From the Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75083
Received for publication, August 14, 2000, and in revised form, October 5, 2000
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ABSTRACT |
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Eukaryotic translation initiation factor eIF2 is
a heterotrimer that binds and delivers
Met-tRNAiMet to the 40 S
ribosomal subunit in a GTP-dependent manner. Initiation requires hydrolysis of eIF2-bound GTP, which releases an eIF2·GDP complex that is recycled to the GTP form by the nucleotide exchange factor eIF2B. The Eukaryotic translation initiation factor
eIF21 is a heterotrimeric
GTP-binding protein that in yeast is encoded by the essential genes
SUI2, SUI3, and GCD11. eIF2 binds
charged initiator tRNA (Met-tRNAiMet) in
a GTP-dependent manner to form a stable ternary complex
that interacts with the small ribosomal subunit and additional
initiation factors (including eIF3 and eIF1A) to form a 43 S initiation
complex (reviewed in Refs. 1 and 2-5). This complex binds at or near the 5'-end of capped mRNAs and "scans" the mRNA in a 5' to
3' direction until an AUG start codon in proper context is encountered. Genetic analyses in yeast have demonstrated a function for eIF2 in the
selection of the translational start site (6, 7). Recognition of the
start codon is accompanied by eIF5-mediated hydrolysis of eIF2-bound
GTP, which facilitates release of an inactive eIF2·GDP binary complex
as well as several other initiation factors. The 40 S/mRNA/Met-tRNAiMet complex
interacts with a 60 S ribosomal subunit to form an 80 S initiation
complex that can then enter the elongation phase of protein synthesis.
Conversion of the inactive eIF2·GDP binary complex to eIF2·GTP,
which is capable of rebinding initiator tRNA, is catalyzed by the
nucleotide exchange factor eIF2B.
To execute its functions in translation initiation, eIF2 must interact
with multiple ligands as well as other components of the translational
apparatus. The former include guanine nucleotides and initiator tRNA,
whereas the latter may include ribosomes, mRNA, and other
initiation factors including eIF2B and eIF5. Several studies have been
conducted to characterize the function of individual eIF2 subunits.
Both yeast and mammalian eIF2 Cross-linking studies have also implicated the Whereas many functions ascribed to eIF2 can be attributed to the We recently demonstrated2
that lethality associated with deletion of the yeast SUI2
gene (eIF2 The construction of strains completely lacking eIF2 Materials and Reagents--
[8,5'-3H]GDP (5-15
Ci/mmol) and
L-[methyl-3H]methionine (70-85
Ci/mmol) were from PerkinElmer Life Sciences;
[ Aminoacylation of Yeast Initiator tRNA--
Yeast methionyl
initiator tRNA was aminoacylated with
L-[3H]methionine as described previously
(14), except that methionine was present at 3 µM, and
tRNA was present at 12.5 µg/µl. Typically, 200-400 pmol (specific
activity 7.8-9.0 × 104 cpm/pmol) of trichloroacetic
acid-precipitable [3H]methionine was obtained from a 1-ml reaction.
Purification of eIF2 and eIF2 Purification of eIF2B and Guanine Nucleotide Exchange
Assay--
Purification of yeast eIF2B from an overexpressing yeast
strain (H2649) and steady-state kinetic analysis of eIF2B-catalyzed guanine nucleotide exchange were performed as described previously (42). In preliminary experiments, the presence of 5 mM GDP
as chase was determined to be saturating for the eIF2 Purification of eIF5--
The wild-type TIF5 (yeast
eIF5) open reading frame plus 290 base pairs of 3'-flanking sequence
was subcloned as a 1.5-kilobase NdeI/BamHI
polymerase chain reaction product into a pET-3A expression vector
(Novagen) such that the NdeI site included the authentic eIF5 start codon. The entire polymerase chain reaction fragment was
sequenced to ensure the absence of polymerase chain reaction-derived mutations. The expression construct or the pET-3A vector alone (for
preparation of a control extract) was introduced into Escherichia coli strain BL21(DE3/pLysS) (43). The purification
procedure was modified from Chaudhuri et al. (23). Cultures
for protein purification (500 ml) were grown at 37 °C, 300 rpm to an
A600 of 1.25, induced with 0.1 mM
isopropyl-1-thio- Nucleotide Binding Assays--
Assays to determine intrinsic
dissociation of guanine nucleotides from eIF2 complexes were performed
at 0 °C as described previously (14).
43 S Complex Stability Assay--
The 43 S complex stability
assay was conducted in a three-stage reaction at 22 °C. The first
stage consisted of ternary complex formation by incubation of 4.4 µg
of yeast eIF2 with 24 pmol of [3H]Met-tRNAiMet for 15 min in a total volume of 110 µl (20 m Tris-HCl, pH 7.5, 100 mM KCl, 0.1 mM Na2EDTA, 1 mg/ml
creatine kinase, 1 mM dithiothreitol, 1 mM GTP
(or Gpp(NH)p), 5 mM MgCl2, 5% glycerol). Stage
two consisted of the addition of 1.0 A260 unit
of 40 S Artemia ribosomes and 0.2 A260 units of AUG codon (Oligos, Etc.,
Wilsonville, OR) followed by a 10-min incubation. For stage 3, the
reaction mixture was split into 2 equal aliquots, and 1 µg of eIF5 or
an equal volume of a control extract without eIF5 (see above) was
added. After a 10-min incubation, the reaction mixture was chilled on
wet ice, layered onto a 5-ml 5-25% sucrose gradient (20 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.1 mM Na2EDTA, 1 mM dithiothreitol, 5 mM MgCl2), and centrifuged at 200,000 × g for 2 h 20 min. Fractions (300 µl) were collected
from the top of the gradient using an ISCO model 640 gradient
fractionator with continuous monitoring at 254 nm. The percentage of
each fraction was measured for radioactivity by scintillation counting
in 5 ml of UltimaGold (Packard Instrument Co.).
Absence of the eIF2
To determine the requirement for the Removal of the eIF2 eIF2
We used steady-state kinetic analysis to better characterize
eIF2 Considerable progress has been made toward understanding the
function of individual eIF2 subunits as relates to the overall activity
of the eIF2 heterotrimer. In the case of the Removal of the Ternary complexes containing eIF2 Although the absence of the Regardless of the precise mechanism, our results indicate that the
-subunit of eIF2 plays a critical role in
regulating nucleotide exchange via phosphorylation at serine 51, which
converts eIF2 into a competitive inhibitor of the eIF2B-catalyzed
exchange reaction. We purified a form of eIF2 (eIF2
) completely
devoid of the
-subunit to further study the role of eIF2
in eIF2
function. These studies utilized a yeast strain genetically altered to
bypass a deletion of the normally essential eIF2
structural gene
(SUI2). Removal of the
-subunit did not appear to
significantly alter binding of guanine nucleotide or
Met-tRNAiMet
ligands by eIF2 in vitro. Qualitative assays to
detect 43 S initiation complex formation and eIF5-dependent
GTP hydrolysis revealed no differences between eIF2
and the
wild-type eIF2 heterotrimer. However, steady-state kinetic analysis of
eIF2B-catalyzed nucleotide exchange revealed that the absence of the
-subunit increased Km for eIF2
·GDP by an
order of magnitude, with a smaller increase in
Vmax. These data indicate that eIF2
is
required for structural interactions between eIF2 and eIF2B that
promote wild-type rates of nucleotide exchange. We suggest that this
function contributes to the ability of the
-subunit to control the
rate of nucleotide exchange through reversible phosphorylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
proteins exhibit a high degree of
amino acid sequence similarity to bacterial EF1A (formerly EF-Tu) and
SELB proteins (8-11), and in vitro cross-linking studies
have implicated the
-subunit in binding of both guanine nucleotide
and Met-tRNAiMet ligands (8, 12, 13).
The implied functional similarity with EF1A-like proteins was
substantiated by mutational analysis, where point mutations in the
structural gene encoding yeast eIF2
(GCD11) conferred
specific biochemical defects in either nucleotide or tRNA binding in
the altered eIF2 proteins (14). These defects were predicted by the
EF1A structural model, confirming the notion that eIF2
provides
EF1A-like function to the eIF2 complex. Other studies demonstrated
cross-linking of the
-subunit to 18 S rRNA, suggesting an
involvement in ribosome interaction and/or 43 S complex formation (15).
Although the role of eIF2
in GTP binding implies a further role in
GTP hydrolysis and nucleotide exchange, there is currently no data to
support direct contact between the
-subunit of eIF2 and either eIF5
or eIF2B.
-subunit of eIF2 in
nucleotide- and Met-tRNAiMet binding (8,
12, 16, 17), indicating at the very least a proximity of this subunit
to binding sites present on eIF2
. A role for eIF2
in tRNA binding
was suggested by analysis of partially purified eIF2 preparations from
yeast strains harboring mutations in the eIF2
structural gene
(SUI3) (18). The function of eIF2
has also been examined
using preparations of mammalian eIF2
obtained as a result of
proteolysis of the
-subunit either during purification or
purposefully at the hands of the experimenter (Ref. 19 and references
therein). These studies demonstrated little effect on the binding of
guanine nucleotides in the absence of intact eIF2
. However, the
eIF2
complex was severely defective in the formation of ternary
complexes, suggesting the
-subunit was involved in
Met-tRNAiMet binding. The eIF2
preparations also showed reduced rates of eIF2B-catalyzed exchange
in vitro, suggesting that the
-subunit plays a role in
catalyzed nucleotide exchange. This is consistent with recent reports
demonstrating interaction between the C terminus of eIF2
and the
- and
-subunits of mammalian eIF2B (20) as well as between the N
terminus of eIF2
and the
-subunit (Gcd6p) of yeast eIF2B (21).
The
-subunit of eIF2 also appears to interact directly with eIF5
(21-24). Curiously, both eIF5 and eIF2B
contain C-terminal
bipartite motifs rich in aromatic and acidic amino acids that are
important for interactions with eIF2
(21).
-
and
-subunits, comparatively little is known about the contribution
of the
-subunit to eIF2 function. Two prior biochemical studies
using
-deficient forms of eIF2 (i.e. containing low
levels of contaminating eIF2
protein) yielded different results in
comparing specific activities of
-deficient and wild-type eIF2
preparations (16, 25). The
-subunit has been cross-linked to 18 S
rRNA, suggesting an involvement in 43 S complex formation (15), and also appears to contain conserved sequences similar to the S1-RM repeated sequence motif present in bacterial ribosomal protein S1 and
thought to be involved in RNA binding (26). However, the
best-characterized aspect of eIF2
is its role in regulating eIF2
function (reviewed in Refs. 5, 27, 28, and 29). Phosphorylation of the
-subunit at a conserved serine residue (Ser-51) converts eIF2 to a
competitive inhibitor of eIF2B-catalyzed nucleotide exchange (30, 31).
This results in functional sequestration of limiting amounts of eIF2B
and leads to decreased levels of active eIF2 and global protein
synthesis. Regulation of eIF2 activity in this manner is conserved
across species (29, 32) and occurs in response to various physiological
stresses including viral infection (33), heat shock (34, 35), heme
deprivation (36), amino acid starvation (37-39), and endoplasmic
reticulum stress (40).
) could be suppressed by cooverexpressing wild-type
SUI3, GCD11, and IMT (initiator
methionyl-tRNA). Suppression was more efficient when the wild-type
GCD11 allele was replaced with gcd11-K250R, which
altered the NKXD element conserved in GTP-binding proteins and
led to increased dissociation of guanine nucleotides in
vitro (14). Because this effect mimics the function of eIF2B in
nucleotide exchange, co-overproduction of
eIF2
K250R and initiator tRNA also bypassed
eIF2B function in vivo and, as with bypass of
SUI2, was more efficient than overproduction of wild-type
eIF2. This suggested that the nucleotide exchange reaction was a
rate-limiting step in translation initiation in these strains. Based
upon these observations, we proposed that the eIF2
complex was
sufficient for providing eIF2 function, whereas eIF2B and the
-subunit of eIF2 served primarily to regulate eIF2 activity.
provides a
unique opportunity to address basic aspects of eIF2
function. In
this study, we purified both eIF2
and eIF2
K250R
and compared their biochemical activities in vitro with
heterotrimeric wild-type eIF2 and eIF2
K250R.
Removal of the
-subunit had little effect on dissociation of guanine
nucleotides and GTP-dependent tRNA binding (ternary complex
formation). In vitro, ternary complexes formed with
eIF2
interacted with the AUG codon and ribosomes to form 43 S
preinitiation complexes, which served as substrates for
eIF5-dependent GTP hydrolysis. Kinetic analysis of
eIF2B-catalyzed nucleotide exchange using eIF2
·GDP as substrate
revealed a 16-fold increase in Km and an 8-fold
increase in Vmax when compared with wild-type
eIF2·GDP. These data suggest that the
-subunit of eIF2 plays a
crucial role in structural interactions with eIF2B that determine
wild-type rates of nucleotide exchange in vivo.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]GTP (800 Ci/mmol) was from ICN Pharmaceuticals.
Protease inhibitors, GDP, and Gpp(NH)p were purchased from Sigma. Bulk
yeast tRNA was from Roche Molecular Biochemicals. 40 S ribosomal
subunits were purified from brine shrimp eggs (Artemia; San
Francisco Bay Brands) as described (41) and stored at 0.1-0.4
A260 units/µl. All buffers and reagents were
prepared with purified water (MilliQ; Millopore Corp.).
Isoforms--
His8-tagged (C terminus of eIF2
) eIF2 and
eIF2
K250R were purified from overexpressing
yeast strains, as described previously (14). The
His8-tagged eIF2
isoforms were overproduced in a derivative of EY841 (MAT
leu2-3 leu2-112
trp1-
63 sui2
gcd11::hisG GAL2+)2 and purified similarly with the
following modifications. After ammonium sulfate precipitation, the
protein pellet was suspended 100 ml of nickel column buffer (20 mM Tris-HCl, pH 7.5, 600 mM KCl, 1 µM GDP, 6.25 mM
-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 1 µg/ml aprotinin) and dialyzed overnight against
4 liters of nickel column buffer. Insoluble material and ribosomes were
pelleted by centrifugation at 200,000 × g for 2 h
at 4 °C. Dialysis after chromatography on nickel nitrilotriacetic
acid-agarose was reduced to 2 h against two liters of heparin
buffer. These modifications reduced the purification time from 3 days
to 2 and also resulted in increased specific activity of the eIF2
preparations.
substrate, since GDP at 10 mM did not lead to further increase in
initial reaction velocities.
-D-galactopyranoside, grown an
additional 3 h, harvested, and rinsed with 0.9% ice-cold NaCl.
Cells (15 g wet weight) were resuspended in 36 ml of buffer (20 mM Tris-HCl pH 8.0, 10 mM MgCl2, 30 mM KCl, 1 mM Na2EDTA, 10 mM
-mercaptoethanol, and 0.5 mM
phenylmethylsulfonyl fluoride) and then lysed by one cycle of freezing
at
20 °C followed by thawing at room temperature. All subsequent
steps were performed at 4 °C using 18 ml of lysate. Cell debris was
removed by centrifugation at 11,000 rpm (SS34 rotor) for 10 min, and 50 µl of a freshly prepared DNaseI (1 mg/ml stock) was added to the
supernatant. After a 30-min incubation on wet ice, the sample was
centrifuged at 200,000 × g for 150 min, and the pellet
was discarded. KCl (2.5 M) was slowly added to supernatant
with constant stirring to bring the salt concentration to 250 mM. The sample was applied to a DE-52 column (Whatman,
28-ml bed volume) at a linear flow rate of 56.5 cm/h with buffer A (50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 0.1 mM Na2EDTA, 10% glycerol) containing 250 mM KCl. The flow-through (140 ml) was collected and
fractionated with ammonium sulfate. The 50-65% ammonium sulfate
pellet was resuspended in 7 ml of buffer B (buffer A with 0.5 mM phenylmethylsulfonyl fluoride), 70 mM KCl
and dialyzed overnight against 1 liter of the same buffer. The
dialysate was applied to a DE-52 column (6.5-ml bed volume) at a linear
flow rate of 33 cm/h and washed with buffer B, 90 mM KCl
until the absorbance of the eluate was below 0.1 A254. The column was developed with a 50-ml
linear KCl gradient (90 to 450 mM) in buffer B. Peak
fractions containing eIF5, identified by SDS-polyacrylamide gel
electrophoresis, were pooled (10 ml) and dialyzed against 1 liter of
buffer B, 100 mM KCl for 3 h. The dialysate was
applied to a heparin-Sepharose (Amersham Pharmacia Biotech) column
(6.4-ml bed volume) at a linear flow rate of 33 cm/h in buffer B, 100 mM KCl. The column was washed with four column volumes of
the same buffer, then developed using a 52-ml linear KCl gradient (100 to 800 mM) in buffer B. eIF5 eluted from the column as a
single peak at ~280 mM KCl. Peak fractions containing eIF5 were dialyzed against 1 liter of storage buffer (20 mM
Tris-HCl, pH 7.5, 1 mM dithiothreitol, 0.1 mM
Na2EDTA, 20% glycerol, 100 mM KCl, 0.5 mM phenylmethylsulfonyl fluoride), spin-concentrated (Centricon 30, Millipore Corp.) to a concentration of 2 mg/ml, then
frozen on dry ice and stored at
70 °C. Preparations of eIF5 were
80-85% pure, as judged by SDS-polyacrylamide gel electrophoresis (data not shown).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Subunit Does Not Alter Dissociation of
Guanine Nucleotides--
We purified eIF2
and
eIF2
K250R heterodimers from overexpressing yeast
strains in which the eIF2
structural gene (SUI2) was
deleted. Yields were comparable with those of heterotrimeric eIF2
proteins reported previously (14). The purified proteins were 80-85%
pure, as judged by densitometric analysis of stained gels (Fig.
1). In addition, the two subunits (
and
) were present in an approximate 1:1 stoichiometry (Fig. 1,
lanes 2 and 4), suggesting that the absence of
the
-subunit does not affect association of
- and
-subunits.
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Fig. 1.
Purification of eIF2 isoforms. 1.5 µg
of eIF2 (lane 1) or eIF2 K250R
(lane 3) and 1.0 µg of eIF2
(lane 2) or
eIF2
K250R (lane 4) were subjected to
SDS-polyacrylamide gel electrophoresis on a 12.5% acrylamide gel
(29.8:0.2 acrylamide to bisacrylamide ratio) followed by staining with
Coomassie Brilliant Blue R-250. The approximate masses (kDa) of
pre-stained molecular weight markers are indicated to the left
(Mr, Bio-Rad pre-stained calibrated
SDS-polyacrylamide gel electrophoresis standards, catalog number
161-0318; soybean trypsin inhibitor, 28.9 kDa; carbonic anhydrase,
34.8 kDa; ovalbumin, 49.1 kDa; bovine serum albumin, 80 kDa;
-galactosidase, 124 kDa; myosin, 209 kDa); eIF2 subunits are
indicated on the right.
-subunit in guanine nucleotide
binding, we measured nucleotide off-rates for eIF2, eIF2
, eIF2
K250R, and eIF2
K250R
using a nitrocellulose filter binding assay. Reactions were performed
at 0 °C to retard the rapid dissociation of nucleotide from
K250R-containing complexes. At 0 °C, wild-type eIF2 complexes
showed little or no dissociation of eIF2·[3H]GDP (Fig.
2A), consistent with
previously reported results (42). The absence of the
-subunit did
not further enhance dissociation of the GDP-containing eIF2 binary
complexes. Dissociation of GTP (Fig. 2B) from wild-type eIF2
complexes, which is more rapid than GDP, was unaffected by the absence
of the
-subunit. As expected, the presence of the K250R form of the
-subunit increased the intrinsic dissociation rate for both GDP and
GTP, and the rate of GDP dissociation was not significantly altered in
the absence of eIF2
(the GTP reaction is essentially complete at the
first time point in both cases). These data suggest that the
-subunit of eIF2 does not play a role in guanine nucleotide binding.
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Fig. 2.
Intrinsic dissociation of guanine nucleotides
from eIF2 isoforms. 35 pmol of each eIF2 isoform was pre-incubated
with 500 pmol (5 µM) of [3H]GDP
(A) or [ -32P]GTP (B). Bound
radioactivity was chased at 0 °C with either water (dotted
lines, open symbols) or 5 mM unlabeled GDP
(solid lines, closed symbols), wild-type eIF2
(
,
), eIF2
(
,
), eIF2
K250R
(
,
), eIF2
K250R (
,
). Assays were
performed in triplicate; vertical bars represent standard
error
-Subunit Has a Mild Effect on
Met-tRNAiMet Binding by eIF2 in
Vitro--
We examined the affinity of the different eIF2 isoforms for
Met-tRNAiMet by measuring equilibrium
binding at various concentrations of [3H]Met-tRNAiMet in the
presence of saturating levels of GTP. Both eIF2
and eIF2
K250R bound
Met-tRNAiMet in a
GTP-dependent manner (Fig.
3A). Scatchard analysis was
used to calculate apparent dissociation constants of 19, 62, and 107 nM for eIF2, eIF2
K250R, and eIF2
,
respectively (Fig. 3B). This compares with previously
reported apparent Kd values of 15 and 25 nM for eIF2 and eIF2
K250R,
respectively (14). The largest difference, approximately 5-fold, was
observed between eIF2 and eIF2
, although this difference in our
filter binding assay is of questionable significance and is discussed
further below. We suggest that the absence of the
-subunit has, at
best, a very mild effect on tRNA binding in vitro.
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Fig. 3.
Met-tRNAiMet binding
by eIF2 isoforms. A, assays were performed as described
previously (14) using the indicated concentrations of
[3H]Met-tRNAiMet and
wild-type eIF2 ( ), eIF2
K250R (
), and eIF2
(
). B, Scatchard analysis of data from A.
Apparent dissociation constants (Kd) were estimated
to be 19, 62, and 107 nM for wild type eIF2,
eIF2
K250R, and eIF2
, respectively.
Forms 43 S Complexes That Are Effective Substrates
for eIF5-dependent GTP Hydrolysis--
In vitro
eIF2·GTP·[3H]Met-tRNAiMet
(ternary complex) interacts with the 40 S ribosomal subunit in an AUG
codon-dependent fashion to form a stable 43 S preinitiation
complex. This complex serves as a substrate for eIF5, which is required
for hydrolysis of eIF2-bound GTP. Under these conditions,
[3H]Met-tRNAiMet is
released from the resulting eIF2·GDP binary complex. We developed an
assay to measure this activity based upon similar assays reported by
Chakrabarti and Maitra (41) and Chakravarti and Maitra (44) using yeast
eIF2, recombinant yeast eIF5, and 40 S ribosomal subunits purified from
brine shrimp eggs. Formation of 43 S complexes in this assay was
dependent upon the presence of ternary complex, 40 S ribosomes, and AUG
codon triplet (data not shown). Complexes were treated using either
purified recombinant yeast eIF5 or extract prepared from recombinant
bacteria harboring the expression vector alone (control extract), then
subjected to centrifugation through sucrose gradients. The addition of
control extract (Fig. 4A) or no treatment (data not shown) resulted in cosedimentation of 40 S
ribosomes and
[3H]Met-tRNAiMet using
wild-type ternary complexes. The addition of yeast eIF5 led to the
disappearance of radioactivity from the 40 S peak, presumably as a
result of GTP hydrolysis. Because eIF2·GDP no longer binds the
initiator tRNA, the weak association between the tRNA, AUG triplet, and
the ribosome forms an unstable complex that is revealed by
centrifugation through the sucrose gradient. That the disappearance of
radioactivity from the 40 S complex is due to eIF5-mediated GTP
hydrolysis is demonstrated by the persistence of radioactivity in the
40 S peak in eIF5-treated 43 S complexes assembled using ternary
complexes containing the nonhydrolyzable GTP analog Gpp(NH)p (Fig.
4C). In the absence of the
-subunit, eIF2
readily
formed 43 S complexes that appeared qualitatively identical to those
formed using the wild-type eIF2 heterotrimer and were also effective
substrates for eIF5-mediated GTP hydrolysis (Fig. 4B). This
suggests that the
-subunit of eIF2 is not required in
vitro for formation of 43 S initiation complexes or for
eIF5-mediated GTP hydrolysis.
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Fig. 4.
43S complex formation and
eIF5-dependent GTP hydroylsis using eIF2 and
eIF2 ternary complexes.
Preinitiation complexes were formed using 40 S ribosomal subunits, AUG
codon triplet, and ternary complexes containing
[3H]Met-tRNAiMet and
eIF2·GTP (A), eIF2
·GTP (B), or
eIF2·Gpp(NH)p (C) and treated with recombinant yeast eIF5
(closed circles, solid lines) or control extract
without eIF5 (open circles, dashed lines).
Complexes were subjected to centrifugation through 5-25% sucrose
gradients; fractions were collected, and radioactivity was measured by
liquid scintillation counting. Curves shown were reproducible in
experiments performed in duplicate or triplicate.
Provides a Structural Function in Catalyzed Nucleotide
Exchange--
Progress curves comparing the eIF2B-catalyzed release of
[3H]GDP from binary complexes indicated that the absence
of the
-subunit in eIF2
appeared to have an effect on the
reaction compared with wild-type eIF2. However, neither the addition of
eIF2B nor the absence of eIF2
had a significant effect on the
intrinsic dissociation of [3H]GDP from binary complexes
formed using eIF2
K250R or
eIF2
K250R (these latter curves were essentially
identical to Fig. 2A) (data not shown).
·[3H]GDP as a substrate in catalyzed
nucleotide exchange. Reaction velocities were measured at early time
points using substrate concentrations between 18.9 and 250 nM. Extrapolation of x and y
intercepts from reciprocal plots of velocity versus
substrate concentration (Fig. 5) yielded
values for Km and Vmax of 193 nM and 1980 fmol/min, respectively. These data, when
compared with our previously reported values of 12 nM and
251 fmol/min using wild-type heterotrimeric eIF2 and the same
preparation and concentration of eIF2B (42) indicated a 16-fold
increase in Km and a smaller 8-fold increase in
Vmax using the
eIF2
·[3H]GDP substrate. We interpret these data
as suggesting that the presence of eIF2
is required for structural
interactions with the eIF2B enzyme that contribute to catalyzed
nucleotide exchange. These interactions may be direct or indirect and
are discussed further below. The 8-fold increase in
Vmax in the absence of eIF2
results in a
similar 8-fold increase in kcat and suggests
that the presence of the
-subunit acts to retard catalyzed
nucleotide exchange (i.e. turnover is less efficient) in a
manner that is not dependent upon phosphorylation. The combined
increase in both Km and Vmax
reduces the specificity constant
(kcat/Km) for
eIF2
·[3H]GDP by 50% compared with the wild-type
heterotrimer, and thus, it remains a relatively good substrate for
catalyzed exchange.
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Fig. 5.
Kinetic analysis of eIF2B-catalyzed
exchange using eIF2 ·[GDP] as
substrate. Substrate concentrations
(eIF2
·[3H]GDP) used were 18.9, 22.2, 27.8, 33.3, 125, 142.9, and 250 nM. Exchange reactions were initiated
by the addition of 5.8 nM eIF2B and 5 mM
unlabeled GDP. Reaction velocities were determined at time points at
which no more than 10% of the substrate had been consumed. All
velocities were determined in triplicate and varied by less than
3%.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit, much effort
has been directed toward understanding its role in regulating
nucleotide exchange via reversible phosphorylation at the conserved
serine 51 (reviewed in Refs. 5, 27, 28, and 29). Genetic analysis in
yeast has suggested an additional function for eIF2
in selecting the
translational start site (45). In this report, we used a biochemical
approach to analyze the function of eIF2
by examining the activity
of purified eIF2
heterodimers in vitro. Our eIF2
proteins were purified from yeast strains in which the normally
essential SUI2 (eIF2
) gene was deleted but in which
lethality was suppressed by increased gene dosage of SUI3
(eIF2
), GCD11 (eIF2
), and IMT (initiator tRNA). These preparations were completely devoid of the
-subunit and
differ from previously reported
-subunit-deficient forms of purified
eIF2 that contained reduced, but measurable, amounts of the
-subunit
(16, 25).
-subunit did not alter the rate of guanine nucleotide
dissociation in the presence of either the wild-type
-subunit or
K250R relative to the corresponding heterotrimeric proteins. This suggests that the
-subunit does not participate in
guanine nucleotide binding and contradicts results from earlier studies
that suggested a direct role for eIF2
in binding guanine nucleotide
(46). Indeed, nucleotide binding by eIF2 appears to be a direct
function of the
-subunit, although participation by eIF2
cannot
be excluded (12, 14, 16, 17, 47). Binding of initiator tRNA also
appears to be a direct function of eIF2
, with additional data
suggesting participation by the
-subunit (8, 14). In the absence of
the
-subunit, the apparent dissociation constant for
Met-tRNAiMet increased 5-fold for
eIF2
(107 nM) compared with the wild-type eIF2
complex (Fig. 3B), with a smaller 2-fold increase for
eIF2
K250R compared with
eIF2
K250R. The apparent
Kd value obtained using eIF2
falls near the
upper limits of values that we have obtained using different wild-type
eIF2 preparations (up to 70 nM).3 Thus,
the differences observed may represent normal variation or, at best, a
mild defect in tRNA binding. In either case, the significance of even a
5-fold difference may be minimal in vivo, as the normal
range of concentrations for initiator tRNA in mammalian cells has been
estimated at 85-310 nM (48), 40-160 nM (49), and 100-200 nM (50). No such estimates are available for
yeast as far as we are aware. Therefore, we cannot exclude the
possibility of a mild tRNA binding defect, which may in part explain
the requirement for increased IMT gene dosage in some
suppressed
sui2 strains.2 A mild
increase in Kd for tRNA binding would
also be consistent with the reduced specific activity of
-deficient
eIF2 reported by Anthony et al. (16).
, GTP, and
Met-tRNAi Met formed stable 43 S
preinitiation complexes in the presence of the AUG codon in
vitro, indicating the absence of an absolute requirement for the
-subunit for interaction between eIF2 and the ribosome. Although
genetic analysis in yeast indicates participation of eIF2
in start
site recognition (45), the viability of our
sui2 strains
and the formation of AUG-dependent preinitiation complexes in vitro using eIF2
indicates that the contribution of
the
-subunit to this function is dispensable. Preinitiation
complexes containing eIF2
ternary complexes were also substrates
for eIF5-mediated GTP hydrolysis. Interaction of eIF5 with eIF2 has
been shown to require the N-terminal lysine-rich region of eIF2
(21,
24), and our data suggest that this interaction is not abolished in the
absence of eIF2
. Because eIF2 GTPase activity is also
ribosome-dependent, it appears that interactions between
eIF2
and the ribosome are sufficient to maintain eIF2 in a
conformation that is recognized by eIF5.
-subunit did not appear to alter
intrinsic dissociation of guanine nucleotides, the
Km for eIF2
·GDP in eIF2B-catalyzed
nucleotide exchange increased by 18-fold compared with eIF2·GDP. This
is the first biochemical demonstration of a contribution of the
-subunit to eIF2B-mediated nucleotide exchange that is independent
of phosphorylation. Hinnebusch and co-workers (51, 52) proposed a
general mechanism for eIF2B-mediated nucleotide exchange based upon
analysis of yeast strains either harboring mutations in eIF2B subunits
or overexpressing various combinations of eIF2 or eIF2B subunit genes.
In their model, an initial interaction between eIF2 and eIF2B is
proposed involving eIF2
and the
(Gcn3p),
(Gcd7p), and
(Gcd2p) eIF2B subunits. This is supported by studies of
overexpressing strains, in which Gcn3p, Gcd2p, and Gcd7p appear to form
an independent subcomplex that is capable of recognizing the
phosphorylation status of eIF2
(52, 53). In addition, specific
mutations in the structural genes encoding these subunits bypass the
toxic effects of hyperphosphorylation at serine 51 (51, 54). The
proposed initial interaction would be nonproductive but, in the absence
of phosphorylation at serine 51, rapidly undergoes isomerization to a
productive (catalytic) mode dominated by interactions involving
eIF2B
(Gcd1p) and eIF2B
(Gcd6p). These subunits comprise the eIF2B
catalytic core, and evidence exists in both yeast and mammalian systems
for the primary role of eIF2B
in catalysis (52, 55, 56). Although
the
-subunit of eIF2 is directly involved in guanine nucleotide
binding, no evidence exists for a direct interaction between this eIF2
subunit and the eIF2B catalytic core. However, there is evidence from two studies for direct interaction between eIF2
and the
- and
-subunits of mammalian eIF2B (20) or the yeast eIF2B
subunit (21), but the region of eIF2
required for the interaction remains controversial. Given the effect on Km for the
exchange reaction upon removal of eIF2
, our results indicate that
structural interactions between eIF2B and eIF2 that require the
presence of eIF2
contribute to wild-type rates of catalysis and are
consistent with the ability of eIF2B to recognize the presence of the
eIF2
subunit. In view of the current model, it is possible that
removal of the
-subunit bypasses the proposed initial nonproductive
interaction, involving eIF2
and the eIF2B
regulatory core,
and proceeds more or less directly to the productive (catalytic)
interaction. This interaction, involving primarily eIF2
(
?) and eIF2B
may in fact be weaker than nonproductive
or productive interactions involving eIF2
. These additional contacts
mediated by the
-subunit may also be inhibitory to nucleotide
exchange, as suggested by the increase in Vmax
observed upon their removal as in the eIF2
·GDP substrate. The
increased Vmax may therefore reflect the absence of a slow step (e.g. involving the nonproductive
interaction) in the exchange reaction. Taken together, our data are
consistent with the idea that the eIF2
-subunit occupies a key
position within the eIF2 complex through which it can mediate
eIF2B-catalyzed nucleotide exchange.
-subunit of eIF2 plays an important role in catalyzed nucleotide
exchange independent of its role in regulation in response to
phosphorylation. Despite the effect on nucleotide exchange, the overall
similarity between the activities of eIF2
and wild-type heterotrimeric eIF2 complexes in vitro is consistent with
the viability of the suppressed
sui2 strains and suggests
that in vivo eIF2
is able to form ternary complexes,
interact with ribosomes, messenger RNA, and (presumably) other
initiation factors, and also serve as a substrate for eIF5-mediated GTP
hydrolysis. The effect on catalyzed nucleotide exchange is apparently
compensated in
sui2 strains by cooverexpression of
initiator tRNA and the
- and
-subunits of eIF2 in addition to the
intrinsic rate of nucleotide dissociation seen for yeast eIF2 (14, 57).
The latter suggestion is supported by the observation of more efficient suppression in the presence of
eIF2
K250R.2 However, the presence of the
-subunit results in a more effective interaction with eIF2B in
vitro. We suggest that this interaction is critical if eIF2B is to
efficiently monitor and respond to the phosphorylation status of the
-subunit in vivo. In this respect, it is likely that a
subset of the GCD2 and GCD7 mutations that render
eIF2B less sensitive to the inhibitory effects of eIF2
phosphorylation might likewise increase Km for the
eIF2 substrate. These studies provide a framework for further analysis of interactions between eIF2 and eIF2B and, in particular, those interactions specifically involving the catalytic or regulatory eIF2B subcomplexes.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Alan Hinnebusch and William Merrick for helpful discussions and valuable comments and suggestions on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the American Cancer Society Grant RPG-97-061-01-NP.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.
To whom correspondence should be addressed: Dept. of Molecular and
Cell Biology, The University of Texas at Dallas, Mail Station FO3.1,
P. O. Box 830688, Richardson, TX 75083-0688. Tel.:
972-883-2505; Fax: 972-883-2409; E-mail: hannig@utdallas.edu.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M007398200
2 F. L. Erickson, J. Nika, and E. M. Hannig, submitted for publication.
3 S. Rippel and E. M. Hannig, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
eIF2, wild-type heterotrimeric eukaryotic initiation factor 2;
eIF2K250R, eIF2 heterotrimer with altered
subunit (K250R);
eIF2
or eIF2
K250R, heterodimeric eIF2 containing
- and
-(or
K250R)
subunits;
eIF2
, heterodimeric eIF2 containing the
- and
-subunits;
Gpp(NH)p, guanosine
5'-(
,
-imido)triphosphate.
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