1 Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; and 2 The Skirball Institute, New York University School of Medicine, New York, New York 10016
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ABSTRACT |
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In eukaryotic cells subjected to
environmental stress, untranslated mRNA accumulates in discrete
cytoplasmic foci that have been termed stress granules. Recent studies
have shown that in addition to mRNA, stress granules also contain 40S
ribosomal subunits and various translation initiation factors,
including the mRNA binding proteins eIF4E and eIF4G. However, eIF2, the
protein that transfers initiator methionyl-tRNAi
(Met-tRNAi) to the 40S ribosomal subunit, has not been
detected in stress granules. This result is surprising because the
eIF2 · GTP · Met-tRNAi complex is thought to
bind to the 40S ribosomal subunit before the
eIF4G · eIF4E · mRNA complex. In the present study, we
show in both NIH-3T3 cells and mouse embryo fibroblasts that stress
granules contain not only eIF2 but also the guanine nucleotide exchange
factor for eIF2, eIF2B. Moreover, we show that phosphorylation of the -subunit of eIF2 is necessary and sufficient for stress granule formation during the unfolded protein response. Finally, we also show
that stress granules contain many, if not all, of the components of the
48S preinitiation complex, but not 60S ribosomal subunits, suggesting
that they represent stalled translation initiation complexes.
eIF4E; eIF4G; eIF3; unfolded protein response; PERK
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INTRODUCTION |
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ONE OF THE
BEST-CHARACTERIZED mechanisms for regulating mRNA translation in
eukaryotic cells involves phosphorylation of the -subunit of
eukaryotic initiation factor, eIF2 (reviewed in Ref. 16).
During initiation, eIF2 forms a complex with GTP and initiator methionyl-tRNAi (met-tRNAi), and this ternary
complex subsequently binds to the 40S ribosomal subunit to form the 43S
preinitiation complex (reviewed in Refs. 10 and 24).
Through the action of a translation initiation factor complex referred
to as eIF4F, which is comprised of eIF4A, eIF4E, and eIF4G, mRNA is
bound to the 43S preinitiation complex, resulting in formation
of the 48S preinitiation complex (reviewed in Ref. 20).
During a later step in initiation, the GDP bound to eIF2 is hydrolyzed
in an eIF5-mediated process and initiation factors are released from the ribosome. Before binding Met-tRNAi and reforming the
ternary complex, the GDP bound to eIF2 must be exchanged for GTP, a
reaction that is catalyzed by the guanine nucleotide exchange factor,
eIF2B. One mechanism for regulating the activity of eIF2B involves
phosphorylation of the
-subunit of eIF2 on Ser51, an event that
converts eIF2 from a substrate into a competitive inhibitor of eIF2B
(reviewed in Ref. 10). Thus, by inhibiting eIF2B,
phosphorylation of eIF2
results in a global inhibition of protein synthesis.
Hyperphosphorylation of eIF2 occurs under a variety of conditions
that result in disruption of normal cell homeostasis. For example,
conditions that impede correct folding of newly synthesized proteins in
the lumen of the endoplasmic reticulum (ER), i.e., the so-called
unfolded protein response, result in activation of the eIF2
kinase,
PERK (also referred to as PEK) (28). PERK is a trans-ER
membrane protein with a luminal domain that exhibits homology to IRE1
and a cytoplasmic domain homologous to other eIF2
kinases (9,
29). Accumulation of misfolded proteins in the lumen of the ER,
such as occurs in response to thapsigargin treatment, promotes the
release of the chaperone protein, BiP, from the IRE1 homology domain,
allowing oligomerization of the protein and subsequent activation of
the cytoplasmic kinase domain (2). The activated kinase
then phosphorylates eIF2
, causing inhibition of eIF2B and a
reduction in protein synthesis rate.
Recent studies by Kedersha and coworkers (14, 15) show
that in cells treated with arsenite or subjected to heat shock, i.e.,
conditions that promote eIF2 phosphorylation, mRNA is sequestered into punctate cytoplasmic aggregates that are referred to as stress granules. Such structures have been observed in plant, yeast, and
animal cells (5, 14, 15, 23), and it has been proposed that they represent sites where mRNA is targeted for either
translation, degradation, or sequestration into untranslated mRNP
complexes (15). In addition to mRNA, stress granules
contain the RNA binding proteins TIA-1 and TIAR, poly(A) binding
protein (PABP), eIF3, the mRNA cap binding protein eIF4E, eIF4G, and
the 40S ribosomal proteins S3 and S19 (13). However,
stress granules reportedly lack eIF2 and the 60S ribosomal proteins L5
and L37 (13). The lack of eIF2 in stress granules is
surprising because binding of the eIF4G · eIF4E · mRNA
complex to the 40S ribosomal subunit does not occur in the absence of
eIF2 (25).
A recent study attempted to answer the question of whether or not
phosphorylation of eIF2 is sufficient to induce stress granule
formation by expression of a phosphomimetic variant of eIF2
, where
Ser51 was changed to aspartic acid (eIF2
S51D) (15). Overexpression of the variant protein promoted stress granule formation. In contrast to the wild-type protein, some, but not all,
stress granules were found to contain eIF2
S51D, although wild-type
eIF2
was not found (13). However, in the study by Kedersha and coworkers (15), the extent to which the
exogenously expressed eIF2
S51D was incorporated into three subunit
eIF2 holoproteins was not assessed. In addition, the phosphorylation
state of endogenous eIF2
in cells expressing eIF2
S51D was not
examined in that study. Thus the exogenously expressed
-subunit
variant may not have been extensively incorporated into the eIF2
holoprotein but instead may have been acting through a mechanism
distinct from that invoked by phosphorylation of the wild-type protein.
The mechanism through which phosphorylation of eIF2
induces stress
granule formation, therefore, remains obscure, and the question of
whether or not eIF2
phosphorylation is sufficient to induce stress
granule formation was incompletely answered.
In the present study, activation of PERK and phosphorylation of eIF2
were shown to be required for stress granule formation during the
unfolded protein response. Moreover, in cells expressing the
cytoplasmic PERK kinase domain as a fusion protein with the extracellular and transmembrane domains of the T-lymphocyte coreceptor CD4 (CD4-PERK) (2), activation of the cytoplasmic
membrane-associated kinase stimulated both eIF2
phosphorylation and
stress granule formation. Thus phosphorylation of eIF2
is necessary
and sufficient for stress granule formation during the unfolded protein
response. In contrast to the results of Kedersha et al.
(13), both eIF2 and eIF2B were associated with stress
granules. Finally, in thapsigargin-treated cells, eIF2 colocalized in
granules with other key regulatory translation initiation factors,
poly(A)+ RNA, and the 40S ribosomal proteins S3 and
S6. In contrast, neither elongation factor eEF2 nor the 60S
ribosomal proteins L5 or L37 accumulated in stress granules under any
of the tested stress conditions. Overall, the results suggest that
stress granules represent sites at which stalled initiation complexes accumulate.
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MATERIALS AND METHODS |
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Materials.
Cy2-conjugated AffiniPure donkey anti-mouse IgG (multiple labeling
grade) and Cy3-conjugated AffiniPure donkey anti-rabbit IgG (multiple
labeling grade) were purchased from Jackson Immunoresearch Laboratories. The Cy2 MAb and Cy3 MAb labeling kits were obtained from
Amersham Pharmacia Biotech. Affinity purified anti-eIF2 (pS51)
antibody was purchased from Biosource International, and antiribosomal
protein S6 and anti-eEF2 antibodies were purchased from Cell Signaling
Technology. Antiribosomal protein S3, L5, and L37 antibodies and
anti-TIA-1 antibody were kind gifts from Dr. Nancy L. Kedersha,
Brigham and Women's Hospital. Antibodies to the
-subunit of
eIF2, the
-subunit of eIF2B, eIF4E, and eIF4G were prepared in our laboratory.
Cell culture.
Cells were plated on coverslips in a six-well dish at an initial
density of 200,000 (NIH/3T3) or 100,000 [cell lines derived from
SV40-transformed mouse embryonic fibroblasts (SVT-MEF)] cells/well. Two days later, coverslips were removed and cells were fixed and permeabilized as described below. Cells remaining in the well were
harvested in SDS sample buffer at 90°C for measurement of eIF2
phosphorylation as described below.
Measurement of protein synthesis. Protein synthesis in NIH/3T3 or SVT-MEF cells was monitored by measuring the incorporation of [35S]methionine and [35S]cysteine into protein as described previously (17).
Cell fixation and permeabilization. Cells on coverslips were fixed in either 1% or 3% paraformaldehyde, as indicated in the figure legends, for 20 min at room temperature and the coverslips were washed twice with phosphate-buffered saline (PBS). The fixed cells were permeabilized by incubation in 0.2% Triton X-100 in PBS. The coverslips were then incubated for 1 h in 10% bovine serum albumin in PBS containing 0.1% Triton X-100.
Microscopy.
After fixation and permeabilization, cells were incubated with primary
antibody overnight at 4°C. For visualization of eIF2 or eIF2B
,
fixed, permeabilized cells were incubated with monoclonal anti-eIF2
or anti-eIF2B
antibodies that had been covalently labeled with Cy2
or Cy3, respectively, using a kit (CyDye MAb labeling kit) from
Amersham Pharmacia Biotech. For visualization of other proteins,
coverslips were incubated with primary antibody followed by a 1-h
incubation with a secondary antibody coupled to either Cy2 or Cy3, as
noted in the figure legends. Phosphorylated eIF2
was visualized by
incubation of permeabilized cells with an affinity-purified
anti-phospho-eIF2
antibody (Biosource International) that had been
covalently labeled with Cy3.5 using a CyDye kit. Coverslips were then
mounted on glass slides using Aquamount. Slides were examined using a
Nikon E-800 fluorescence microscope equipped with a Nikon PCM-2000
multiline laser confocal system equipped with argon ion and helium/neon
lasers. All images were acquired using a Nikon 60X plan apo objective
and were processed as TIFF files in Canvas (Deneba) using standard
image processing techniques.
Measurement of eIF2 phosphorylation.
Cells were maintained in culture as described above, with the exception
that they were harvested by scraping in SDS sample buffer at 90°C.
The relative amount of eIF2
in the phosphorylated form was
quantitated by protein immunoblot analysis using an affinity-purified antibody that specifically recognizes eIF2
phosphorylated at Ser51
[eIF2(
P)] (Biosource International). For this analysis, samples
were resolved by electrophoresis using a 12.5% SDS-polyacrylamide gel
and the proteins in the gel were electrophoretically transferred to a
polyvinylidene difluoride (PVDF) membrane. The membranes were incubated
with the rabbit polyclonal antibody that specifically recognizes
eIF2(
P), and blots were developed as described previously (18). The horseradish peroxidase coupled to the
anti-rabbit secondary antibody was then inactivated by incubating the
membrane in 15% hydrogen peroxide for 30 min at room temperature. The
total amount of eIF2
in the samples was determined by reprobing the blot with a monoclonal antibody that recognizes equally the
phosphorylated and unphosphorylated forms of eIF2
followed by an
anti-mouse secondary antibody. Values obtained using the
anti-eIF2(
P) antibody were normalized for the total amount of
eIF2
present in the sample.
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RESULTS |
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Previous studies have shown that arsenite treatment and heat
shock, both of which promote phosphorylation of eIF2, result in the
accumulation of TIA-1, TIAR, and poly(A)+ RNA into stress
granules (14, 15). To establish whether activation of the
eIF2 kinase PERK can likewise promote stress granule formation, NIH/3T3 cells were incubated in the presence or absence of thapsigargin and the distribution of TIA-1 and poly(A)+ RNA was assessed
by immunofluorescence microscopy. In cells maintained in the absence of
thapsigargin, TIA-1 was present in both the nucleus and cytoplasm (Fig.
1). However, in the presence of
thapsigargin, TIA-1 accumulated in punctate foci in the cytoplasm, a
distribution reminiscent of that reported for stress granules.
Likewise, poly(A)+ RNA was shown to accumulate in punctate
foci in thapsigargin-treated but not control cells, suggesting that the
cytoplasmic foci are indeed stress granules. Because conditions that
promote stress granule formation also stimulate eIF2
phosphorylation, the possibility that eIF2 might colocalize with TIA-1
in stress granules was examined. As shown in Fig. 1, both the
-subunit of eIF2 and the
-subunit of eIF2B accumulate in stress
granules in response to thapsigargin treatment. Moreover, both
translation factors colocalize with TIA-1, showing that under these
conditions both eIF2 and eIF2B accumulate in stress granules.
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Although previous studies have reported that a portion of eIF2
localizes to the nucleus (6, 12), the high proportion of
eIF2 present in the nucleus observed in Fig. 1 was surprising. A
possible explanation for this finding is that under the fixation conditions used above, some of the cytoplasmic eIF2 was lost, leading
to an apparently low cytoplasmic content. To assess this possibility,
cells were fixed with 3% rather than 1% paraformaldehyde and then
probed with anti-eIF2
or anti-eIF2B
antibodies. As shown in Fig.
2, following fixation with 3%
paraformaldehyde, the cytoplasmic signal is greatly increased relative
to the nuclear signal for both proteins. Importantly, cytoplasmic foci
are still observed in stressed cells fixed with 3% paraformaldehyde.
However, due to the overall stronger cytoplasmic staining, foci are not as easily visualized. Therefore, for the remainder of the studies reported herein, cells were fixed with 1% paraformaldehyde.
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To determine whether or not the eIF2 that accumulates in stress
granules is phosphorylated, an affinitypurified antipeptide antibody that only recognizes eIF2 when it is phosphorylated on
Ser51 [eIF2
(P)] was covalently labeled with Cy3.5 and used to
probe fixed cells. As shown in Fig. 3,
thapsigargin causes accumulation of eIF2
(P) in granules. Whether or
not all of the eIF2
in the granules is phosphorylated could not be
determined by using these antibodies.
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Phosphorylation of eIF2 in cells treated with thapsigargin occurs
through activation of PERK. If eIF2
phosphorylation is required for
stress granule formation, then cells lacking PERK should not exhibit
either eIF2
phosphorylation or granule formation. To examine the
requirement for eIF2
phosphorylation in the response, stress granule
formation in wild-type SVT-MEF (PERK +/+) cells was compared with
formation in SVT-MEF cells containing a chromosomal disruption of the
PERK gene (PERK
/
cells; Ref. 8). As in NIH/3T3 cells, thapsigargin
treatment of SVT-MEF (PERK +/+) cells results in formation of granules
that contain eIF2
(Fig. 4,
bottom). In contrast, granule
formation is not observed in SVT-MEF (PERK
/
) cells treated with
thapsigargin. Similarly, thapsigargin treatment inhibits protein
synthesis and enhances eIF2
phosphorylation in SVT-MEF (PERK +/+),
but not SVT-MEF (PERK
/
), cells (Fig. 4, top). Like
thapsigargin, arsenite inhibits protein synthesis by stimulating
eIF2
phosphorylation but does not induce the unfolded protein
response or activate PERK (8). As shown in Fig. 4, arsenite inhibits protein synthesis, enhances eIF2
phosphorylation, and promotes stress granule formation to a similar extent in SVT-MEF (PERK
/
) as in SVT-MEF (PERK +/+) cells. This result suggests that
it is eIF2
phosphorylation, and not ER stress, that promotes foci
formation.
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If eIF2 phosphorylation is sufficient to induce stress granule
formation, then induction of eIF2
phosphorylation through a
mechanism that does not otherwise impose a stress on the cell should
cause stress granule assembly. To further define the sufficiency of
eIF2
phosphorylation in the induction of stress granule formation, studies were performed using NIH/3T3 cells stably expressing fusion proteins consisting of the extracellular and transmembrane domains of
the CD4 coreceptor with either the wild-type PERK kinase domain [CD4-PERK(wt)] or a kinase-dead K618A variant [CD4-PERK(ka)]. As
shown in Fig. 5 (top),
treating cells expressing CD4-PERK(wt) with anti-CD4 antibody causes a
reduction in protein synthesis to 67% of the untreated control value.
In contrast, anti-CD4 antibody has no effect on protein synthesis in
CD4-PERK(ka)-expressing cells. Similarly, anti-CD4 antibody causes a
greater than fivefold increase in eIF2
phosphorylation in cells
expressing CD4-PERK(wt) but has no effect in cells expressing
CD4-PERK(ka). Furthermore, treating cells expressing CD4-PERK(wt), but
not CD4-PERK(ka), with anti-CD4 antibody results in accumulation of
eIF2
in stress granules (Fig. 5, bottom). The lack of
effect of anti-CD4 antibody in cells expressing CD4-PERK(ka) is not due
to an artifact of the transfection procedure because
thapsigargin inhibits protein synthesis, enhances eIF2
phosphorylation, and causes granule formation equally in cells
expressing either CD4-PERK(wt) or CD4-PERK(ka) (Fig. 5). Thus
activation of exogenously expressed PERK in the absence of ER stress
promotes stress granule formation.
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Because both eIF2 and eIF2B localize to stress granules, the
localization of the two proteins with each other and with other initiation factors was examined in control and thapsigargin-treated cells. As shown in Fig.
6A, the
cellular distribution of eIF2B exhibits a pattern similar to that of
eIF2
, and thapsigargin treatment causes accumulation of both
proteins in stress granules. Labeling cells simultaneously with both
antibodies reveals colocalization of eIF2
and eIF2B
in foci. To
determine whether other translation initiation factors exhibit a
similar redistribution during the unfolded protein response, the
subcellular distribution of two proteins that regulate mRNA binding to
the 43S preinitiation complex, eIF4G and eIF4E, were examined. In
contrast to eIF2
and eIF2B
, eIF4G and eIF4E are predominantly
localized to the cytoplasm, with less intense staining observed in the
nucleus (Fig. 6, B and C). Thapsigargin treatment
promotes the accumulation of eIF4G and eIF4E in foci and colocalization
of eIF4G with eIF2
and eIF4E with eIF4G, indicating that all four of
the proteins are present together in foci.
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During formation of the translation initiation complex, the
met-tRNAi · eIF2 · GTP ternary complex and
the eIF4G · eIF4E · mRNA complex both bind to the 40S
ribosomal subunit (reviewed in Ref. 24). Because this is
the only known mechanism through which eIF2 and eIF4F associate, the
possibility that 40S ribosomal subunits might localize to stress
granules in thapsigargin-treated cells was examined. As shown in Fig.
7, A and B, in
thapsigargin-treated cells, two proteins that are integral members of
the 40S ribosomal subunit, S3 and S6, colocalize with eIF2 into
cytoplasmic foci, suggesting that 40S ribosomal subunits accumulate in
stress granules under such conditions. In contrast, neither 60S
ribosomal proteins L5 or L37 nor eEF2 are detected in stress granules.
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DISCUSSION |
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In previous studies (14, 15), arsenite was shown to promote the accumulation of poly(A)-containing RNA, PABP, and two other RNA binding proteins, TIA-1 and TIAR, into what were referred to as stress granules. Results of a more recent study (13) revealed that stress granules also contain the translation initiation factors eIF3, eIF4E, and eIF4G, but eIF2, eIF2B, and eIF5 were not detected in stress granules. Finally, ribosomal proteins S3 and S19 were detected in stress granules, but L5 and L37 were not, suggesting that 40S, but not 60S, ribosomal subunits are components of stress granules. Thus stress granules contain most of the components of the 48S preinitiation complex. However, because eIF2 was not detected, it was proposed that stress granules represent sites of accumulation of inactive 48S preinitiation complexes (13).
In the present study, stress granule formation induced by arsenite, the
unfolded protein response, and activation of the protein kinase domain
of PERK at the plasma membrane all promoted the accumulation of eIF2
and eIF2B
into stress granules. These results demonstrate
that under a variety of conditions that promote eIF2
phosphorylation, eIF2 is associated with stress granules. The reason
that Kedersha et al. (13) failed to detect either eIF2
or eIF2B
in stress granules may be related to the conditions that
they used to visualize the proteins. In the study by Kedersha et al.
(13), eIF2
and eIF2B
were visualized using primary antibodies followed by secondary antibodies coupled to fluorescent dyes. In contrast, in the present study, the eIF2
and eIF2B
monoclonal antibodies were directly labeled with fluorescent dye to
eliminate the need for a secondary antibody [note that in both the
present study and the study by Kedersha and coworkers
(13), the same eIF2B
monoclonal antibody was used]. In
this regard, we found that when eIF2
was visualized in
thapsigargin-treated NIH/3T3 cells using the secondary antibody
approach, little if any eIF2
was detected in stress granules
(unpublished observations). The basis for this discrepancy is unknown
but could be a result of steric hindrance, in which the secondary
antibody is not able to bind to the primary antibody associated with
the 48S preinitiation complex. A second reason that Kedersha et al.
(13) might not have detected eIF2
in stress granules
could be related to the different cell types that were utilized in the
two studies. However, it should be noted that in the present study,
eIF2
was detected in stress granules in a variety of cell types,
including NIH/3T3 (Fig. 2), MEF (Fig. 5), and L6 myoblasts (unpublished
observations), suggesting that localization of eIF2
to stress
granules is not a finding unique to a single cell type.
The results of the present study extend earlier studies to show that
induction of stress granules requires phosphorylation of eIF2;
stress in the absence of eIF2
phosphorylation does not cause stress
granule formation. Thus, in PERK
/
cells, thapsigargin does not
cause eIF2
phosphorylation but does induce other endpoints of the ER
stress response (8). In fact, IRE1
activation occurs earlier and persists longer in PERK
/
cells than in wild-type cells
following induction of ER stress (8), suggesting that PERK
/
cells undergo a heightened stress response compared with wild-type cells. Therefore, stress in the absence of eIF2
phosphorylation is not sufficient to induce assembly of stress
granules. Further evidence that eIF2
phosphorylation is sufficient
to induce granule formation is provided by studies in which CD4-PERK
was ectopically expressed. In such cells, ER stress does not activate
the expressed kinase because it is localized to the cytoplasmic rather
than ER membrane, but treatment with anti-CD4 antibody does
(2). In the present study, it was found that activation of
the ectopically expressed kinase also promotes stress granule formation
and inhibits protein synthesis. In contrast, in cells expressing
kinase-dead CD4-PERK, anti-CD4 antibody had no effect on eIF2
phosphorylation or granule formation. Because protein synthesis is not
affected by anti-CD4 antibody treatment in cells expressing kinase-dead CD4-PERK, it is unlikely that either antibody treatment by itself or
expression of the CD4 chimeric proteins are inducing significant levels
of stress. Thus these results also suggest that eIF2
phosphorylation and not some other manifestation of the stress response is sufficient for stress granule formation. It should be noted that stress granule formation has been observed in the absence of eIF2
phosphorylation (13). In that study, agents that deplete intracellular
energy stores (i.e., 2-deoxyglucose, FCCP, or oligomycin) were shown to
cause stress granule formation in the absence of changes in eIF2
phosphorylation. However, the reduced ATP-to-ADP ratio that occurs in
response to treatment with such agents would likely lead to a decrease
in the GTP-to-GDP ratio. Because formation of the
eIF2 · GTP · met-tRNAi ternary complex is
sensitive to changes in the GTP-to-GDP ratio (see Ref. 11
and references therein), energy depletion would mimic eIF2
phosphorylation in that both result in reduced formation of the ternary
complex. The latter point is key because it is most likely reduced
ternary complex availability rather than eIF2
phosphorylation per se that is responsible for stress granule formation.
The finding that eIF2, eIF4E, eIF4G, poly(A)+ RNA, and 40S ribosomal subunits all localize to stress granules indicates that the structures contain 48S preinitiation complexes. However, because initiation factors are released (reviewed in Ref. 24) from the 48S preinitiation complex upon binding of the 60S ribosomal subunit to form the active initiation complex, and neither 60S ribosomal subunits nor eEF2 are observed in stress granules, it appears that granules do not contain either the final initiation complex (i.e., the complete 80S ribosome bound to mRNA) or polysomes. This result suggests that the preinitiation complexes present in stress granules may be defective at either the 60S subunit joining step or an earlier step, such as hydrolysis of GTP bound to eIF2. Thus, if joining of the 60S ribosomal subunit to the 48S preinitiation complex is slowed under stress conditions, then 48S preinitiation complexes would accumulate. In support of this premise, agents that inhibit protein synthesis by stalling the ribosome on mRNA, e.g., emetine and cycloheximide, minimize stress granule formation, whereas agents, e.g., puromycin, that inhibit protein synthesis by causing premature termination enhance granule formation (14). Thus it was proposed that puromycin enhances the formation of both preinitiation complexes and stress granules by increasing the availability of free (i.e., nonpolysome associated) 40S ribosomal subunits. In contrast, emetine and cycloheximide would cause sequestration of ribosomal subunits in polysomes, making them unavailable to form preinitiation complexes, which would lead to a reduction in stress granule formation. Thus the availability of free 40S ribosomal subunits is crucial for stress granule formation.
The finding that preinitiation complexes accumulate in stress granules suggests that such structures might represent assembly points for initiation complexes. In this regard, stress granules might functionally be the cytoplasmic equivalent of nuclear speckles. Speckles are intranuclear structures characterized by a localized concentration of proteins involved in pre-mRNA splicing. They persist for long periods of time, but the proteins within speckles are in continuous flux and exchange rapidly with proteins in the nucleoplasm (26). The function of speckles is controversial, but a variety of studies suggest that they may act as a storage site for splicing components and/or may represent an assembly site for splicing components. Furthermore, speckles become larger when transcription is inhibited but become smaller and more diffuse after stimulation of transcription (3, 4, 21, 30). Thus both stress granules and speckles may represent stalled complexes, translation initiation complexes in stress granules, and splicing complexes in speckles.
On the basis of the results of the present and previous studies, it
seems likely that stress granules contain preinitiation complexes not
actively involved in protein synthesis but, instead, similar to nuclear
speckles, may represent a storage or assembly site for translation
initiation complexes. The possibility that stress granules form at
intracellular sites where initiation complexes normally assemble is
particularly intriguing because the RNA binding proteins TIA-1 and TIAR
accumulate in stress granules (Fig. 1 and Ref. 15). In
this regard, both proteins continuously shuttle between the nucleus and
cytoplasm (1), suggesting that they may serve as carrier
proteins that help to ferry mRNA from the nucleus to the cytosolic
location where initiation occurs. When translation initiation is
inhibited by eIF2 phosphorylation, mRNA with the accompanying TIA-1
and TIAR proteins accumulates at the putative assembly sites, resulting
in formation of visible stress granules. It has also been suggested
that TIA-1 and TIAR may have a role in the transfer of mRNA from
polysomes to stress granules (1), although the mechanism
through which this might occur is unknown. Overall, the available
evidence suggests that translation is initiated at discrete points
within the cell as opposed to occurring randomly throughout the
cytoplasm. Such a finding is not surprising based on the multitude of
components (e.g., initiation factors, 40S ribosomes, and mRNA) that
must be assembled to form the 48S preinitiation complex. Having the required components present in a localized environment should allow
translation initiation to occur in a more efficient manner.
The mechanism(s) involved in the accumulation of preinitiation
complexes in cytoplasmic foci in response to cellular stress is
unknown. However, previous studies by others have shown that under
conditions that increase eIF2 phosphorylation, eIF2
(P) and eIF2B
accumulate on 48S preinitiation and 80S initiation complexes (19,
27). Furthermore, Matts and coworkers (19) have
suggested that eIF2 is released from the ribosome upon its interaction
with eIF2B. Thus phosphorylation of eIF2
may result in accumulation of initiation factors in inactive preinitiation complexes by inhibition of the step that occurs immediately before joining of the 60S ribosomal
subunit, release of initiation factors from the 48S preinitiation
complex. The resulting inactive preinitiation complexes would then
accumulate at putative assembly sites, resulting in stress granule
formation. In this regard, stress granules may be viewed as possible
storage sites for mRNAs and translation initiation factors that
allow the cell to rapidly reinitiate translation once it has recovered
from the stress. One might also speculate that mRNAs that are
preferentially translated during the unfolded protein response, such as
ATF4 mRNA, may be excluded from foci under such conditions. The
mechanism through which such mRNAs are preferentially expressed when
eIF2
is phosphorylated involves the multiple short open reading
frames (uORF) present in the 5'-untranslated region of the mRNA. When
eIF2
phosphorylation is low, the uORFs act as translational
repressors and limited ATF4 expression occurs (7). In
contrast, hyperphosphorylation of eIF2
permits the scanning
translation initiation complex to bypass the translationally repressive
uORF(s) and allows the initiation complex to access the AUG start codon
of the protein and initiate translation (reviewed in Ref.
22).
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ACKNOWLEDGEMENTS |
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We acknowledge the generosity Dr. Nancy L. Kedersha for helpful discussions.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-15658 (L. S. Jefferson), DK-13499(L. S. Jefferson), and ES-08681 (D. Ron).
Address for reprint requests and other correspondence: S. R. Kimball, Dept. of Cellular and Molecular Physiology, Pennsylvania State Univ. College of Medicine, P.O. Box 850, Hershey, PA 17033 (E-mail: skimball{at}psu.edu).
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.
First published October 3, 2002;10.1152/ajpcell.00314.2002
Received 8 July 2002; accepted in final form 17 September 2002.
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