From the Division of Genome Biology, Cancer Research
Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan and § Human Genome Center, Institute of Medical
Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku,
Tokyo 108-8639, Japan
Received for publication, December 29, 2000, and in revised form, January 31, 2001
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ABSTRACT |
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When starved for a single amino acid, the budding
yeast Saccharomyces cerevisiae activates the
eukaryotic initiation factor 2 Protein synthesis in eukaryotic cells is suppressed by
stress-induced phosphorylation of eukaryotic initiation factor 2 Mammalian cells have four eIF2 The molecular mechanism underlying general control response is
currently considered as follows. When the budding yeast starves for a
single particular amino acid, free tRNAs, which are not charged with
amino acids, accumulate within the cells and bind to a bipartite domain
composed of the histidyl tRNA synthetase-related domain and the
C-terminal ribosome-binding domain of GCN2 (11). This bipartite domain
forms an inhibitory interaction with the kinase domain, which is
disrupted upon binding of tRNAs (11). In addition to uncharged tRNAs,
which unmask the kinase domain, genetic evidence suggests that in
vivo activation of GCN2 requires another gene, GCN1,
encoding a protein bearing a region homologous to translation
elongation factor 3 (12). GCN1 forms a stable complex with the
ATP-binding cassette protein GCN20 and functions on an elongating
ribosome (13, 14). GCN2 is activated by uncharged tRNAs in the presence
of GCN1 and phosphorylates eIF2 In contrast to the action of uncharged tRNAs and the mechanism for
derepressed translation of GCN4 mRNA, how GCN1
participates in the activation of GCN2 was poorly understood at the
molecular level. Recently, we and others showed that a direct
interaction between GCN1 and GCN2 is necessary for general control
response, thereby providing the first insight into the underlying
mechanism (15, 16).
In this study, we determine the minimal essential regions of GCN1 and
GCN2 for the complex formation and demonstrate that phosphorylation of
eIF2 Yeast Strains--
The strains used in this study are summarized
in Table I.
Two-hybrid Assay and Other General Yeast Methods--
The
two-hybrid vectors, pGBK and pGAD424g, were described previously (17,
18). For high efficiency transformation, the protocol of Gietz and
Schiestl (19) was adopted except for the addition of 10% dimethyl
sulfoxide prior to the heat shock step (20).
PCR-based Random Mutagenesis of GCN1--
A
GCN1 DNA fragment (nucleotides 6142-7146) was cloned
between the segments encoding the GAL4 activation domain (AD) and the C-terminal 75-amino acid (aa) region of CDC24, which bears a PC motif
and is called the PC motif-containing region (PCCR) (18). We subjected
the GAL4 AD-GCN1-PCCR fragment to error-prone PCR in 50 µl of 1× PCR
buffer containing 1 unit of Taq DNA polymerase, 200 µM dATP, 2 mM dCTP, 2 mM dGTP, 2 mM dTTP, 2 mM MgCl2, 5 pmol of each
primer under the following thermal cycling: 94 °C for 3 min,
followed by 30 cycles of 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 2 min. The amplified products of error-prone PCR were cloned into pGAD424g by a transformation-associated recombination technique (21) using PJ69-4A Construction of a gcn1-F2291L-T7 Mutant--
The
gcn1-F2291L DNA fragment (nucleotides 6538-7401) was cloned
in pUC-URA3, a pUC13 derivative bearing the URA3 marker
(15), to obtain pUC-URA3-gcn1-F2291L, which was subsequently linearized with Bglll, and introduced into the yeast MB758-5B cells.
The Ura+ transformants were tested for successful targeted
integration by diagnostic colony PCR. These clones were then selected
for 5FOA resistance and examined for the desired allele replacement to
obtain the strain JBZ1 (MATa ura3
gcn1-F2291L).
For epitope tagging of GCN1, we first constructed a pUC-URA3 derivative
bearing a DNA fragment encoding the 3'-end portion of GCN1
ORF with its flanking region (nucleotides 7705-8214) followed by
Ashbya gossypii TEF2 terminator, which is derived from the kanMX cassette (22). Following the insertion of a T7
epitope-encoding sequence by an inverse PCR-mediated procedure to
C-terminally tag GCN1, the plasmid was linearized and transformed into
MB758-5B and JBZ1 to obtain the strains JBZ2 (MATa
ura3 GCN1-T7::URA3) and JBZ3
(MATa ura3 gcn1-F2291L-T7::URA3),
respectively. These strains were spotted onto agar plates of SC Reporter Assay for Derepression of GCN4 mRNA--
The p180
reporter construct is a low copy number plasmid, which encodes an
mRNA bearing the GCN4-lacZ fusion ORF preceded by the
entire 5' leader region of GCN4 mRNA containing four
upstream ORFs to monitor the translational derepression (23). This
plasmid was introduced into MB758-5B, JBZ1, JBY4, and JBY5 cells, and the transformants were grown to midlogarithmic phase and shifted to SC
or synthetic dextrose (SD) medium (0.67% yeast nitrogen base
without amino acids, 2% glucose) containing 10 or 20 mM
3AT. Following the incubation at 30 °C for 3 h, cells were
collected and subjected to Immunoblotting Analysis of eIF2 The GI Domain Is the Minimal Essential Region to Interact with
GCN1--
We previously showed that the characteristic GI domain (aa
1-125) occurring at the N-terminal extremity of GCN2 directly binds to
GCN1 (15). Others also demonstrated that GCN2 interacts with GCN1 via
its N-terminal 272-aa region, which contains the GI domain followed by
an acidic region (16). To delimit the minimal essential region to
interact with GCN1, we prepared a series of truncated mutants for the
N-terminal region of GCN2 and tested them for binding with GCN1 using
the yeast two-hybrid system.
All of the mutants bearing intact GI domain (aa 1-125) showed
two-hybrid interactions with GCN1 (Fig.
1). The yeast cells with the longest GCN2
hybrid protein (aa 1-598), which includes the GI domain, acidic
region, and degenerate protein kinase domain ( Mapping the GCN2-binding Segment of GCN1--
Two-hybrid screening
using the GI domain as bait had revealed the C-terminal portion (aa
1925-2552) of GCN1 as its binding partner. This is coincident with the
results recently reported by others, which identified the region
spanning aa 2052-2428 as the sole GCN2-binding region of GCN1 (24). To
pinpoint the minimal binding region, we prepared variously truncated
fragments around this region using PCR and tested their binding to the
GI domain using the yeast two-hybrid system (Fig.
2). Two-hybrid interactions were examined
for growth on the medium lacking adenine and histidine. The
segment spanning aa 2064-2382 of GCN1 supported the growth as
efficiently as those by longer constructs when expressed as a
DNA-binding domain fusion. However, we failed to detect the interaction
in the opposite orientation for unknown reason. Further deletion either
from its N- or C-terminal end completely abolished the interaction
(Fig. 2). From these results, we concluded that the region spanning
amino acids 2064-2382 is sufficient for binding to the GI domain of
GCN2.
Isolation of gcn1 Mutants Defective in Interaction with
GCN2--
We next intended to isolate gcn1 mutants
defective in association with GCN2, because they would highlight
critical residues for the recognition of the GI domain and because they
can be used to examine the role of this interaction. For this purpose,
we used a PCR-based random mutagenesis. However, since we had
already pinpointed the minimal essential GCN1 region for GCN2 binding, we do not need nonsense mutations to truncate the protein anymore. To
selectively obtain missense mutants, we developed a novel strategy described below (Fig. 3A).
We first modified the pGAD-GCN1-(2048-2382) plasmid so that the
GAL4 AD-GCN1 fusion protein is further tailed with the PCCR of CDC24
(18), which specifically interacts with the PB1 domain occurring at the
C-terminal end of BEM1.2 Following random
mutagenesis to GCN1 by error-prone PCR, clones were selected
for two-hybrid interaction with the PB1 domain, which guarantees that
the hybrid protein retains the C-terminal PCCR and hence is not
truncated within the GCN1 portion. From these untruncated populations,
clones incapable of interacting with GCN2 were identified using the
reverse two-hybrid selection based on URA3 reporter gene and
5FOA (25). Notably, this screening may be useful not only for the
elimination of truncated proteins but also for the identification of
those with missense mutations leading to protein instability, because
such clones display weaker two-hybrid signals than the wild-type
parental clone.
In practice, we used a dual bait two-hybrid system to perform both
selections simultaneously. We prepared a mutagenized GAL4 AD-GCN1-PCCR
library in PJ69-4A
From the clones displaying resistance to both 3AT and 5FOA, we
identified three single amino acid substitutions, namely F2291L, S2304P, and L2353P. While showing two-hybrid interactions with the PB1
domain comparable with that of the parental clone, these three
gcn1 single point mutants all failed to interact with GCN2 (Fig. 3B). These results suggest that the mutations affect
the ability of GCN1 to interact with GCN2 but not the stability of hybrid proteins (see below). We also identified seven mutants, each
bearing two amino acid substitutions, namely (L2303S,V2329D), (F2291S,V2376A), (K2317R,L2319P), (S2304P,L2356S), (F2291I,T2307N), (F2281L, Q2294R) and (F2299A,R2328D). Although we did not
determine which of the two is critical for binding, it is intriguing to note that all of these mutations occurred in the C- terminal half of
the pinpointed GCN1 segment, as do those in the three single point
mutants (Fig. 4).
Yeast Cells with gcn1-F2291L Fail to Show General Control
Response--
To examine the role of GCN1-GCN2 interaction, we
intended to generate a yeast strain bearing GCN1 incapable of
interacting with GCN2. For this purpose, we chose F2291L substitution,
because it occurs within a cluster of identically conserved amino acid residues among GCN1 from various species (Fig. 4). We also tagged GCN1
in this mutant and its parental strain with the T7-epitope at their
C-terminal ends to facilitate detection by anti-T7 antibody.
As shown in Fig. 5A,
comparable amounts of GCN1 were detected in wild type and
gcn1-F2291L cells, thereby demonstrating that the mutation
does not destabilize the full-length protein in vivo. We
then examined these cells for sensitivity to 3AT, which is an inhibitor
of HIS3, a typical GCN4 target, and has been used as an indicator of
general control response. The mutant cells displayed remarkably higher
3AT-sensitivity than the parental strain (Fig. 5B). A
similar phenotype was reported for yeast cells bearing the
gcn2-Y74A allele, which encodes GCN2 defective in interaction with GCN1 (15). Taken together, these results indicate that
the interaction between GCN1 and GCN2 is necessary for general control
of amino acid synthesis.
Mutants with Defective GCN1-GCN2 Interaction Fail to Derepress
Translation of GCN4 mRNA--
The Gcn Phosphorylation of eIF2 GCN1 is required for the activation of GCN2 in the budding yeast
under amino acid starvation; deletion or mutations of GCN1 were
reported to abolish phosphorylation of eIF2 The minimal essential region of GCN2 to interact with GCN1 was mapped
to its N-terminal 125 residues (Fig. 1), which we had designated as the
GI domain (15). The N-terminal 272 residues involving the GI domain
followed by an acidic region were reported necessary for the
interaction by others (16). However, our data shown here and in a
previous report (15) clearly indicate that the GI domain per
se, but not the acidic region, serves as the core for the binding.
As pointed out previously (15), the GI domain is found in various
proteins other than GCN2. They include Impact (a product of an
evolutionarily conserved gene that is genetically imprinted in mice)
(29-31), AO7 (a RING finger protein interacting with
ubiquitin-conjugating enzymes) (32), ARA54 (a coactivator of androgen
receptor) (33), YDR152W (a yeast hypothetical protein), YLR419W (a
member of the DEAH-box RNA helicase family), and so forth. It remains
to be elucidated whether these GI domains also function in protein binding.
We also found that amino acid residues 2064-2382 comprise the minimal
essential region of GCN1 to recognize the GI domain of GCN2 (Fig. 2).
In accordance with this result, the segment spanning residues
2052-2428 of GCN1 was recently reported responsible for binding to
GCN2 (24). In contrast to GCN2, the primary sequence of the GI
domain-binding region of GCN1 lacks any apparently characteristic feature (Fig. 4). We thus took a random mutagenesis approach to identify critical residues for the recognition of GI domain.
For this purpose, we developed a unique dual bait two-hybrid strategy,
which allows one to selectively identify missense mutations leading to
defective interaction (Fig. 3A). Furthermore, this strategy
would be also useful to eliminate mutants encoding unstable proteins,
which can be identified as clones with weaker interaction between the
C-terminally attached domain and its binding partner (i.e.
PCCR and PB1). Therefore, it provides a versatile tool for fine
analysis of protein-protein interactions.
Using this strategy, we have so far identified three single amino acid
substitutions, namely F2291L, S2304P, and L2353P, each of which
abolishes interaction between GCN1 and GCN2 (Fig. 3B). Others reported that GCN1(R2259A) also fails to associate with GCN2
(24). Intriguingly, all of these mutations occur in the C-terminal half
of the GCN2-binding region. In addition, 14 amino acid substitutions in
the seven mutants that we identified as bearing two mutations were also
mapped to this portion (Fig. 4). These results suggest that the
C-terminal half functions as the interaction surface with the GI
domain. The apparent lack of N-terminal mutants may indicate that the
N-terminal half is not involved in target recognition
and rather plays a role in structural integrity of the domain.
Then, most mutations in this region lead to disordered and unstable
proteins and hence would be excluded by our screening strategy as
discussed above. More exhaustive screening to fully uncover the
important residues is necessary to obtain deeper insight into the
mechanism for recognition of the GI domain. It would also be
informative to compare the sequence of GCN1 with those of the binding
partners for other GI domains, which remain to be identified.
We and others showed that yeast cells defective in GCN1-GCN2
interaction display 3AT-sensitive growth, indicative of impaired induction of HIS3, which is under the regulation of the
transcription factor GCN4 (15, 16, 24) (Fig. 5). Indeed, the
derepression of GCN4 translation was impaired in these
mutants (Fig. 6).
However, note that the derepression of GCN4 translation is
not always caused by the activation of GCN2. Mutations in subunits of
eIF2 and eIF2B can mimic the effect of phosphorylation of eIF2 We thus directly examined the phosphorylation of eIF2 Residual phosphorylation of eIF2 Based on these observations, we conclude that the GI domain-mediated
association of GCN2 to GCN1 is necessary for the full activation of
GCN2 kinase in vivo upon amino acid starvation and hence for
efficient derepression of GCN4 translation, leading to
general control response.
(eIF2
) kinase GCN2 in a
GCN1-dependent manner. Phosphorylated eIF2
inhibits
general translation but selectively derepresses the synthesis of the
transcription factor GCN4, which leads to coordinated induction of
genes involved in biosynthesis of various amino acids, a phenomenon
called general control response. We recently demonstrated that this
response requires binding of GCN1 to the GI domain occurring at the N
terminus of GCN2 (Kubota, H., Sakaki, Y., and Ito, T. (2000)
J. Biol. Chem. 275, 20243-20246). Here we provide the
first evidence for the involvement of GCN1-GCN2 interaction in
activation of GCN2 per se. We identified a C-terminal segment of GCN1 sufficient to bind the GI domain and used a novel dual
bait two-hybrid method to identify mutations rendering GCN1 incapable
of interacting with GCN2. The yeast bearing such an allele,
gcn1-F2291L, fails to display derepression of GCN4
translation and hence general control response, as does a GI
domain mutant, gcn2-Y74A, defective in association with
GCN1. Furthermore, we demonstrated that phosphorylation of eIF2
is
impaired in both mutants. Since GCN2 is the sole eIF2
kinase in
yeast, these findings indicate a critical role of GCN1-GCN2 interaction
in activation of the kinase in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(eIF2
)1 on a serine
residue at position 51 (1). The phosphorylation converts eIF2
from
the substrate to an inhibitor of eIF2B, the guanine nucleotide exchange
factor of eIF2; phosphorylated eIF2-GDP forms a stable complex with
eIF2B to hamper recycling of eIF2-GDP to eIF2-GTP (2). Scarcity of
eIF2-GTP accordingly decreases the level of the ternary complex
composed of eIF2, GTP, and the charged initiator tRNA, a prerequisite
for translational initiation, and hence leads to general suppression of
protein synthesis. Thus, eIF2
kinases play pivotal roles in this
famous translational control.
kinases, each of which is activated
in response to a distinct stress. Heme-regulated inhibitor is
activated by hemin deprivation (3); double-stranded
RNA-dependent kinase is activated by double-stranded RNA
(3); RNA-dependent kinase-like endoplasmic reticulum kinase
is activated by unfolded proteins (4, 5); and GCN2 is activated by
serum or amino acid starvation (6, 7). In contrast, the budding yeast
Saccharomyces cerevisiae has the sole eIF2
kinase, GCN2, the founding member of this family. The yeast GCN2 is
activated by starvation for amino acids, glucose deprivation, purine
limitation, and impaired tRNA synthetase activity (8-10). The gene for
this kinase was originally identified in the studies of a response to
amino acid starvation called general control of amino acid synthesis,
and hence was termed GCN2 (general
control nonderepressible 2).
to suppress protein synthesis via
the mechanism described above. However, the mRNA encoding GCN4 is
selectively translated by a unique mechanism, which depends on the four
short open reading frames (ORFs) preceding the one for GCN4 (10). The
transcription factor GCN4 induces the expression of genes involved in
various amino acid synthetic pathways.
, translational derepression of GCN4 mRNA, and
general control response are impaired in the gcn1 and
gcn2 mutants defective in this interaction. These results
provide the first direct evidence for a crucial role of GCN1-GCN2
interaction in the activation of the eIF2
kinase.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Genotypes of yeast strains used in this study
(MATa) as a
host. Transformants were then mated with Mav
(MAT
)
bearing pGBK-GCN2 and pHLZ-BEM1-PB1. The former plasmid encodes a
hybrid protein between the GAL4 DNA-binding domain and GI domain of
GCN2, and the latter one encodes a protein between LexA and the PB1
domain of BEM1 (aa 472-551), which specifically binds to PCCR of
CDC24.2 Diploid cells were
then plated onto synthetic complete medium (20) lacking Trp,
Leu, and His (SC
Trp
Leu
His) supplemented with 20 mM
3-aminotriazole (3AT) and 0.2% 5-fluoro-orotic acid (5FOA).
Ura or
SC
Ura
His plus 20 mM 3AT as described previously
(15).
-galactosidase assay as described
previously (18).
Phosphorylation--
The
yeast JBY2, JBY3, JBZ2, and JBZ3 cells were grown to midlogarithmic
phase and shifted to YPAD (1% yeast extract, 2% peptone, 0.004%
adenine sulfate, 2% glucose) or SD medium supplemented with 10 or 20 mM 3AT. Following incubation at 30 °C for 4 h, ~107 cells were collected, resuspended in 1.0 ml of
distilled H2O, and broken by the addition of 150 µl of
breaking buffer (2 M NaOH, 2 M
-mecaptoethanol). Following incubation on ice for 10 min, the lysate
was neutralized by the addition of 130 µl of 60% trichloroacetic acid. Proteins were then collected by centrifugation at 15,000 × g and washed twice with 700 µl of acetone. Protein extract
equivalent to 106 cells was subjected to each lane of
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose filters. Filters were blocked by PBS-T (137 mM NaCl, 8.1 mM
Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4, 0.1% Tween 20, 4%
nonfat dry milk). Phosphorylated eIF2
was visualized by using PBS-T
containing an antibody that specifically recognizes eIF2
phosphorylated at serine 51 (BIO-SOURCE).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PK), showed higher
-galactosidase activity than those lacking
PK (aa 1-272).
Although many factors affecting two-hybrid interactions, including
intracellular level of hybrid proteins and efficiency of nuclear
transport, make it difficult to evaluate strength of binding by this
method, the result described above is in accordance with the one
obtained using an in vitro pull-down binding assay (16).
Notably, removal of the acidic region did not diminish
-galactosidase activity any more, and we failed to detect any evidence for this region to bind GCN1 (Fig. 1). In contrast, further deletion from either end of the GI domain (aa 1-125) completely abolished the interaction (Fig. 1). We had also shown that substitution of conserved residues in the GI domain abrogated the interaction (15).
These results indicated that the GI domain itself serves as the minimal
essential region for the binding to GCN1 and that the acidic region has
little if any role in the interaction.
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Fig. 1.
The GI domain of GCN2 is sufficient to
interact with GCN1. The schematic domain structure of GCN2 is
shown at the top. Variously truncated N-terminal regions of
GCN2 were examined for interaction with GCN1 using the yeast two-hybrid
system. The yeast PJ69-4A cells (37) co-transformed with the indicated
two-hybrid plasmids were examined for adenine- and
histidine-independent growth and for the induction of -galactosidase
activity (in units (U)) driven by the lacZ
reporter gene.
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Fig. 2.
Pinpointing the minimal essential segment of
GCN1 to interact with the GI domain of GCN2. The structure of GCN1
is schematically depicted at the top. A C-terminal fragment
of GCN1 (aa 1925-2552), which had been identified in a two-hybrid
screening using the GI domain as bait, was variously truncated by a
PCR-mediated procedure and examined for the interaction with the GI
domain of GCN2. Two-hybrid interactions were examined for growth on the
medium lacking adenine and histidine. Note that the segment
spanning aa 2064-2382 showed a two-hybrid interaction with GCN2
comparable with those of longer constructs when expressed as a
DNA-binding domain fusion but not as an AD fusion.
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Fig. 3.
Isolation of gcn1 mutants
incapable of interacting with GCN2 using a dual bait two-hybrid
system. A, the principle of dual bait two-hybrid
selection schematically shown. The GCN2-binding segment of GCN1
(aa 2048-2382) was fused at its C terminus with the PCCR of CDC24,
which specifically binds to the PB1 domain of BEM1. The GCN1-PCCR
hybrid protein was expressed as a GAL4 AD fusion from pGAD-GCN1-PCCR,
the insert of which was amplified by error-prone PCR. Clones bearing
these plasmids were subjected to two-hybrid selection using dual baits,
namely GAL4 DNA-binding domain-GCN2 and LexA-BEM1-PB1, which induce
URA3 and HIS3, respectively, when interacting
with GAL4 AD-GCN1-PCCR. Clones that showed two-hybrid interaction with
the PB1 domain but not with GCN2 were selected as those resistant to
both 3AT and 5FOA. The products of such mutants bear intact PCCR and
hence would not be truncated within the GCN1 moiety, thereby allowing
us to identify missense mutations abolishing the interaction with GCN2
(see "Results"). B, the parental clone and the
three single point mutants obtained by the dual bait screening
(A) were examined for two-hybrid interactions with GCN2 and
the PB1 domain, both expressed as GAL4 DNA-binding domain fusions from
pGBK, in PJ69-4A cells (37). Transformants bearing the indicated
plasmids (bottom) were streaked onto agar plates of
SC Trp
Leu (top left) and SC
Trp
Leu
Ade
His
(top right).
(MATa). These cells were then mated with Mav
(MAT
) cells that bear two plasmids
expressing GAL4 DNA-binding domain-GCN2 hybrid protein and LexA-PB1
hybrid protein. Note that the genome of Mav
has URA3 and
HIS3 reporter genes driven by GAL4 and LexA, respectively
(Table I). The diploid cells formed by the mating were selected for
resistance to both 3AT and 5FOA. The resistance to 3AT indicates that
the HIS3 reporter gene is induced and that an interaction is
occurred between the GCN1-hybrid protein and the PB1 domain. On the
other hand, 5FOA resistance is an indicative of failed induction of
URA3 or impaired association of the GCN1-hybrid protein with GCN2.
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Fig. 4.
GI domain-binding region of GCN1 and
mutations abolishing interaction. The amino acid sequence of the
GI domain-binding region of GCN1 (aa 2048-2382) is aligned with the
corresponding regions of its homologs from fission yeast
(GenBankTM accession number CAA92385),
Arabidopsis (AAD38254), nematode (AAF60721), fruit fly
(AAF45332), and human (BAA13209). Residues identically conserved among
at least four species are boxed in black. #, the
positions of amino acid substitutions found in the three single-point
mutants defective in interaction with GCN2, namely F2291L, S2304P, and
L2353P. *, R2259A substitution, which was also reported to abrogate the
binding (24). The letters above the budding yeast
sequence represent amino acid substitutions found in the seven double
mutants, namely L2303S,V2329D, F2291S,V2376A, K2317R,L2319P,
S2304P,L2356S, F2291I,T2307N, F2281L,Q2294R, and F2299A,R2328D. Note
that we did not determine which of the two substitutions in each mutant
is responsible for defective interaction.
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Fig. 5.
General control response of
gcn1-F2291L mutant defective in interaction with
GCN2. A, T7-tagged GCN1 and GCN1(F2291L) were
visualized by immunoblotting with an anti-T7 tag antibody.
B, the yeast cells JBZ2 (GCN1-T7) and JBZ3
(gcn1-F2291L-T7) were spotted onto agar plates for SC Ura
(top) or SC
Ura
His supplemented with 20 mM
3AT (bottom).
phenotype
described above suggests that the derepression of GCN4
translation is impaired in both gcn1-F2291L and
gcn2-Y74A mutants. We thus examined the translation of
GCN4 mRNA using a reporter construct bearing
lacZ preceded by the characteristic GCN4 leader
region, which is responsible for the derepression (23). The wild-type
cells showed a remarkable induction of
-galactosidase activity under
starved or derepressed conditions (Fig.
6). In contrast, the induction was
severely impaired in both gcn1-F2291L and
gcn2-Y74A cells (Fig. 6). Thus, the interaction between GCN1 and GCN2 is required for efficient derepression of GCN4
translation under amino acid-starved conditions.
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Fig. 6.
Derepression of GCN4
translation in mutants defective in GCN1-GCN2 interaction.
A, the yeasts MB758-5B (GCN1) and JBZ1
(gcn1-F2291L), each bearing the GCN4-lacZ
reporter plasmid p180 (23), were cultured in the indicated
medium, and -galactosidase (
-gal) activities
were measured (in units (U)). B, the yeasts JBY4
(GCN2-T7) and JBY5 (gcn2-Y74A-T7), which had been
generated from JBY2 and JBY3 (15) by popping out the integrated
URA3 marker, were transformed with p180 and examined for the
induction of
-galactosidase activities.
Is Impaired in Mutants with Defective
GCN1-GCN2 Interaction--
Finally, we intended to determine whether
the eIF2
kinase is activated in these mutants upon amino acid
starvation, because GCN2-independent mechanisms to derepress
GCN4 translation are also possible (9, 10, 26-28). The
phosphorylated eIF2
in mutants and their parental strains were
examined under rich or poor conditions using an antibody specific to
eIF2
phosphorylated at Ser-51. While phosphorylated eIF2
was
barely detected under rich or repressed conditions, substantial
phosphorylation of eIF2
was readily observed in the wild-type cells
subjected to amino acid starvation (Fig.
7). In contrast, the induction of the
phosphorylation was substantially impaired in the cells bearing
gcn1-F2291L or gcn2-Y74A compared with their
respective parental strains, although residual levels of
phosphorylation were detected (Fig. 7). Since GCN2 is the sole eIF2
kinase in the budding yeast, these results indicate that the
interaction with GCN1 is necessary for the GCN2 to be fully activated
in amino acid-starved cells.
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Fig. 7.
Phosphorylation of eIF2
in mutants defective in GCN1-GCN2 interaction. A,
the yeast cells JBZ2 (GCN1-T7) and JBZ3
(gcn1-F2291L-T7) cultured under the indicated conditions
were subjected to immunoblotting analysis using an anti-phospho-eIF2
antibody. B, the yeast cells JBY2 (GCN2-T7) and
JBY3 (gcn2-Y74A-T7) were similarly analyzed as in
A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, which is mediated by
the sole eIF2
kinase GCN2 (12, 14). It had, however, remained totally unknown how GCN1 activates GCN2 until we and others provided evidence for their direct association and its requirement for a general
control response (15, 16). In this study, we determined the minimal
essential regions for GCN1-GCN2 association and demonstrated, for the
first time, that the interaction is critical to the activation of GCN2
itself, which leads to the selective derepression of GCN4 translation and subsequent general control response.
as
exemplified by several GCD genes (9, 10). The reduction in
amounts (10) or impaired base modification (26) of initiator tRNA also
induces a similar phenotype. Other studies demonstrated that
GCN2-independent derepression of GCN4 translation is
elicited by overexpression of NME1 (27), encoding the RNA
component of RNase MRP (34), or PUS4, encoding the tRNA
pseudouridine 55 synthase (28).
in these
mutants and demonstrated its considerable impairment (Fig. 7); the
Gcn
phenotype of mutants defective in GCN1-GCN2
interaction is associated with impaired phosphorylation of eIF2
.
Therefore, these mutants fail to fully activate GCN2, because it is the
sole eIF2
kinase of the budding yeast.
in these mutants (Fig. 7) indicates
that weakened GCN1-GCN2 interactions can still support minimal
activation of the kinase. Notably, GCN1 and GCN2 are anchored onto
ribosome through their respective ribosome-binding domains (14, 24, 35,
36), presumably, in such close proximity that they can interact. It is
thus conceivable that the two proteins occasionally take a
configuration leading to activation of the kinase although the
interaction is considerably impaired by the mutations. In this context,
it is intriguing to note that overexpression of GCN2 can suppress the
Gcn
phenotype of gcn1-R2259A, also defective
in binding to GCN2, but not that of gcn1-
D,
which encodes GCN1 totally lacking the GCN2-binding region (24).
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ACKNOWLEDGEMENTS |
---|
We thank R. C. Wek (Indiana University) for the generous gift of plasmid p180 and H. Sumimoto (Kyushu University) for critical reading of the manuscript and encouragement.
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FOOTNOTES |
---|
* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan, Science, the Technology Agency of Japan, and the Japan Society for the Promotion of Science.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. Tel.: 81-76-265-2726; Fax: 81-76-234-4508; E-mail: titolab@kenroku.kanazawa-u.ac.jp.
Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M011793200
2 T. Ito and H. Sumimoto, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
eIF2, eukaryotic
initiation factor 2;
ORF, open reading frame;
aa, amino acid(s);
PCR, polymerase chain reaction;
AD, activation domain;
PCCR, PC
motif-containing region;
SC, synthetic complete;
SD, synthetic
dextrose;
3AT, 3-aminotriazole;
5FOA, 5-fluoro-orotic acid;
PK, degenerate protein kinase domain.
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REFERENCES |
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