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INTRODUCTION |
Eukaryotic translation initiation factor 5 (eIF5),1 a monomeric protein
of about 49 kDa in mammals (1-3) and 46 kDa in the yeast
Saccharomyces cerevisiae (4, 5), in conjunction with GTP and
other initiation factors, plays an essential role in initiation of
protein synthesis in eukaryotic cells. In vitro studies
using purified initiation factors have shown that the overall
initiation reaction proceeds with the initial binding of the initiator
Met-tRNAf as the Met-tRNAf·eIF2·GTP ternary
complex to a 40 S ribosomal subunit followed by the positioning of the
40 S preinitiation complex (40 S·eIF3·Met-tRNAf·eIF2·GTP) at the initiation AUG
codon of the mRNA to form the 40 S initiation complex (40 S·eIF3·mRNA·Met-tRNAf·eIF2·GTP). The
initiation factor eIF5 then interacts with the 40 S initiation complex
to promote the hydrolysis of ribosome-bound GTP. Hydrolysis of GTP
causes the release of eIF2·GDP (and Pi) as well as eIF3 from the 40 S subunit, an event that is essential for the subsequent joining of the 60 S ribosomal subunit to the 40 S complex to form a
functional 80 S initiation complex (80 S·mRNA·Met-tRNAf) that is active in peptidyl
transfer (for a review, see Refs. 6-8). eIF5-promoted GTP hydrolysis
has also been shown to play an important role in the selection of the
AUG start codon by the 40 S preinitiation complex (9). The mammalian
cDNA and the yeast gene, designated TIF5, encoding eIF5
have been cloned and expressed as functional proteins in
Escherichia coli (4, 10).
Biochemical characterization of eIF5-promoted GTP hydrolysis
reaction has shown that hydrolysis of GTP occurs only when eIF5 interacts with GTP bound to eIF2 in the 40 S initiation complex and
that eIF5, by itself, does not hydrolyze either free GTP or GTP bound
to eIF2 as a Met-tRNAf·eIF2·GTP ternary complex in the absence of 40 S ribosomal subunits (10, 11). These observations suggested that eIF5 interacts with one or more components of the 40 S
initiation complex to cause hydrolysis of GTP. Subsequent studies
showed that eIF5 forms a complex with eIF2 (10), a component of the 40 S initiation complex, and that eIF5·eIF2 complex formation occurs by
interaction between the conserved lysine residues at the N-terminal
region of eIF2
and the conserved glutamic acid residues at the
C-terminal region of eIF5 (12-14). More importantly, mutational
analysis of the conserved glutamic acid residues in the C-terminal
eIF2
-binding region of eIF5 showed that eIF5·eIF2
interaction
plays an essential role in eIF5 function in vitro (14) as
well as in vivo (13, 14). However, an important question
remained as to whether the interaction of eIF5 with eIF2
is
sufficient for eIF5-promoted GTP hydrolysis or other regions in eIF5
are also required for its function.
In the work presented in this paper, using deletion analysis we show
that, in addition to the eIF2
-binding region at the C terminus of
eIF5, the N-terminal region of eIF5 is also required for eIF5-promoted
GTP hydrolysis. Furthermore, consistent with the hypothesis that eIF5
functions as a GTPase-activating protein (GAP), we observed that, like
typical well characterized GAPs, e.g. RasGAPs and RhoGAPs
(15, 16), eIF5 also contains an invariant arginine residue (Arg-15) at
the N terminus that is essential for eIF5 function both in
vitro and in vivo. Mutational analysis of this arginine
residue as well as of Lys-33 and Lys-55 of rat eIF5 showed that these
residues are critical for eIF5-promoted GTP hydrolysis as well as in
the ability of rat eIF5 to functionally substitute for the homologous
yeast protein in a
TIF5 yeast strain. The implications of
these results in understanding the mechanism of
eIF5-dependent GTP hydrolysis reaction are discussed.
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EXPERIMENTAL PROCEDURES |
tRNA, Ribosomes, Purified Proteins, and Antibodies--
The
preparation of 35S-labeled rabbit liver initiator
Met-tRNAf (30,000-50,000 cpm/pmol), 40 S and 60 S
ribosomal subunits from Artemia salina eggs, purified eIF2
from rabbit reticulocyte lysates, and rabbit anti-rat eIF5 antibodies
was described previously (2, 10, 11, 17). The mixture of protease
inhibitors added to buffer solutions used during purification of
recombinant proteins from bacterial cell extracts consisted of
leupeptin (0.5 µg/ml), pepstatin A (0.7 µg/ml), aprotinin (2 µg/ml), and freshly prepared phenylmethylsulfonyl fluoride (1 mM).
Construction of Plasmids and Yeast Strains Expressing Wild-type
or Mutant Rat eIF5 Proteins--
Deletion mutants of eIF5 were
generated by one-stage PCR amplification of eIF5 ORF sequences using
pGEX-KG-eIF5 as the template and appropriate oligonucleotide primers
containing BamHI/EcoRI overhangs. A
BamHI/EcoRI restriction fragment of each
PCR-amplified deletion mutant was inserted at the same restriction
sites of the vector pGEX-KG. The construct
pGEX-KG-
-(18-58)eIF5 was generated by three-fragment
ligation of a N-terminal fragment obtained by annealing two
single-stranded oligonucleotides containing
NdeI/ClaI overhangs that correspond to amino
acids 1 to 17, a C-terminal PCR fragment digested with
ClaI/EcoRI that corresponds to amino acids 59 to
430, and the vector pGEX-KG digested with
NdeI/EcoRI. The resulting constructs expressed
deleted eIF5 mutants as GST fusion proteins. The construction of yeast
centromeric plasmids pRS316-TIF5, pTM100-EIF5, and pUB-TIF5R and the
haploid yeast strains TMY101 (MAT
leu2-3,
112 his3-11, 15 ade2-1 trp1-1 ura3-1 can1-100
tif5::TRP1[pRS316-TIF5) and TMY201R (MAT
leu2-3, 112 his3-11, 15 ade2-1 trp1-1
ura3-1 can1-100 tif5::TRP1[pUB-TIF5R]) and preparation
of media for yeast cell growth have been described previously (18).
Point mutations within the coding sequence of eIF5 present in the yeast
centromeric plasmid pTM100-EIF5 or the bacterial expression plasmid
pGEX-KG-eIF5 were constructed as described previously (14). Expression
of wild-type or mutant rat eIF5 proteins in yeast cells was detected by
immunoblot analysis as described previously (18).
Expression and Purification of Recombinant Wild-type or Mutant
Rat eIF5 Proteins--
E. coli XLI Blue cells transformed
with recombinant pGEX-KG plasmids containing either the wild-type or
mutant eIF5 coding sequences (expressing wild-type or mutant eIF5 as
GST fusion proteins) were grown as described previously (14). Untagged
wild-type and mutant eIF5 proteins were purified from these cells
following the procedure described by Das et al. (14). The
purified proteins were stored in a buffer containing 20 mM
Tris-HCl, pH 7.5, 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, and 60% glycerol at
20 °C.
GST-eIF2
Fusion Protein Binding Assay--
The binding assays
were carried out as described previously (14). A typical binding
reaction mixture contained 200 µl of 20 mM potassium
phosphate (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, 0.5 mM
phenylmethylsulfonyl fluoride, 6 µl of a 30% suspension of GSH beads
containing bound GST-eIF2
(2 µg of total protein), and about 1 µg of either purified wild-type or mutant eIF5 protein.
Other Methods--
Yeast cell-free extracts for
eIF5-dependent translation of mRNAs were prepared from
TMY201R yeast cells as described previously (18). The 40 S initiation
complex containing bound [
-32P]GTP was prepared and
isolated free of unreacted reaction components by sucrose-density
centrifugation as described (10, 11). Yeast transformations were
performed as described by Rose et al. (19). Methods for
plasmid and genomic DNA preparations, restriction enzyme digestion, DNA
ligation, cloning, and bacterial transformations were according to
standard protocols (20).
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RESULTS |
Effect of N-terminal Deletion of eIF5 on Its Function in
Vitro--
We have shown previously (12, 14) that mammalian eIF5
interacts with mammalian eIF2
through a conserved acidic amino acid-rich C-terminal region and that this interaction is necessary for
eIF5-promoted hydrolysis of GTP bound to eIF2 in the 40 S initiation
complex (40 S·AUG·Met-tRNAf·eIF2·GTP). However, the possibility exists that the interaction between the C-terminal acidic
amino acid-rich domain of eIF5 with eIF2
alone is not sufficient for
eIF5-promoted GTP hydrolysis.
To determine whether other regions of eIF5 are also essential for eIF5
function, we retained the C terminus of eIF5 containing the
eIF2
-binding region and carried out truncations at the N terminus
(Fig. 1A). The deletion
mutants and wild-type eIF5 were expressed as GST fusion proteins in
E. coli XL1-Blue cells and purified as untagged proteins to
apparent electrophoretic homogeneity (Fig. 1B), as described
under "Experimental Procedures." These purified mutants were then
assayed for their ability to promote hydrolysis of GTP bound to the 40 S initiation complex. Wild-type eIF5 was able to promote rapid and
quantitative hydrolysis of GTP as expected (Fig. 1D).
However, when the first 98 amino acids from the N terminus of eIF5 were
deleted, the resultant mutant
-(1-98)eIF5 failed to promote
hydrolysis of GTP (Fig. 1D), suggesting that the N-terminal
end of eIF5 was also essential for eIF5 activity. The mutant
-(1-98)eIF5 was, however, able to bind to eIF2
as expected (Fig.
1C). To further map the region at the N terminus of eIF5
that is essential for eIF5 function, we constructed two additional
deletion mutants. In the first mutant
-(1-17)eIF5, the first 17 amino acids at the N terminus of eIF5 were deleted whereas in the other
mutant
-(18-58)eIF5, the region between amino acids 18 to 58 was
deleted, keeping the rest of the eIF5 ORF intact (Fig. 1A).
Both mutants failed to promote hydrolysis of GTP bound to eIF2 in the
40 S initiation complex (Fig. 1D) but were able to bind
eIF2
(Fig. 1C). These results indicate that, although the
C terminus of eIF5 is involved in binding to eIF2 (via the
subunit
of eIF2), this interaction alone is not sufficient for eIF5-promoted
GTP hydrolysis. The N terminus of eIF5 is also required for eIF5
activity.

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Fig. 1.
Effect of N-terminal truncations on the
ability of eIF5 to bind eIF2 and promote GTP
hydrolysis. A, schematic representation of N-terminal
deletion mutants of eIF5 expressed as GST fusion proteins in E. coli XLI-Blue cells. B, approximately, 3 µg each of
recombinant wild-type (WT) and the indicated deletion mutant
eIF5 proteins, purified from
isopropyl-1-thio- -D-galactopyranoside-induced XL1-Blue
cell lysates as described under "Experimental Procedures," were
subjected to SDS-polyacrylamide gel electrophoresis (15% gel) and
visualized by Coomassie Blue staining. The arrow indicates
the position of purified wild-type rat eIF5. C, 1 µg each
of purified recombinant wild-type (lane b) and the indicated
mutant eIF5 proteins were separately incubated with 2 µg of
GST-eIF2 fusion protein immobilized on GSH beads. In lane
c, wild-type eIF5 was incubated with 2 µg of GST alone.
Following incubation at 4 °C with gentle shaking, reaction mixtures
were centrifuged, the beads were washed, suspended in 1× Laemmli
buffer, and subjected to Western blot analysis using polyclonal
anti-eIF5 antibodies. In lane a, purified rat eIF5 was
electrophoresed as a marker and probed with anti-eIF5 antibodies.
D, eIF5-promoted GTP hydrolysis from the 40 S initiation
complex. A 120-µl aliquot of isolated 40 S initiation complex
(Met-tRNAf·eIF2·[ -32P]GTP·40
S·AUG) containing 2.5 pmol of bound [ -32P]GTP
(38,500 cpm/pmol) in buffer R (20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 100 mM KCl, 1 mM dithiothreitol) was incubated with 20 ng of purified
recombinant wild-type or mutant eIF5 proteins as indicated at 25 °C.
Aliquots (15 µl) were removed at various times as indicated, and the
amount of 32Pi released by the hydrolysis of
[ -32P]GTP was measured by the ammonium
phosphomolybdate method as described (11). A reaction lacking eIF5 was
also included, and the amount of 32Pi released
in this control reaction mixture is also shown. The results shown
represent the total amount of 32Pi formed per
120-µl reaction mixture.
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The requirement of both the N terminus and the C terminus of eIF5 for
its activity is analogous to that of other well characterized GAPs.
Typical GAPs, e.g. RhoGAPs and RasGAPs, contain sequence motifs that are necessary for their GTPase-stimulating activity in
addition to motifs that are necessary for binding to their respective
GTPase proteins (16). In the case of eukaryotic translation initiation,
it is the eIF2 molecule that binds GTP and presumably acts as a GTPase,
whereas eIF5 acts as the GAP. The C-terminal domain of eIF5 contains
the sequence motifs required for binding of eIF5 to eIF2, the GTPase
protein (12-14). It is likely that the activation domain of eIF5 is
present at the N terminus and contains sequence motifs required for the
GTPase-stimulating activity of eIF5.
Strategy for Mutational Analysis of the N-terminal Region of
eIF5--
Comparison of the N-terminal region of eIF5 from different
species shows that this region of eIF5 is highly conserved (Fig. 2).

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Fig. 2.
Comparison of the N-terminal region of eIF5
from different species and location of alanine substitution mutations
of conserved residues. The amino acid sequences of the N-terminal
region of rat eIF5 (amino acids 1-58) were aligned with the
corresponding regions in human, phavu, S. cerevisiae, S. pombe, and maize eIF5 for maximum
homology using the program DNASTAR. The sequence of rat, human, and
S. cerevisiae eIF5 are from a previous study (29). The
sequences of S. pombe, maize, and phavu eIF5 were
obtained from SWISS-PROT (accession numbers Q09689, P55876, and P41375,
respectively). The highly conserved amino acid residues between eIF5 of
all species are highlighted with dark shading and
the moderately conserved residues are highlighted with
light shading. Broken lines represent gaps.
Arrowheads indicate residues in rat eIF5 that were targeted
for mutagenesis in this study to generate the eIF5 mutants.
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The activation domains of GAPs belonging to the well characterized
families of RasGAPs and RhoGAPs have been shown to contain "arginine-finger" motifs consisting of an invariant arginine
residue at the N terminus of their catalytic domain that are required for their ability to stimulate GTP hydrolysis (Ref. 16, see also Table
I). In rat eIF5, there are two invariant
arginine residues at the N-terminal end at positions 15 and 48. The
importance of these arginine residues in eIF5 function was investigated
by alanine substitution mutagenesis of these residues. These mutant eIF5 proteins were initially examined for their ability to substitute for yeast eIF5 in a
TIF5 haploid yeast strain. The
rationale behind this strategy was that, if these invariant arginine
residues were required for eIF5 function, eIF5 proteins containing
mutations at these residues would be lethal in yeast cells. All
mutations were carried out in rat eIF5, because we use mammalian
factors in our in vitro assay systems to measure eIF5
function. It should be noted that mammalian eIF5 functionally
substitutes for yeast eIF5 in sustaining yeast cell growth and
viability (18).
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Table I
Sequence homology of arginine-finger motif in eIF5 and other GAPs
The sequences of "arginine-finger" motifs of RasGAPs and RhoGAPs
are from Ref. 16. The invariant arginine residue is shown as
boldface letters.
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Mutational Analysis of Putative Arginine-finger Motif in Rat eIF5:
Effect on eIF5 Function in Yeast Cells--
To identify the invariant
arginine residue in eIF5, the eIF5-Ala mutants R15A and R48A were
generated using a PCR-based site-directed mutagenesis protocol using
the LEU2-based yeast expression plasmid pTM100-EIF5 as the
template as described under "Experimental Procedures." The
wild-type rat eIF5 ORF (gene designation EIF5) in the CEN plasmid pTM100-EIF5 is under the transcriptional control of the galactose-inducible GAL1 promoter (18). To test the function of the rat eIF5-Ala mutants in yeast cells, both the wild-type and the
mutant recombinant expression plasmids and the parental vector plasmid
pTM100 were transformed separately into the haploid yeast strain
TMY101. (The strain TMY101 carries an inactive TIF5 allele
disrupted with the TRP1 marker gene and is kept viable by
maintenance of an URA3-based CEN plasmid
pRS316-TIF5 in which yeast eIF5 is expressed from its natural
promoter.) Trp+Ura+Leu+
transformants were selected on SGal-Trp-Leu-Ura plates and then replica-plated onto similar SGal plates, which also contained uracil
and 5-fluoroorotic acid (5-FOA) to select against retention of the
URA3-based plasmid pRS316-TIF5, expressing wild-type yeast eIF5.
Fig. 3A shows that the yeast
strain TMY101 transformed with plasmid pTM100-EIF5, expressing
wild-type rat eIF5, grew on 5-FOA plates, in agreement with the results
reported previously (18), while cells transformed with the parental
vector plasmid pRS315 failed to grow, as expected. Under the same
conditions TMY101 cells transformed with pTM100-EIF5(R15A), carrying a
single point mutation at Arg-15 (Arg to Ala) in rat eIF5, failed to
grow on 5-FOA plates (Fig. 3A, right panel) while
cells expressing alanine substitution mutation at Arg-48 of rat eIF5
from pTM100-EIF5(R48A) grew as well as the cells expressing wild-type
eIF5 from the plasmid pTM100-EIF5. It is known that even a conservative
mutation of the invariant arginine residue in RasGAP to lysine
dramatically affects its GAP activity (16). We also observed that when
TMY101 cells were transformed with the recombinant plasmid
pTM100-EIF5(R15K), which expressed the mutant eIF5 protein in which
Arg-15 was mutated to Lys, and the resulting transformants were tested
for their ability to grow on 5-FOA plates, they failed to grow (Fig.
3A, right panel). Taken together these results
suggest that Arg-15 in rat eIF5 plays a critical role in eIF5 function
in maintaining yeast cell growth and viability.

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Fig. 3.
Effect of mutations in rat eIF5 on growth of
haploid yeast transformants expressing rat eIF5. A,
haploid yeast strain TMY101 (18) that carries inactive TIF5,
disrupted with TRP1 and harboring the URA3
plasmid pRS316-TIF5 that expresses the yeast wild-type TIF5
gene under the control of its natural promoter, was transformed
separately with different mutant eIF5-expressing plasmids as indicated.
Transformants selected on SGal-Trp-Leu-Ura plates (left
panel) were replica-plated on SGal-Trp-Leu+Ura + 5-FOA plates
(right panel). Cells were allowed to grow on 5-FOA plates
for 5 days. B, immunoblot analysis of eIF5 in lysates of
yeast cells expressing both yeast eIF5 and wild-type or mutant
mammalian eIF5 proteins from the recombinant plasmids. Yeast cells
harboring both the URA3 plasmid pRS316-TIF5 and the
different recombinant LEU2-expression plasmids expressing
either the wild-type or mutant mammalian eIF5 were grown to
mid-logarithmic phase in synthetic medium containing 2% galactose as
the sole source of carbon. Cell lysates were prepared as described
previously (18) and analyzed by Western blotting using rabbit
polyclonal anti-rat eIF5 antibodies. In lane a, purified
recombinant rat eIF5 was electrophoresed as a marker.
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In RasGAPs and RhoGAPs, a "secondary" positively charged residue
(Arg in RasGAP and Lys in RhoGAP) is also necessary for their GTP
hydrolysis-stimulating activity. To identify such a residue in eIF5, we
carried out mutagenesis of other invariant positively charged residues
at the N-terminal region of rat eIF5 (Fig. 2). The residues Lys-24,
Lys-33, Asn-38, and Lys-55 in rat eIF5 were each mutated to alanine.
Once again, plasmid shuffling technique was used to test the effect of
these mutations on yeast cell growth and viability. Yeast cells
expressing either mutant eIF5 K33A (Lys-33 was mutated to Ala) or
mutant eIF5 K55A (Lys-55 was mutated to Ala) failed to grow on 5-FOA
plates (Fig. 3A, right panel). In contrast,
alanine substitution mutation at Lys-24 and Asn-38 of rat eIF5 did not
affect cell growth on 5-FOA plates (data not shown). These results
indicate that Lys-33 and Lys-55 in rat eIF5 may also play an important
role in eIF5 function.
The functional defect of mutant rat eIF5 proteins R15A, R15K, K33A, and
K55A was not due to lack of expression of mutant eIF5 in yeast cells.
When cell extracts were prepared from
Trp+Ura+Leu+ transformants
harboring both pRS316-TIF5 and pTM100-EIF5(wild-type or mutant)
plasmids and analyzed by Western blotting using rabbit anti-rat eIF5
antibodies, the mutant eIF5 proteins R15A (lane d), R15K
(lane e), K33A (lane f), and K55A (lane
g) were expressed at levels comparable to wild-type eIF5
(lane b). Extracts prepared from TMY101 cells harboring the
plasmid pRS316-TIF5 and the vector plasmid pRS315 and expressing only
wild-type yeast eIF5 did not show any immunoreactive polypeptide band
with anti-rat eIF5 antibodies, as expected (lane c). It
should be noted that rabbit anti-rat eIF5 antibodies do not recognize
yeast eIF5 (18).
eIF5 Mutants R15A, R15K, K33A, and K55A Are Able to Bind eIF2
but Are Unable to Promote GTP Hydrolysis--
The eIF5 mutants R15A,
R15K, K33A, and K55A, which were unable to complement in
vivo a genetic disruption in the chromosomal copy of
TIF5, were initially tested to confirm that these mutations did not cause a defect in the ability of eIF5 to bind eIF2
. For this
purpose, the mutant eIF5 proteins were expressed in bacteria and
purified to apparent homogeneity (Fig.
4A) as described under "Experimental Procedures." The ability of each purified mutant protein and wild-type eIF5 to bind eIF2
fused to GST and immobilized on GSH-Sepharose beads was examined. Fig. 4B shows that
mutant eIF5 protein R15A (lane d), R15K (lane e),
K33A (lane f), and K55A (lane g) were all able to
bind GST-eIF2
, although the binding efficiency of K55A is somewhat
reduced (see legend to Fig. 4B). As expected, wild-type eIF5
did not bind to GST immobilized on GSH-Sepharose beads (lane
c).

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Fig. 4.
Analysis of eIF5 point mutants for their
ability to bind eIF2 and promote hydrolysis of
GTP bound to the 40 S initiation complex. A,
recombinant wild-type and mutant eIF5 proteins R15A, R15K, K33A, and
K55A were purified from
isopropyl-1-thio- -D-galactopyranoside-induced XL1-Blue
cell lysates as described previously (14). Purified recombinant eIF5
proteins (3 µg each) were subjected to SDS-polyacrylamide gel
electrophoresis (15% gel) and visualized by Coomassie Blue staining.
The arrow indicates the position of purified eIF5.
B, purified recombinant wild-type and mutant eIF5 proteins
(1 µg each) were separately incubated with 2 µg of GST-eIF2
fusion protein immobilized on GSH beads as indicated. Following
incubation at 4 °C with gentle shaking, reaction mixtures were
centrifuged and the beads were washed, suspended in 1× Laemmli buffer,
and subjected to Western blot analysis using polyclonal anti-eIF5
antibodies. In lane a, purified rat eIF5 was electrophoresed
as a marker and probed with anti-eIF5 antibodies. In lane c,
wild-type rat eIF5 was incubated with GST alone. It should be noted
here that densitometric scanning using NIH IMAGE 1.62b7 (not shown)
indicated that mutants R15A and K33A bound GST-eIF2 with similar
efficiency. Mutant R15K bound GST-eIF2 with about 30% more
efficiency, whereas mutant K55A bound GST-eIF2 with about 30% less
efficiency. C, eIF5-promoted GTP hydrolysis. Each reaction
mixture (90 µl) was prepared as described under the legend to Fig. 2
using 20 ng of either purified or mutant eIF5 proteins as indicated.
Following incubation at 25 °C, aliquots (10 µl) were removed at
each indicated time point and the amount of
32Pi released by the hydrolysis of
[ -32P]GTP was measured by the ammonium
phosphomolybdate method as described (11). A reaction lacking eIF5 was
also included, and the amount of 32Pi released
in this control reaction mixture is also shown. The results shown
represent the total amount of 32Pi formed per
90-µl reaction mixture. It should be noted here that the results
shown are representative of three independent experiments. The average
margin of error is ±5%.
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The purified mutant eIF5 proteins were also tested for their ability to
promote in vitro hydrolysis of GTP bound to the 40 S
initiation complex. As expected, wild-type rat eIF5 promoted rapid
hydrolysis of [
-32P]GTP bound to the 40 S initiation
complex (Fig. 4C). In contrast, under similar experimental
conditions the mutant eIF5 protein R15A was unable to hydrolyze
[
-32P]GTP bound to the 40 S initiation complex (Fig.
4C). When the mutant eIF5 proteins R15K, K33A, and K55A were
tested in the GTP hydrolysis reaction, we observed between 2- and
6-fold reduction in GTP hydrolysis activity (Fig. 4C). Taken
together, these results suggest that the eIF5 mutants R15A, R15K, K33A,
and K55A were defective in their ability to hydrolyze GTP bound to the
40 S initiation complex, although they retained the ability to bind eIF2
.
The small reduction in binding efficiency of mutant K55A as compared
with wild-type eIF5 (see legend to Fig. 4B) is presumably due to some conformational change induced in this eIF5 mutant. However,
it should be noted that these binding analyses are at best
semi-quantitative and stoichiometric, whereas eIF5-promoted GTP
hydrolysis is highly catalytic. Additionally, it is known that the N
terminus of eIF5 is not required for binding to eIF2
(14). Thus, the
small reduction in the binding efficiency of K55A to eIF2
is
unlikely to account for the reduced rate of GTP hydrolysis promoted by
this eIF5 mutant.
eIF5 Mutants R15A, R15K, K33A, and K55A Are Also Defective in
Translation of mRNAs in Vitro--
We have previously demonstrated
that, in a yeast cell-free translation system depleted of endogenous
eIF5, translation of mRNAs in vitro can be restored by
the addition of exogenous yeast or mammalian eIF5 (18). We used such an
eIF5-depleted yeast cell-free translation system to test the ability of
the eIF5 mutants to restore translation of yeast mRNAs in
vitro. Fig. 5 shows that these
extracts showed poor translation activity in the absence of exogenously
added eIF5. As expected, translation in these extracts was restored by
the addition of purified wild-type rat eIF5 (Fig. 5). In the absence of
mRNA, the addition of eIF5 had virtually no effect (data not
shown). In contrast to wild-type rat eIF5, the mutant eIF5 protein R15A
was virtually inactive in restoring translation of mRNAs in these
extracts, whereas the mutant eIF5 proteins R15K, K33A, and K55A were
about 50, 40, and 20% as active as the wild-type protein, respectively
(Fig. 5).

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Fig. 5.
Effect of eIF5 mutations on in
vitro translation of total yeast RNA. eIF5-depleted
cell-free translation extracts were prepared from TMY201R cells (18),
incubated and analyzed for [35S]methionine incorporation
into proteins as described in Ref. (18). Each reaction mixture (50 µl) contained 15 µCi of [35S]methionine (11 Ci/mmol)
and 25 µg of total yeast RNA and where indicated 100 ng of either
purified recombinant rat eIF5 or purified mutant eIF5 proteins.
Following incubation at 25 °C for 40 min, aliquots (10 µl) were
withdrawn and analyzed for [35S]methionine incorporation
into proteins. A control reaction mixture lacking total yeast RNA and
exogenously added eIF5 was also incubated and analyzed. The amount of
[35S]methionine incorporated into proteins in this
control reaction mixture was subtracted from the results shown. It
should be noted that data similar to these presented in the figure were
obtained in several independent experiments.
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DISCUSSION |
Several lines of evidence presented in previous reports (1,
11-14) and summarized below suggested that eIF5 functions as a
GTPase-activating protein (GAP) in translation initiation. First, eIF5
promotes GTP hydrolysis only when the protein interacts with the 40 S
initiation complex (40 S·AUG·Met-tRNAf·eIF2·GTP).
eIF5, by itself, neither binds nor hydrolyzes either free GTP or GTP bound as a Met-tRNAf·eIF2·GTP ternary complex in the
absence of 40 S ribosomal subunits. Second, eIF5 interacts with the
heterotrimeric GTP-binding protein eIF2 (10). This interaction, which
occurs between the conserved lysine residues at the N-terminal region of eIF2
and the conserved glutamic acid residues at the C-terminal region of eIF5 (12-14), is essential for eIF5 activity both in vitro (14) and in vivo in yeast cells (13, 14).
In this paper, we present additional evidence that shows that eIF5
indeed functions as a GAP in translation initiation. Typical GAPs like
RhoGAPs and RasGAPs, which have been characterized extensively, contain
"arginine-finger" motifs consisting of an invariant arginine residue at the N terminus of their catalytic domains that are necessary
for their GTPase-stimulating activity in addition to motifs that are
necessary for interacting with their respective G proteins (Ref. 16 and
Table I). Biochemical and structural studies carried out with RasGAP
have shown that in RasGAP the arginine at position 789 is the
"primary" element required for stimulating GTP hydrolysis (16). The
positive charge of the invariant arginine residue stabilizes the
transition state of the GTP hydrolysis reaction by neutralizing the
negative charges developing in the transition state (16). In addition,
hydrogen bonding between Ras-GDP and GAP334 (the catalytic domain of
RasGAP) also stabilizes the transition state (21). A secondary
arginine residue, Arg-903 in GAP334 (Lys-122 in the case of
RhoGAP) stabilizes the finger loop carrying the primary arginine
residue (16).
Comparison of the N-terminal amino acid sequences of eIF5 from
different species revealed the presence of invariant arginine residues
at positions 15 and 48 that are conserved in all species of eIF5
identified so far (Fig. 2). Like RasGAP and RhoGAP (22), the Arg-15
residue in eIF5 is preceded by two conserved hydrophobic residues,
phenylalanine and tyrosine. It has been suggested that the function of
these two hydrophobic residues is to anchor the catalytic loop into the
hydrophobic core of the GAP (22). Thus, in eIF5, Arg-15 is in a better
sequence context than Arg-48. Based on this analysis, we observed that
mutation of Arg-15 in rat eIF5 to Ala or even to conservative Lys
resulted in mutant proteins that were unable to substitute for yeast
eIF5 in maintaining yeast cell growth and viability of a
TIF5 yeast strain (Fig. 3). In contrast,
TIF5 yeast cells expressing eIF5 mutant R48A were able to
maintain growth and viability (Fig. 3). In agreement with these results
obtained in vivo, we observed that the purified eIF5 mutants R15A and R15K were severely defective in their ability to promote GTP
hydrolysis. Whereas mutant R15A showed virtually no activity, mutant
R15K showed a low level of activity (<20%). Thus, although the lysine
residue at position 15 of eIF5 mutant R15K was able to compensate for
the positive charge of arginine, it was probably unable to correctly
position itself with respect to the guanine nucleotide or the active
site of the GTPase protein (presumably the conserved histidine residue
at position 138 in the
subunit of human eIF2 (23)) in the
transition state. We also observed that mutation of Lys-33 and Lys-55
of rat eIF5 to alanine also caused a severe defect in eIF5 function
both in vitro and in vivo in yeast cells. In
analogy with RasGAP and RhoGAP, it is likely that either Lys-33 or
Lys-55 or both in rat eIF5 constitute the secondary element required
for eIF5 GAP function.
An important property of eIF5-dependent GTP hydrolysis
reaction is that, in addition to eIF2 and eIF5, 40 S subunits also play
an essential role in GTP hydrolysis. This is analogous to the essential
requirement of 50 S ribosomal subunits in IF2-, EF-Tu-, and
EF-G-catalyzed GTP hydrolysis reaction in prokaryotes (7, 8). A similar
requirement of ribosomes in GTP hydrolysis reaction catalyzed by the
signal recognition peptide receptor subunit SR
has been reported
(24). Biochemical studies of GAP mutants and crystal structure analysis
of GTPase·GAP complexes (22) have shown that GAPs provide two
functions to the transition state of a GTP hydrolysis reaction. First,
it physically binds to the G protein and causes a conformational change
in the G protein resulting in the stabilization of the switch I and
switch II regions in the G protein (22). This results in the correct
positioning of the active site glutamine residue of the G protein in
the transition state and its activation. Second, GAP also provides an
arginine residue to the pentacoordinate transition state and stabilizes it. In the case of eIF5-dependent GTP hydrolysis reaction,
although eIF5 provides the essential arginine residue of the GAP, the
question arises: how are the switch regions in eIF2
(the polypeptide
that contains the consensus GTP binding domains (23, 25) and is the
presumed GTPase) stabilized in the absence of direct physical interaction between eIF5 and eIF2
? Clearly, although interaction between eIF5 and eIF2
anchors eIF5 to eIF2 and eIF2
is also known
to physically interact with eIF2
(26), the fact remains that these
interactions, although necessary, are not sufficient for GTP
hydrolysis, because eIF5 cannot promote hydrolysis of GTP bound to eIF2
in the Met-tRNAf·eIF2·GTP ternary complex in the
absence of 40 S ribosomal subunits. Thus, the possibility exists that
the 40 S subunit directly interacts with eIF2
and causes the
conformational change necessary to stabilize the switch regions in
eIF2
. In this respect, the 40 S ribosomal subunit also behaves as a
GAP in eIF5-dependent GTP hydrolysis reaction during
translation initiation. Such a distribution of dual functions of a GAP
in two different proteins has also been suggested for GTP hydrolysis
reaction mediated by ADP-ribosylation factor and G
signal
transduction protein (27).
Finally, both mammalian and yeast eIF5 have been reported (3, 4) to
contain sequence motifs that are somewhat homologous to G1-G4 domains
that are characteristic of members of the GTPase superfamily (28).
However, unlike the well characterized GTPases, these domains in eIF5
are quite "imperfect" (Table II). The
conserved G1 domain GXXGXGK(S/T) is present in
rat eIF5 as 27GKGNGIKT34 and in yeast eIF5 as
27GRGNGIKT34. Clearly, insertion of an extra
amino acid, isoleucine, into the consensus phosphate-binding loop makes
the G1 domain an imperfect consensus sequence. Additionally, the first
four amino acids in the G1 domain in eIF5 are
GXGX instead of the consensus GXXG. Furthermore, the spacings between the four domains are also not conserved in eIF5 as they are in members of the GTPase superfamily. Finally, unlike GTPases like Ras, where a conserved glutamine residue
(histidine in some GTPases) in the G3 domain is the active site of the
GTPase (28), such a conserved positively charged residue at a similar
position is absent in eIF5. These observations along with the results
of our mutational analysis (data not shown) showing that mutation of
conserved residues in the G1 and G4 domains of eIF5 did not affect eIF5
function in vitro and in vivo in yeast cells
suggested that these domains are not critical for eIF5 function.
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Table II
Sequence homology of eIF5 with proteins of the GTPase superfamily
Comparison of the putative G1-G4 GTP-binding domains in the eIF5 amino
acid sequence with the conserved sequence motifs in the GTPase
superfamily (28). The sequences of rat and yeast eIF5 are from Refs. 3
and 4, respectively. The other sequences are from Ref. 28.
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