GTP molecule is then hydrolyzed to
GDP and Pi in a process that requires a further factor,
eIF5. The inactive eIF2·GDP complex is released from the ribosome.
The rate of release of GDP from eIF2 is very slow under physiological
conditions, and the exchange of bound GDP for GTP, to regenerate active
eIF2·GTP, requires an additional factor, eIF2B. The activity of eIF2B
plays a key role in regulating the initiation of translation and can be
controlled in several ways. For example, phosphorylation of the
eIF2B is an unusually complicated guanine nucleotide exchange factor
(GEF), those acting on small G-proteins such as Ras, for example, being
much smaller monomeric proteins. The reasons for this complexity and
the roles of the five subunits are still not completely clear. A major
step toward addressing these issues has been the cloning of cDNAs
encoding subunits of eIF2B. This has now been achieved for several
species, including yeast (see, e.g., Refs. 10 and 11),
mammals (see, e.g., Refs. 12-16), and Drosophila
(17). Studies for all three sources have shown that the There is also disagreement over the catalytic mechanism of eIF2B; early
studies supported the so-called "ping-pong" or substituted enzyme
mechanism (Mechanism A in Fig. 1; Ref. 24), which
is the one utilized by another GEF involved in mRNA
translation, the bacterial elongation factor EF-Ts (25) and by factors
that promote nucleotide exchange on other GTP-binding proteins
(26-29). These factors act to stimulate the release of bound guanine
nucleotide and are thus termed "GDP dissociation stimulator
proteins" (GDS proteins, Ref. 29).Other studies have suggested that
eIF2B may operate via the alternative sequential mechanism
(Mechanism B in Fig. 1; Refs. 30 and 31).
Here we demonstrate that the properties of eIF2B match the operation of
the ping-pong mechanism and suggest that eIF2B acts as a GDS protein.
We also show that eIF2B Chemicals and Biochemicals--
Chemicals and biochemicals were
obtained from BDH-Merck (Poole, Dorset, United Kingdom (UK)) or Sigma
Alrich (Gillingham, Dorset, UK), unless otherwise stated.
[3H]GDP (10Ci/mmol), [ Purification of eIF2 and eIF2B--
These proteins were purified
as described previously (8), but using HeLa cell cytoplasm as the
starting material (from Computer Cell Culture Center, Seneffe,
Belgium). The cytoplasmic fraction was subjected to ammonium
sulfate fractionation, and the material precipitating between 30% and
60% saturation was subjected to ion exchange chromatography
successively on Q-Sepharose, S-Sepharose, MonoQ, and MonoS (all from
Pharmacia Nycomed).
Assay of eIF2B Activity--
eIF2B was assayed by its ability to
promote the release of [3H]GDP from preformed
eIF2·[3H]GDP complexes (8), except that time allowed
for formation of the eIF2·[ 3H]GDP binary complexes was
extended to 20 min. In some cases, assays were performed with
modifications (e.g. in the absence of unlabeled nucleotide)
as described in the figure legends.
UV Cross-linking--
UV cross-linking reactions were performed
using a Stratalinker 1800 (Stratagene). Reactions were 10 µl and
contained 20 mM Hepes/NaOH, pH 7.6, 100 mM KCl,
5 µl of eIF2B (2 pmol), either 2.5 µCi of
[ Cloning and Expression of His-eIF2B Dependence of eIF2B on Free Nucleotide for Catalytic Guanine
Nucleotide Exchange--
eIF2B was purified from HeLa cell extract
using a slightly modified version of the method used earlier for its
isolation from reticulocyte lysates (see "Materials and Methods").
This yielded protein displaying five polypeptides migrating on
SDS-PAGE. These polypeptides displayed expected apparent molecular
weights for subunits of mammalian eIF2B. This material was devoid of
eIF2, as assessed by staining with Coomassie Brilliant Blue (Fig.
2A) and by the much more stringent criterion of Western
blotting with a sensitive monoclonal antibody against eIF2
We (8) and others (30, 31) have previously reported that eIF2B failed
to catalyze the release of labeled GDP from
eIF2·[3H]GDP complexes unless excess unlabeled GTP was
present. In view of the aims of this study, it was important to show
that this was also true for this preparation of eIF2B, which is indeed
the case as shown in Fig. 2C; whereas rapid release of bound
[3H]GDP was observed when unlabeled GTP was present, very
little release was observed in the absence of added GTP. A small degree of release of [3H]GDP was seen when the higher
concentration of eIF2B was used, and this effect is discussed in
greater detail below. Two different models have been proposed to
explain the catalytic mechanism of eIF2B. These are depicted in Fig.
1. Briefly, in the ping-pong or
substituted enzyme model, the binding of eIF2B to eIF2·GDP complexes
elicits the release of the GDP, allowing GTP to bind to eIF2 to form
active eIF2·GTP complexes and resulting in the release of the eIF2B
(Fig. 1A). In this model, eIF2B is acting as a GDS.
Complexes between eIF2, GDP, and eIF2B presumably do form, but only
transiently, decaying as the GDP is released to yield eIF2·eIF2B
binary complexes. The second model (sequential, Fig. 1B)
involves the formation of a "ternary complex" containing eIF2·GDP, eIF2B and GTP. The data showing a requirement for added guanine nucleotide in order to observe the release of bound GDP from
eIF2 have been taken as support for the sequential mechanism (B in Fig. 1) (30, 31). However, consideration of the two mechanisms reveals that they are also entirely consistent with mechanism A. Indeed, such behavior is a prediction of this mechanism as
displayed in Fig. 1A. eIF2B is included in the assays used by ourselves and others at catalytic rather than stoichiometric amounts; in the assays depicted in Fig.
2C, typically the ratio of
eIF2:eIF2B is about 1000:1. Inspection of the mechanism shown in Fig.
1A shows that for eIF2B to act catalytically, it must be
released from the intermediate eIF2·eIF2B complex and that this
requires free nucleotide. Free nucleotide would act to regenerate eIF2B, thus allowing it to function as a catalyst and thus to mediate
further release of labeled GDP from the eIF2·[3H]GDP
complexes, which are the substrate for this reaction.
Since eIF2 can bind either GDP or GTP, one would expect that, under
this model, catalytic exchange would proceed with either free GTP or
GDP present. This is indeed the case for mammalian eIF2B (Fig.
2C and data not shown), as reported earlier for the yeast
factor (11). Consistent with the earlier findings of Cigan et
al. (11) for yeast eIF2B, ATP did not facilitate release of bound
GDP from eIF2 (data not shown).
Guanine Nucleotides Cause Dissociation of eIF2·eIF2B
Complexes--
A prediction of mechanism A, the ping-pong mechanism,
but not of mechanism B, is that incubation of eIF2·eIF2B complexes
with guanine nucleotides should cause such complexes to dissociate. The
eIF2·eIF2B complex was purified by ion-exchange chromatography on
MonoQ (Fig. 3A) and then
incubated with GDP, followed by addition of MgCl2 and
re-chromatography on MonoQ. During this second run, eIF2 was resolved
from eIF2B (which eluted later from the column; see also Ref. 8 and
Fig. 3 (B and C)). Chromatography of complexes that had not been pre-treated with GDP did not result in separation of
the two proteins, which instead again eluted as the eIF2·eIF2B complex (data not shown). To study this further, we performed similar
experiments using gel permeation chromatography rather than ion
exchange to resolve eIF2 or eIF2B from eIF2·eIF2B complexes. When
eIF2·eIF2B complexes were applied to a Superose 6 column without GDP
treatment, as expected, eIF2 and eIF2B eluted together (Fig.
3D). In contrast, when another sample of the eIF2·eIF2B complexes was incubated with GDP and then applied to a Superose 6 column, eIF2 (seen in fractions 27-29) clearly eluted later than eIF2B
(mainly in fractions 24-26), indicating that eIF2 and eIF2B had
dissociated from one another (Fig. 3E). These data are again
consistent with mechanism A, but not mechanism B, and support the
contention that the ability of free GDP or GTP to facilitate the
release of [3H]GDP bound to eIF2 is due to the
regeneration of free, active eIF2B. The data again support the idea
that eIF2B acts as a GDS.
Stoichiometric Amounts of eIF2B Can Mediate Nucleotide Release in
the Absence of Free Nucleotide--
An additional prediction of
mechanism A is that stoichiometric amounts of eIF2B should lead to
extensive release of GDP even in the absence of free nucleotide. That
this might be the case was suggested by the modest release of
[3H]GDP observed in Fig. 2C in the presence of
the higher amount of eIF2B. To test this further,
eIF2·[3H]GDP complexes were formed and subjected to gel
filtration to remove free nucleotide. Purified eIF2B was then added in
increasing amounts similar to or greater than the amount of
eIF2·[3H]GDP complexes present. Under these conditions,
release of labeled GDP was observed (Fig.
4A). Release was not complete,
but in each case leveled out at a maximum extent (as the reaction
attained equilibrium). As expected, the extent of release increased as larger amounts of eIF2B were used, since this will drive the
equilibrium further toward completion. This is precisely the behavior
predicted from the ping-pong mechanism, where eIF2B can bring about the release of amounts of GDP similar to the amount of eIF2B added, but
cannot act catalytically as it becomes locked into the eIF2·eIF2B complexes. The release of eIF2B from these complexes requires free GDP
or GTP, enabling the eIF2B now to act catalytically rather than
stoichiometrically. Consistent with this, upon addition of free
nucleotide, rapid complete loss of bound labeled GDP occurred, due to
the catalytic action of the eIF2B (Fig. 4B).
Cross-linking of Purine Nucleotides to eIF2B
To verify that GTP bound directly to eIF2B
eIF2B is also known to interact with NAD+ and related
compounds (5, 8). To examine whether the binding site for these compounds was also located in eIF2B eIF2B The data presented here show that eIF2B can elicit release of
[3H]GDP from eIF2 even in the absence of GDP or GTP. This
apparently contradicts earlier data from ourselves (8) and (1, 30) others; however, these earlier experiments were mostly performed using
catalytic amounts of eIF2B (i.e. low amounts compared with the substrate eIF2·[3H]GDP). We have reported
previously that approximately stoichiometric amounts of eIF2B prepared
from a different source did not elicit release of bound GDP from eIF2;
in fact, the present data show that a roughly equal amount of eIF2B
displaces approximately 50% of the nucleotide bound to eIF2. The
extent release of bound GDP at any given ratio of eIF2B to eIF2 depends
of course on the rate constants for the reaction eIF2·GDP + eIF2B Other studies have provided evidence that eIF2B can bind guanine
nucleotides, which is a requirement of the substituted enzyme mechanism. Wahba and colleagues (30) reported GTP binding to mammalian
eIF2B, although the stoichiometry was low. Similarly, recent data from
Hannig's group (31) for yeast eIF2B show only a stoichiometry of only
0.2 mol/mol, even at a nucleotide concentration almost 3 orders of
magnitude above the apparent dissociation constant. In this work we
were unable to detect significant binding of GTP to purified eIF2B in
filter binding assays. In addition, eIF2B does not bind to GDP-agarose
(data not shown), although this could reflect steric problems due
either to the means of attachment of the GDP to the resin (via the
ribose hydroxyls) or to the large size of the eIF2B complex. It is
possible that either the off-rates for guanine nucleotides are very
high, despite the low apparent Kd reported by
others, so that little or no stable binding is obtained, or that the
low stoichiometry of binding seen by others reflects the presence of a
contaminating nucleotide-binding protein, although none was evident on
the gels shown by earlier workers (1, 30). We do nonetheless observe
cross-linking of GTP to eIF2B, specifically to eIF2B We found that addition of NAD+ did not reduce the labeling
of eIF2B As noted above, the substituted enzyme mechanism requires that eIF2B
possesses a binding site for GTP. Although our data suggest that there
may be such a site, it is important to note that this resides in
eIF2B It has previously been suggested that purine nucleotides might act as
allosteric modulators of eIF2B (39). Indeed, Kimball and Jefferson (9)
have shown that ATP inhibits the activity of eIF2B. This effect is
surprising in physiological terms, as one might expect enhanced
cellular energy status, if anything, to activate eIF2B rather than
inhibit it. Their data suggested that the effect might be mediated by
antagonizing the activating effect of NADPH (9), which, based on our
findings, is presumably mediated by a different binding site in eIF2B
(since NAD+ did not reduce the labeling of eIF2B A third area of conflicting data relating to eIF2B is the role of its
smallest 32 (-
. The largest of these (
) displays catalytic
activity in the absence of the others. The catalytic mechanism of eIF2B
and the functions of the other subunits remain to be clarified. Here we
show that, when present at similar concentrations to eIF2, mammalian
eIF2B can mediate release of eIF2-bound GDP even in the absence of free nucleotide, indicating that it acts as a GDP dissociation stimulator protein. Consistent with this, addition of GDP to purified eIF2·eIF2B complexes causes them to dissociate. The alternative sequential mechanism would require that eIF2B
itself bind GTP. However, we show
that it is the
-subunit of eIF2B that interacts with GTP. This
indicates that binding of GTP to eIF2B is not an essential element of
its mechanism. eIF2B preparations that lack the
-subunit display
reduced activity compared with the holocomplex. Supplementation of such
preparations with recombinant eIF2B
markedly enhances activity,
indicating that eIF2B
is required for full activity of
mammalian eIF2B.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunit of eIF2 inhibits eIF2B and serves to inhibit translation
under a range of stressful conditions (1). Conversely, eIF2B is
activated by many stimuli, which stimulate protein synthesis, including insulin, and this appears to involve changes in the state of
phosphorylation of the largest (
) subunit of eIF2B (3, 4). eIF2B has
also been reported to be regulated, in vitro, by a number of
allosteric modulators, including nicotinamide adenine nucleotides
(5-9). Studies using photoactivatable nucleotide analogs suggested
that eIF2B contains binding sites for GTP and ATP, although the
functions of these sites are unclear.
-subunit
alone can catalyze nucleotide exchange on eIF2 (17-19), although its
activity is enhanced by the others (18-20). eIF2B
forms a catalytic
subcomplex with eIF2B
, with which it shares some sequence
similarity. The other three subunits also display mutual sequence
similarity and form a second subcomplex, which appears to have a
regulatory function (19). There is some disagreement over the role of
the
-subunit. This polypeptide is encoded by a non-essential gene in
yeast (GCN3), although it is required for regulation of
eIF2B by phosphorylation of eIF2
(21). Similarly, studies in which
mammalian eIF2B polypeptides were expressed in insect cells concluded
that eIF2B
was dispensable for activity (22). However, others have
found that preparations of eIF2B lacking the
-subunit showed reduced
activity in nucleotide exchange (23), implying that this component was
required for full activity.
contains a binding site for purine
nucleotides and that eIF2B
is required for the full activity of
mammalian eIF2B.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]GTP (5000 Ci/mmol), and [
-32P]ATP (30 Ci/mmol) were from
Amersham International. X-ray film was from Konica. Restriction enzymes
were from Promega.
-32P]GTP or 2.5 µCi of [
-32P]ATP
(as indicated), and either 0.5 or 2.5 µM unlabeled
nucleotide (also as indicated). Reactions were incubated at 30 °C
for 5 min prior to exposure to UV radiation (1.2 J/cm2) for
20 min on ice. Samples were then subjected to SDS-PAGE, and gels were
stained with Coomassie Brilliant Blue and dried prior to exposure to
x-ray film. Control reactions were either incubated at 30 °C for 5 min and then on ice for 20 min without exposure to UV radiation, or
incubated on ice only, as detailed in the figure legend.
and
His-eIF2B
--
eIF2B
in pET15b was provided by Dr. J. K. Tyzack
(a former member of the laboratory at Dundee and Canterbury), and
eIF2B
in pET11a was provided by Dr. B. Craddock (a former member of our laboratory at Bristol). The regions encoding the open
reading frames for both subunits were excised (using NdeI
and BamHI for eIF2B
and NdeI and
XhoI for eIF2B
) and ligated into pET28c (Novagen). Vectors were then transformed into Escherichia coli (BL21
DE3 pLysS) and grown overnight in LB containing 100 µg/ml kanamycin. They were then diluted 10-fold and grown to an
A600 of 0.6. Cultures were cooled on ice for 15 min, and expression was induced with 1 mM
isopropylthio-
-D-galactoside for 2.5 h. Cells were
the harvested by centrifugation (3500 × g) and lysed
in 5% glycerol, 500 mM KCl, 20 mM Tris/HCl, pH
7.6, 3 mM MgCl2, 5 mM
-mercaptoethanol, 10 mM imidazole, 0.1% Triton X-100, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml protease inhibitors (leupeptin, pepstatin, aprotinin, and
benzamidine) at
80 °C. Following lysis cells were defrosted and
sonicated to shear any DNA present. Purification of proteins was
performed using nickel-nitrilotriacetic acid-agarose (Qiagen).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(Fig.
2B). Such preparations are highly active in catalyzing
exchange of labeled GDP bound to eIF2 for unlabeled GTP (Fig.
2C).
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Fig. 1.
Proposed mechanisms for the
eIF2B-mediated exchange of nucleotides on eIF2. A,
ping-pong or substituted enzyme-mechanism; binding of eIF2B to
eIF2·GDP brings about release of GDP. Subsequent binding of GTP to
eIF2 leads to formation of active eIF2·GTP binary complexes and
release of eIF2B, regenerating free eIF2B for further rounds of
catalysis (dotted line). B,
sequential mechanism; this involves formation of a ternary complex
(eIF2·GDP·eIF2B·GTP) and subsequent release of GDP and active
eIF2·GTP.
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Fig. 2.
Characterization of purified human eIF2 and
eIF2B. A, gel of purified eIF2 and eIF2B preparations
to show relative concentrations of protein. Samples of purified eIF2 or
eIF2B (as indicated) were applied at differing dilutions to an
SDS-polyacrylamide gel, which was then stained with Coomassie Brilliant
Blue and thoroughly destained. Shown are the regions corresponding to
eIF2 and eIF2B
. We chose to use these polypeptides for
comparative purposes as they possess almost identical molecular masses.
B, purified eIF2 and eIF2B were diluted as indicated and 10 µl were analyzed by SDS-PAGE, followed by Western blotting using
antisera to eIF2
and eIF2B
. The positions of these proteins on
the resulting immunoblot are indicated. C, binary
eIF2·[3H]GDP complexes were formed using fraction and
gel-filtered over Sephadex G-50 to remove free labeled nucleotide.
Complexes (4 pmol) were then incubated with no further addition, or
with eIF2B (75 or 750 fmol, as indicated) and, where indicated,
GTP/MgCl2 (to give final concentrations of 0.5 and 1.5 mM, respectively). Reaction mixtures (total volume, 60 µl) were incubated at 30 °C. At the times indicated, samples were
removed and filtered through nitrocellulose membranes. Residual
[3H]GDP binding was determined by scintillation
spectrometry. Zero time values were obtained by processing samples of
the binary complex, which had not been incubated with eIF2B or
GTP/MgCl2. D, Coomassie-stained gel of the
purified eIF2B used in these experiments; positions of its five
subunits are indicated.
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Fig. 3.
Addition of GDP causes dissociation of
eIF2·eIF2B complexes. eIF2·eIF2B complexes were isolated as
described under "Materials and Methods." A, Western blot
of consecutive fractions from MonoS containing eIF2/eIF2B complexes as
demonstrated using antisera for eIF2 and eIF2B
(positions shown).
B, a Coomassie-stained gel of the complex is shown
(eIF2/eIF2B). GDP (final concentration 200 µM)
was added to the complex (in Buffer A: 20 mM HEPES-KOH, pH
7.4, 100 mM KCl, 5% (v/v) glycerol, 1 mM
dithiothreitol), and this was incubated for 30 min at 30 °C.
MgCl2 was then added (final concentration, 5 mM), and the material was applied to a MonoQ column
attached to a fast protein liquid chromatography system equilibrated in
the same buffer supplemented with GDP (1 µM) and
MgCl2 (5 mM). The column was developed with a
salt gradient (KCl), and fractions were collected. Selected fractions
were analyzed by SDS-PAGE followed by Coomassie staining or
immunoblotting (shown in panel C). Fractions 20 and 29 correspond to KCl concentrations of 250 and 350 mM,
respectively. In panel B, the migration positions
of the subunits of eIF2 (
-
) and eIF2B (
-
) are shown. The
single bands in lanes 23-26 are contaminating
proteins resolved on the MonoQ column and are not subunits of eIF2 or
eIF2B. Vertical lines indicate separate gels,
which have been combined for presentation. It should be noted that
eIF2B
stains relatively weakly with Coomassie Blue when compared
with the other subunits of the complex. C, this panel shows
immunoblots developed with antisera against eIF2B
or eIF2
as
indicated. Note that, on the immunoblot for eIF2
, the peak
fraction (28) was loaded at one-tenth the amount of the others. The
last lane in each case shows the signal from the
initial eIF2·eIF2B complex run in parallel. eIF2 is eluted at
240-260 mM KCl, whereas eIF2B, free of eIF2, was eluted
between 335 and 350 mM KCl. When a sample of eIF2·eIF2B
was subjected to ion exchange on MonoQ without prior treatment with
GDP, and without GDP in the buffer, as a control, it was eluted at
~290-315 mM KCl as the complex, and no separation of
eIF2 from eIF2B was observed (data not shown). D, purified
eIF2·eIF2B complexes were applied to a Superose 6 column equilibrated
in Buffer A containing 5 mM MgCl2. Fractions
were collected, and the figure shows a Coomassie Blue-stained gel of
those containing eIF2 and eIF2B. E, a second sample of the
same material was preincubated with GDP (1 µM) for 15 min
on ice prior to addition of MgCl2 (to 5 mM) and application to the
same column now equilibrated in Buffer A with 5 mM
MgCl2 and 1 µM GDP. Again, a
Coomassie-stained SDS-polyacrylamide gel is shown. The migration
positions of the subunits of eIF2 and eIF2B are indicated by
labeled arrows. Similar data, showing separation
of eIF2 from eIF2B in the presence of GDP but not in its absence, were
obtained in four independent experiments.
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Fig. 4.
eIF2B can mediate release of GDP from eIF2 in
the absence of free nucleotide. A,
eIF2·[3H]GDP complexes were formed and subjected to gel
filtration to remove free GDP. Reaction mixtures (60 µl) contained 3 pmol of eIF2·[3H]GDP complexes and were incubated at
30 °C with no added eIF2B (control) or 1, 2.5, or 5 pmol eIF2B as
indicated. GTP (0.5 mM final concentration, plus same
concentration of MgCl2) was added to one reaction as
indicated. Samples were removed at the times shown and processed to
determine the [3H]GDP remaining associated with the eIF2.
Zero time values are for eIF2·[3H]GDP complexes that
had not been incubated. B, as panel A
but using 4.5 pmol of eIF2·[3H]GDP/incubation and
different amounts of eIF2B, as indicated. GTP was added after 20 min of
incubation to a final concentration of 0.5 mM for another
10 min, and a final sample was taken.
--
Mechanism B
requires that eIF2B contains a binding site for GTP and two earlier
reports (for eIF2B from mammals (30) and yeast (31), respectively, have
provided data indicating that eIF2B can bind GTP, albeit only to a low
stoichiometry (~0.2 mol of GTP/mol of eIF2B). Since eIF2B
displays
nucleotide exchange activity when expressed alone (17-19), one would
expect that, if mechanism B were the mechanism of action of eIF2B,
eIF2B
should possess a binding site for GTP. However, we were unable
to observe any significant binding of [
-32P]GTP to our
purified preparations of eIF2B complex in filter binding assays
(binding <10% of the level seen for eIF2 on a molar basis; data not
shown). Nevertheless, we pursued this issue further by using UV
irradiation to cross-link [
-32P]GTP to purified eIF2B.
(This material is free of eIF2, thus obviating the problem of GTP
binding to any eIF2 present.) We elected to perform these studies using
unmodified GTP rather than 8-azido-GTP (where the photoactivatable
label is in the base), as used previously (32), since use of nucleotide
analogs can give misleading results due to cross-linking to adjacent
subunits in a protein complex rather than to the subunit which actually binds the nucleotide. An excellent example of this is eIF2 itself, where 8-azido-GTP (32) or other GTP analogs label eIF2
or -
(33,
34) rather than, or as well as, eIF2
, the subunit that actually
contains the GTP-binding site (35, 36). It could therefore be that the
earlier studies, where 8-azido-GTP was cross-linked to eIF2B
, rather
than eIF2B
as expected for mechanism B, were also misleading. Our
cross-linking studies using [
-32P]GTP resulted in
labeling of a polypeptide migrating at ~40 kDa, i.e. in
the position of eIF2B
(Fig.
5A), confirming the earlier study of Haley et al. (32), who found that this polypeptide was also labeled by azido-GTP. However, in contrast to that study, labeling was reduced by the presence of cold ATP as well as by cold GTP
(Fig. 5A). Similar labeling experiments using
[
-32P]ATP led to labeling of the same polypeptide,
rather than eIF2B
and -
reported by Haley et al. (32).
To rule out the possibility that labeling occurred by transfer of the
-phosphate (e.g. by a contaminating kinase), reactions
were also performed without UV irradiation. Under this condition, no
labeling was indeed observed (Fig. 5B, lane
2). Labeling was decreased by addition of cold GTP or ATP
(Fig. 5C), GTP being slightly more effective than ATP. This
suggests that ATP and GTP bind to the same (or overlapping) sites in
eIF2
.
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Fig. 5.
Ultraviolet cross-linking of eIF2B to
nucleoside triphosphates. A, cross-linking with
[ -32P]GTP with competitors. eIF2B (0.5 pmol) was
incubated with [
-32P]GTP as described under
"Materials and Methods." Unlabeled nucleotides were included in the
reactions at either 2.5 µM or 0.5 µM as
indicated. None indicates that no unlabeled nucleotide was
added. Samples were analyzed by SDS-PAGE and autoradiography. The
figure shows an autoradiograph. The position of migration of eIF2B
is marked by a labeled arrow. B,
samples of eIF2B complex were subjected to UV cross-linking with
[
-32P]GTP (lane 1), incubated in
the same manner except without exposure to UV radiation
(lane 2), or denatured immediately after addition
of [
-32P]GTP (lane 3), followed
by SDS-PAGE. The figure is an autoradiograph of the dried gel. The
position of eIF2B
is indicated. C, as panel
A but [
-32P]ATP was employed in place of
[
-32P]GTP. D, His-eIF2B
was expressed in
E. coli and purified as described under "Materials and
Methods." Lane 1 shows a Coomassie-stained gel
of the eIF2B
obtained. Lanes 2-4 show results
of experiments in which samples of His-eIF2B
were subjected to UV
cross-linking with [
-32P]GTP (lane
2), incubated in the same manner except without exposure to
UV radiation (lane 3), or incubated on ice only
(lane 4) followed by SDS-PAGE. The figure is an
autoradiograph of the dried gel. The position of the His-eIF2B
is
indicated.
, we expressed this
polypeptide in E. coli as a His-tagged protein, purified it on nickel-agarose, and tested whether [
-32P]GTP became
cross-linked to it. As shown in Fig. 5D (lane
2), UV irradiation did indeed result in cross-linking of
labeled GTP to recombinant eIF2B
. This confirms that GTP interacts
directly with this subunit and thus that the cross-linking to this
polypeptide observed for the complex is not a consequence of its
binding to another subunit at a site adjacent to eIF2B
in the
holoprotein. As a negative control, reactions were again performed in
the same way but without UV irradiation. In this case, no
cross-linking was observed, once more ruling out the theoretical
possibility that labeling by [
-32P]GTP was due to
phosphorylation of eIF2B
by a contaminating kinase (Fig.
5D, lanes 3 and 4).
, we performed UV cross-linking experiments with radioactive NAD+. However, this failed to
result in labeling of any of the polypeptides of eIF2B, although
lactate dehydrogenase, used as a positive control, was labeled (data
not shown). In another approach, we examined whether NAD+
or NADPH competed with GTP or ATP in the UV cross-linking experiments (Fig. 5). It did not; in fact, a reproducible increase in cross-linking was observed. It therefore appears that the nucleotide-binding site in
eIF2B
is not the site responsible for binding NAD+,
which presumably resides elsewhere in the protein. The ability of
NAD+ to enhance labeling by [
-32P]GTP or
ATP may reflect allosteric interactions between the binding sites for
these ligands.
Is Required for Full Activity of Mammalian eIF2B--
As
reported previously (23), isolation of mammalian eIF2B from rabbit
reticulocytes yields a proportion of the protein as a four-subunit
complex lacking the
-subunit. The same is true of eIF2B prepared
from HeLa cells (Fig. 6A).
Chromatography on MonoS yielded two pools of eIF2B; the material
eluting at 290-320 mM KCl showed only four subunits on
SDS-PAGE (Fig. 6, A and B), whereas the material
eluting at 330-370 mM showed five. The absence of eIF2B
from the former material was confirmed using an antibody specific for
the
-subunit of eIF2B (Fig. 6A). No signal for eIF2B
was observed in these fractions, whereas a clear signal was seen for
the factor displaying all five subunits on SDS-PAGE (Fig. 6B). When the activities of the four- and five-subunit
preparations were compared, the former was found to display only about
20-25% of the activity of the five-subunit material in the standard
nucleotide exchange assay (Fig. 6C). This is consistent with
our earlier data for the four- and five-subunit preparations of the
factor from rabbit reticulocytes (23). To confirm that the absence of
eIF2B
from the four-subunit material was the explanation of its low
activity, mammalian eIF2B
(rat) was expressed in
E. coli (as a His-tagged polypeptide) and purified. This
yielded material showing a single protein band on SDS-PAGE (Fig.
6C) that displayed no nucleotide exchange activity on its
own (Fig. 6C). Supplementation of four-subunit eIF2B with
recombinant rat eIF2B
markedly stimulated its activity, almost to
the levels observed for the five-subunit protein (Fig. 6C).
Thus, the deficiency in activity of eIF2B
relative to the
heteropentamer does indeed appear to be due to lack of the
-subunit
rather than any other defect.
View larger version (32K):
[in a new window]
Fig. 6.
The activity eIF2B lacking the
-subunit is enhanced by adding recombinant
eIF2B
. A, immunoblots of
purified eIFB lacking the
-subunit (lane 1),
five-subunit eIF2B (lane 2), and eIF2/eIF2B
complex probed with the anti-eIF2B
and -eIF2B
antisera.
B, Coomassie-stained gel of the samples shown in
A; eIF2 is also included (lane 4).
C, guanine nucleotide exchange assay showing the activity of
four subunit eIF2B, the activity of the four subunit eIF2B following a
pre-incubation of 10 min at 30 °C with recombinant rabbit His-tagged
eIF2B
and the activity of five subunit eIF2B. Also shown is an
immunoblot of the recombinant His-eIF2B
using the anti-His antisera
(inset). (Data show S.E. where n = 3.)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
eIF2·eIF2B + GDP. The ping-pong mechanism does indeed predict that
the catalytic function of eIF2B requires free nucleotide in order to
regenerate eIF2B from the eIF2·eIF2B complexes, which are an
intermediate in this mechanism (Fig. 1A). When added at
stoichiometric amounts, as in this study, this mechanism predicts that
GDP will be released from eIF2, as eIF2B displaces the nucleotide from
eIF2. In complete accordance with this, we show that the addition of
GDP to eIF2·eIF2B complexes causes them to dissociate, again as
expected from the ping-pong mechanism. Neither of these effects is
consistent with the other mechanism proposed for eIF2B (sequential
mechanism, Fig. 1B). In our study we also failed to detect
eIF2B bound to eIF2·GDP, an intermediate that may be formed in the
substituted enzyme mechanism, but that cannot arise in the ping-pong
mechanism. Thus, our data are in agreement with the ping-pong mechanism
but do not, of course, prove that this is the mechanism by which eIF2B functions. Another GEF involved in translation, EF-Ts, also uses the
substituted enzyme mechanism (25). RCC1 and Cdc25Mm,
exchange factors for the small GTP-binding proteins Ran and Ras,
respectively, have been shown to operate by an analogous mechanism
(26-28), and recent data support such a mechanism for the guanine
nucleotide dissociation factor that acts on Rho (29). It seems
plausible that the substituted enzyme mechanism is a general one by
which such proteins function, although the fine details of the
mechanism at the structural level are likely to vary (37, 38). The data
in Figs. 2 and 6 suggest that eIF2B acts as a GDS (by promoting the
release of GDP from eIF2 rather than the exchange of GDP for GTP; GTP
binds spontaneously to the unliganded eIF2).
. This suggests
that the protein does indeed bind guanine nucleotides and that this
subunit is involved in the interaction, as concluded by the earlier
studies of Haley et al. (32), who used a
photoaffinity-labeling reagent (8-azido-GTP) rather than normal GTP as
used here. In our study, cross-linking of [
-32P]GTP
was competed by ATP, and, consistent with this,
[
-32P]ATP also became cross-linked to this subunit.
These findings differ from the earlier results (32) where labeling by
GTP was not affected by ATP, and where 8-azido-ATP became cross-linked to the
and
subunits. It is not clear how this discrepancy is to
be explained. Our data suggest that the binding site in eIF2B
interacts with both ATP and GTP. The observation that the isolated
recombinant
-subunit is also labeled by [
-32P]GTP
strongly supports the idea that this subunit directly binds guanine
nucleotides, and that its labeling is not merely a consequence of the
binding of GTP to an adjacent subunit in the complex. Since the
sequence of eIF2B
contains no identifiable nucleotide-binding motifs, its interaction with GTP and ATP must involve a non-canonical binding site.
by GTP or ATP, indicating that the binding site in eIF2B
is distinct from that through which nicotinamide adenine dinucleotides exert their allosteric effects upon eIF2B activity. We were unable to
achieve labeling of eIF2B using radiolabeled NAD+ and were
thus also unable to try to locate the binding site in eIF2B for this nucleotide.
, and not in the catalytic
-subunit. This is a key point,
since if eIF2B utilized the substituted enzyme mechanism, this site
would necessarily be present in eIF2B
, which has been shown to
catalyze nucleotide exchange on its own, based on data from three
different groups for the protein from yeast, insects, and mammals
(17-19). These findings thus also argue against the substituted enzyme
mechanism. Taken together, the present data suggest that eIF2B mediates
release of GDP from eIF2, allowing the subsequent binding of GTP (the
more abundant of these two nucleotides in the cell). If this is the
case, then eIF2B acts as a GDS protein rather than a nucleotide
exchange factor (a name which could be taken to imply catalysis of the
GDP/GTP exchange step itself). The physiological role of eIF2B is of
course to promote this exchange process, but our data suggest that this is a consequence of its ability to bring about the release of GDP from
eIF2, with binding of GTP occurring to the resulting nucleotide-free eIF2.
by
GTP). The function of nucleotide binding to eIF2B
therefore remains unclear.
) subunit. Earlier studies agree that this subunit is
required for the regulation of eIF2B activity by phosphorylation of
eIF2
(22, 40, 41). However, genetic analysis in yeast or expression
of mammalian eIF2B subunits in insect cells suggested that eIF2B
was
not required for activity in vivo or in vitro,
respectively. In contrast, our data for preparations of eIF2B that lack
this subunit indicated it was necessary for full activity (23). The
findings reported here confirm this for eIF2B isolated from a different
source (HeLa cells versus reticulocyte lysate) and, most
importantly, demonstrate that addition of recombinant eIF2B
almost
completely restores eIF2B activity. This eliminates the possibility
that the low activity of four-subunit eIF2B is due to other defects in
the protein and demonstrates that, under in vitro assay
conditions, the mammalian factor does require eIF2B
for full
activity. Possible explanations for the differences between our data
and those of earlier studies include (i) for the yeast studies,
complementation of GCN3 by the product of another gene, or
differences between the function of eIF2B in vitro and
in vivo and (ii) for the studies in Spodoptera
cells, the presence of endogenous insect eIF2B
in the complexes
containing the four subunits of eIF2B from mammals, although no band is
apparent at this position in the authors' SDS-polyacrylamide gels.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Linda Campbell and Dr. Xuemin Wang for valuable assistance with the preparation of initiation factors.
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FOOTNOTES |
---|
* This work was supported by a committee studentship (to D. D. W.) and a project grant from the Biotechnology and Biological Sciences Research Council (to C. G. P.) and by Project Grant 037005 and Program Grant 046110 from the Wellcome Trust (to C. G. P.).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.
§ Present address: Avidex Ltd., 57c Milton Park, Abingdon OX14 4RX, United Kingdom.
Present address: Dept. of Cell Biochemistry, Hannah Research
Inst., Ayr, Scotland KA6 5HL, United Kingdom.
Present address: Dept. of Biological Sciences, Open University,
Walton Hall, Milton Keynes MK7 6AA, United Kingdom.
§§ To whom correspondence should be addressed: Div. of Molecular Physiology, School of Life Sciences, Medical Sciences Institute/Wellcome Trust Biocentre Complex, University of Dundee, Dow St., Dundee DD1 5EH, Scotland, United Kingdom. Tel.: 44-1382-344919; Fax: 44-1382-322424; E-mail: c.g.proud@dundee.ac.uk.
Published, JBC Papers in Press, April 25, 2001, DOI 10.1074/jbc.M011788200
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ABBREVIATIONS |
---|
The abbreviations used are: eIF, eukaryotic initiation factor; GDS, guanine nucleotide dissociation stimulator; GEF, guanine nucleotide exchange factor; Met-tRNAi, initiator methionyl-tRNA; PAGE, polyacrylamide gel electrophoresis.
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