(Received for publication, January 29, 1996, and in revised form, September 19, 1996)
From the Department of Biochemistry, University of
California, Riverside, California 92521-0129 and the
¶ Department of Chemistry and Biochemistry, University of Texas,
Austin, Texas 78712-1096
Several translation initiation factors in mammals
and yeast are regulated by phosphorylation. The phosphorylation state
of these factors is subject to alteration during development,
environmental stress (heat shock, starvation, or heme deprivation), or
viral infection. The phosphorylation state and the effect of changes in
phosphorylation of the translation initiation factors of higher plants
have not been previously investigated. We have determined the
isoelectric states for the wheat translation initiation factors eIF-4A,
eIF-4B, eIF-4F, eIF-iso4F, and eIF-2 and the poly(A)-binding protein in
the seed, during germination, and following heat shock of wheat
seedlings using two-dimensional gel electrophoresis and Western
analysis. We found that the developmentally induced changes in
isoelectric state observed during germination or the stress-induced changes were consistent with changes in phosphorylation. Treatment of
the phosphorylated forms of the factors with phosphatases confirmed that the nature of the modification was due to phosphorylation. The
isoelectric states of eIF-4B, eIF-4F (eIF-4E, p26), eIF-iso4F (eIF-iso4E, p28), and eIF-2 (p42) were altered during germination, suggesting that phosphorylation of these factors is developmentally regulated and correlates with the resumption of protein synthesis that
occurs during germination. The phosphorylation of eIF-2
(p38) or
poly(A)-binding protein did not change either during germination or
following a thermal stress. Only the phosphorylation state of two
factors, eIF-4A and eIF-4B, changed following a heat shock, suggesting
that plants may differ significantly from animals in the way in which
their translational machinery is modified in response to a thermal
stress.
Exposure to heat shock results in profound changes at almost every level of gene expression including transcription, splicing, nucleocytoplasmic transport, translation, and protein turnover (for reviews, see Refs. 1, 2, 3). Although many of the molecular events involved in heat shock gene induction are remarkably conserved in eukaryotes, the control of translation following heat shock varies considerably among species. In yeast, the heat shock response appears to involve only transcriptional mechanisms, whereas in Xenopus oocytes, the heat shock response is mediated entirely at the translational level (4, 5). As with mammals, the heat shock response in plants lies between these extremes, with gene regulation involving both transcriptional and translational mechanisms. In addition to a specific set of heat-induced genes, i.e. those encoding the heat shock proteins, a low level of non-heat shock mRNA translation continues in plants following heat shock (6, 7, 8, 9). We have shown that heat shock causes a reduction in translational efficiency and an increase in the mRNA half-life of non-heat shock mRNAs in plants that are proportional to the severity of the stress (10). Under these conditions, the translational machinery loses its ability to discriminate between capped and uncapped mRNAs. As a consequence, translation becomes less cap-dependent, and the functional co-dependence between the cap and the poly(A) tail is reduced (10, 11).
In animals, the reprogramming of translation following thermal stress
correlates with changes in phosphorylation for several initiation
factors (12). Two of the best studied examples are 1) the
dephosphorylation of the cap-binding protein subunit of eukaryotic
initiation factor (eIF)1 4F (also known as
eIF-4E) (13) and 2) the phosphorylation of eIF-2 (reviewed in Refs.
14, 15, 16, 17). eIF-4F binds to the cap structure at the 5
-terminus of the
mRNA and stimulates the binding of eIF-4A and eIF-4B. eIF-4A is an
RNA-dependent RNA helicase that, together with eIF-4B and
eIF-4F, unwinds any secondary structure present in the 5
-untranslated
leader, thereby preparing the mRNA for binding to the 40 S
ribosomal subunit. The smallest of the three subunits that constitute
mammalian eIF-4F is eIF-4E (p25). The site of phosphorylation in eIF-4E
is at serine 209, near the C terminus (18, 19). Dephosphorylation of
mammalian eIF-4E occurs following serum starvation (20), mitosis (21), viral infection (22), or heat shock (12) and correlates with reduced
eIF-4F binding to the cap and protein synthesis activity (23, 24, 25, 26).
eIF-2 is a three-subunit complex that is responsible for bringing
Met-tRNAiMet to the 40 S subunit in both
plants and animals (reviewed in Ref. 27). The -subunit is subject to
phosphorylation in animal cells by a number of physiological events,
including heat shock (13), viral infection, and heme deprivation
(reviewed in Ref. 28), or in yeast following amino acid starvation
(reviewed in Refs. 27, 28, 29). Phosphorylation of the
-subunit prevents
GDP/GTP exchange by eIF-2B and consequently inhibits eIF-2 activity
(30). Overexpression of an eIF-2
mutant that is resistant to
phosphorylation partially protected Chinese hamster ovary cells from
inhibition of protein synthesis following a heat shock (30), suggesting that the control of this initiation factor constitutes an important regulatory point following a heat shock in animal cells.
Additional eIFs that are modified following a stress include the dephosphorylation of mammalian eIF-4B during heat shock (12) and the phosphorylation of eIF-4A in hypoxic maize roots. Both of these events correlate with reduced translational activity (31). In addition, heat shock causes an increase in the synthesis of the poly(A)-binding protein in HeLa cells (32), a protein essential for efficient translation (33). Although heat shock profoundly impacts translation in plants, the modifications that initiation factors undergo following this stress have not been investigated. We demonstrate that plants differ from other eukaryotes in the way that several key components of the translational machinery are modified following thermal stress. In addition, we show that modification of several initiation factors occurs during seed germination, a developmental process unique to plants.
eIF-4F and eIF-iso4F (34), eIF-4B (35), and eIF-4A and eIF-2 (36) were purified from wheat germ (commercially prepared wheat embryos) as described. The recombinant p86 (eIF-iso4G) and p28 (eIF-iso4E) subunits of eIF-iso4F were purified as described (37). Polyclonal antibodies to the initiation factors were produced in rabbits or mice and purified as described (38, 39).
PAB was purified essentially as described by Yang and Hunt (40), except that KAc was substituted for KCl, and fractionation on a Mono Q column immediately followed Affi-Gel blue chromatography. Antibodies raised against purified PAB were purified as described previously (41).
Plant Extract Preparation and Two-dimensional Gel Electrophoresis/Western Blot AnalysisWheat seeds were germinated
aseptically for 4 days. Wheat seedling leaves were excised and placed
in 3 ml of MS medium (42) in a plastic 60-mm Petri dish. The leaves
were heat-shocked by placing the dish in a 45 °C shaking water bath
for 15 or 90 min. Control leaves were kept at 24 °C for 90 min.
Total soluble protein extracts were prepared by grinding the tissue in
a mortar first with liquid nitrogen, followed by 10% trichloroacetic
acid, 0.07% -mercaptoethanol at
20 °C. The samples were
pelleted by centrifugation at 12,000 × g for 15 min,
thoroughly washed in acetone with 0.07%
-mercaptoethanol, and
resuspended in 9.5 M urea with 0.5% dithiothreitol. Cell
debris was then pelleted by centrifugation, and the protein concentration was determined (43). 20-30 µg of protein was loaded on
IEF tube gels (3.6% acrylamide, 9 M urea, 2% ampholytes,
2% Nonidet P-40) and run at 400 V for 4.5 h, followed by 0.5 h at 500 V. The protein was then resolved in the second dimension using standard SDS-PAGE cooled to 4 °C during electrophoresis, and the protein was transferred to 0.22-µm nitrocellulose membrane by electroblotting. Equal protein loading was confirmed by staining replicate gels with Coomassie Brilliant Blue. Following transfer, the
nitrocellulose membranes were blocked overnight in 5% milk, 0.01%
thimerosal in TPBS (0.1% Tween 20, 13.7 mM NaCl, 0.27 mM KCl, 1 mM Na2HPO4,
0.14 mM KH2PO4), followed by
incubation with primary antibodies diluted typically from 1:500 to
1:2000 in TPBS with 1% milk for 1.5 h. The blots were then washed
twice with TPBS and incubated with goat anti-rabbit (used for those
antibodies raised against eIF-4A, eIF-4B, eIF-4F, eIF-iso4F, eIF-2
,
eIF-2
, or PAB) or anti-mouse (used for those antibodies raised
against recombinant p28, p86, or p26) horseradish peroxidase-conjugated antibodies (Southern Biotechnology Associates, Inc.) for 1 h. The
blots were washed twice with TPBS, and the signal was detected typically between 1 and 15 min using chemiluminescence (Amersham Corp).
In some cases, purified protein was resolved using two-dimensional gel
electrophoresis and stained with Coomassie Brilliant Blue. The range of
ampholytes used is indicated in the legend to each figure. The pH range
of the IEF tube following isoelectric focusing was determined from the
measurements of 5-mm sections of a control tube gel soaked in 1 ml of
15 mM NaCl. Phosphatase treatments were performed with the
phosphorylated form of each factor prior to analysis on two-dimensional
Western blots. Phosphorylation studies of eIF-4B were carried out using
30-50 units of casein kinase II for 1 h.
Wheat eIF-4A, a single polypeptide (46,932 Da) (44),
is an ATP-dependent RNA helicase that, in conjunction with
eIF-4B and eIF-4F, is thought to remove secondary structure present
within a 5-leader (45, 46). It is also an RNA-dependent
ATPase in plants, animals, and yeast (47, 48). Although there is no evidence that eIF-4A is phosphorylated in mammalian cells, a
phosphorylated form has been demonstrated in Drosophila (49)
and in maize (31). Phosphorylation of maize eIF-4A occurs in roots
following oxygen deprivation (31); consequently, phosphorylation of
eIF-4A is part of the hypoxic stress response. To examine whether heat
shock also results in the appearance of phosphorylated eIF-4A,
4-day-old wheat leaves were treated at 45 °C for 15 or 90 min.
Control tissue was maintained at 24 °C for 90 min. The tissue was
frozen in liquid nitrogen immediately following the treatment. Extracts
made from the tissue were displayed on two-dimensional gels,
transferred to nitrocellulose membrane, and probed with anti-wheat
eIF-4A antibodies. eIF-4A purified from wheat embryos was also analyzed using the same approach.
A single species (pI 5.6) was observed for eIF-4A purified from wheat
embryos (Fig. 1). In control wheat leaves, a second, more acidic form (pI 5.5) was present at ~10% the level of the basic
form. The acidic and basic isoforms correspond well with the
monophosphorylated (pI 5.75) and nonphosphorylated (pI 5.9) forms,
respectively, of eIF-4A observed in maize that had been shown
previously to be modified by phosphorylation (31). The nonphosphorylated and phosphorylated isoforms were present in an
approximate 9:1 ratio (nonphosphorylated to phosphorylated) in wheat
leaves subject to a 15-min treatment at 45 °C. When leaves were
treated for 90 min at 45 °C, however, the amount of phosphorylated eIF-4A increased such that both isoforms were present in equal amounts.
Therefore, as in hypoxically treated maize roots, heat shock can result
in the phosphorylation of wheat eIF-4A, suggesting that phosphorylation
of this initiation factor may be a characteristic response to stress in
plants.
We have shown previously that translational efficiency was severely reduced following only a 15-min heat shock at 45 °C (10). Our observation that a 15-min heat treatment at 45 °C was insufficient to induce phosphorylation of eIF-4A and that a far longer heat shock (90 min) was required suggests that phosphorylation of eIF-4A is not responsible for the immediate reduction in translation following a heat shock. Instead of being a part of the initial response to heat shock, phosphorylation of eIF-4A might be part of an adaptive response to prolonged stress.
eIF-4BMammalian eIF-4B is a single subunit of 69,843 kDa
(deduced from the amino acid sequence) present in 8-10 isoforms (12). The acidic cluster (four to six isoforms) is composed of phosphorylated isoforms, whereas the basic cluster (approximately four isoforms) results from an as yet undetermined modification (50). Although the
actual eIF-4B phosphorylation sites have not been identified, five
serine phosphorylation sites have been reported (51). The dephosphorylation of mammalian eIF-4B that is observed following heat
shock (51), serum depletion (52), or mitosis (21) correlates with a
reduction in translation, whereas the stimulation of eIF-4B phosphorylation by insulin treatment (53) correlates with an increase
in translation. The addition of phosphorylated eIF-4B partially
restored translation to an in vitro translation lysate prepared from heat-shocked HeLa cells (12, 51). eIF-4B, a 59-kDa
polypeptide in wheat (35), is present in wheat leaves in several
isoelectric states similar to those observed in mammalian cells (Fig.
2). By analogy with animal eIF-4B, the acidic cluster (pI 5.9-7.0) is consistent with a phosphorylated form of the basic cluster (pI 7.2-8.0). The acidic cluster is completely absent when
eIF-4B is purified from commercially prepared wheat embryos (wheat
germ), suggesting that either eIF-4B is developmentally regulated or
the modifications to eIF-4B are lost during purification. We have
observed that the acidic cluster is also missing when crude extracts
from freshly excised embryos are analyzed,2
supporting the possibility that the modification of eIF-4B is developmentally regulated. Upon even a brief exposure to heat shock (15 min at 45 °C), the acidic cluster of eIF-4B is lost in leaves (Fig.
2). Similar observations were made for leaves treated for 90 min at
45 °C (Fig. 2). The appearance of the acidic cluster of eIF-4B
correlates with the resumption of active translation during
germination, and its loss correlates with the repression of translation
following heat shock.
The acidic isoforms of eIF-4B are modified by phosphorylation (Fig.
3). eIF-4B from wheat leaf extract, in which the acidic cluster of eIF-4B isoforms is predominant (Fig. 3A), was
treated with alkaline phosphatase and resolved on a two-dimensional
gel, followed by Western analysis. Treatment with the phosphatase
resulted in the complete conversion of the acidic isoforms to the basic isoforms (Fig. 3B), suggesting that eIF-4B is multiply
phosphorylated. No dephosphorylation of eIF-4B was observed when potato
acid phosphatase was used (data not shown). eIF-4B purified from
embryos (i.e. hypophosphorylated) was treated with casein
kinase II to determine whether the acidic isoforms could be generated.
Treatment with a high level of the kinase resulted in complete
conversion (Fig. 3D) to the acidic isoforms, whereas a lower
level of the enzyme resulted in a partial conversion of the basic
cluster to the acidic isoforms (Fig. 3E). As
[32P]ATP was used in the phosphorylation of eIF-4B in
Fig. 3E, the membranes could also be exposed to film to
detect which eIF-4B isoforms were radiolabeled (Fig. 3F).
The basic isoforms observed in Fig. 3E were not
radiolabeled, as expected for isoforms that are not phosphoproteins.
The intermediate isoforms resulting from the phosphorylation of the
basic eIF-4B isoforms were observed as radiolabeled phosphoproteins in
Fig. 3F. In addition, a small amount of the most acidic
isoforms was detected as radiolabeled phosphoproteins in Fig.
3F that had not been detected by Western analysis in Fig.
3E.
eIF-4F and eIF-iso4F
Mammalian eIF-4E (the 25-kDa cap-binding
protein subunit of eIF-4F) is normally phosphorylated in animal cells
and is required for activity (reviewed in Refs. 14, 15, 16, 17).
Dephosphorylation of mammalian eIF-4E occurs following serum starvation
(20), mitosis (21), and viral infection (22). As dephosphorylation of
eIF-4E correlates with a reduced rate of translation, the small subunit
of eIF-4E is thought to function as a key control point in the
regulation of translation in animal cells. Heat shock also causes
dephosphorylation of eIF-4E (12, 23, 24, 51), resulting in a reduced
interaction between eIF-4F and the 5-cap structure (25, 54) and
reduced eIF-4F complex formation (26). Reduced eIF-4F activity would
mean that the 5
-cap would offer less of a translational advantage to
an mRNA, and consequently, translation would become less
cap-dependent. This prediction was borne out in
serum-starved 3T3-L1 cells, in which eIF-4E underwent dephosphorylation (20) and translation became less cap-dependent (55).
In plants, heat shock causes a loss in cap-dependent translation that is proportional to the severity of the stress such that a severe heat shock can result in the complete loss of cap function (10). Such observations suggest that the activity of one or more of the cap-associated initiation factors, e.g. eIF-4B or eIF-4F, may be altered following thermal stress. The translational machinery of plants differs significantly from that of animals and yeast in that plants contain not only eIF-4F, but also an isoform, designated eIF-iso4F (34, 38), which are encoded by separate genes (56, 57). Like mammalian eIF-4F, wheat eIF-4F and eIF-iso4F can be purified as a three-subunit (containing eIF-4A) or two-subunit (no eIF-4A) complex depending on the purification scheme employed (31, 38). eIF-4E (p26) and eIF-iso4E (p28) share homology and are functionally analogous (38, 56). The same is true for the large subunits of eIF-4F (i.e. eIF-4G) and eIF-iso4F (i.e. eIF-iso4G), which are also referred to as p220 and p86, respectively. As in mammalian cells, eIF-4F and eIF-iso4F are the least abundant of the initiation factors (39). Whether they play a key role in the regulation of translation in plants remains unknown.
The large and small subunits of eIF-4F and eIF-iso4F can be seen when
these factors purified from wheat embryos are displayed on
two-dimensional gels and stained with Coomassie Brilliant Blue (Fig.
4A). p220 and p86 are large basic proteins
that do not resolve well on a two-dimensional gel. p220 has a pI range
of ~6.5-7.0 (Fig. 4A, left panel), whereas p86
has a pI range of 7.1-7.7 (right panel). eIF-4E (p26) from
eIF-4F in embryos is present as two pairs (Fig. 4A,
left panel), whereas two isoforms of eIF-iso4E (p28) from
eIF-iso4F are observed (right panel). Note that the basic
p26 and p28 isoforms are very faint. When antibodies raised against
recombinant p26 and p28 were used to probe eIF-4E and eIF-iso4E from
embryos, the two pairs of p26 isoforms (pI values of 6.15/6.25 and
6.75/6.85) and the two p28 isoforms (pI values of 6.1 and 6.6) could be
more clearly seen (Fig. 4B). The basic pair of p26 and the
basic isoform of p28 were observed in only some fractions following the
final purification of eIF-4F or eIF-iso4F on
m7GTP-Sepharose, suggesting a differential affinity for
m7GTP or a reduced abundance for the basic forms of these
subunits. In contrast to the observations in embryos, only the acidic
pair of p26 isoforms and the acidic isoform of p28 were observed when soluble protein extracts from leaves were probed with the anti-p26 and
anti-p28 antibodies (Fig. 4C). The presence of the basic
isoforms of p26 and p28 in embryos and their absence in leaves suggests that eIF-4E and eIF-iso4E may undergo modification that is
developmentally regulated.
An additional eIF-4E isoform could be detected in young shoots and
roots as a minor isoform (Fig. 5, left
panels) that exhibited a more acidic pI than the two more
predominant isoforms observed in Fig. 4. This isoform was detected only
in shoots and roots of 3-day-old seedlings, which are composed of
rapidly dividing cells in which translation is highly active. It was
not observed in later stages of leaf development (Fig. 4C,
left panel) even upon long exposure of the chemiluminescent
signal (data not shown). The basic p26 isoforms observed in wheat
embryos (Fig. 4B, left panel) were not detected
in the 3-day-old shoots and roots, suggesting that these basic isoforms
disappear early during germination. As with eIF-4E, an additional
acidic eIF-iso4E (p28) isoform was also observed in young shoots and
roots (Fig. 5, right panels). Like the most acidic isoform
of eIF-4E, this additional eIF-iso4E isoform was not detected in older
leaves (Fig. 4C, right panel), and the basic
eIF-iso4E isoform observed in embryos (Fig. 4B, right
panel) was not detected in young shoots and roots (Fig. 5,
right panels).
To examine further whether eIF-4E and eIF-iso4E are subject to
modification in wheat, recombinant p26 and p28 were overexpressed in
Escherichia coli and purified (37), and their isoelectric points were compared with those of p26 and p28 isolated from wheat (Fig. 6). Recombinant p26 was composed of a major
species and a minor, more acidic species (Fig. 6A,
left panel). The nature of the difference between these two
recombinant isoforms is unknown. An additional minor species of
slightly smaller size then the acidic species was also observed and may
represent a degradation product of recombinant p26. Recombinant p26
(Fig. 6A, left panel) exhibited a more basic
isoelectric point than did wheat-purified p26 (Fig. 6B,
left panel). This is most clearly seen when the recombinant
p26 and wheat-purified p26 proteins were analyzed together (Fig.
6C, left panel). Similar results were observed for eIF-iso4E (p28) (Fig. 6, middle panels). A single
species was observed for recombinant p28 with a smaller species of
identical pI that may be a degradation product (Fig. 6A,
middle panel). Recombinant p28 was more basic than the
acidic isoform of wheat-purified p28 (Fig. 6B, middle
panel). By resolving both recombinant p28 and wheat-purified p28
on the same two-dimensional gel, the difference in their isoelectric
points can be clearly seen (Fig. 6C, middle panel).
The cDNA for eIF-iso4G (the p86 subunit of eIF-iso4F) has also been
isolated (56), and recombinant p86 can be overexpressed and purified
from E. coli (37). Recombinant p86 ran as a single, highly
basic isoform (Fig. 6A, right panel) compared
with the smear of wheat-purified p86 isoforms (Fig. 6B,
right panel). These observations suggest that eIF-4E,
eIF-iso4E, and eIF-iso4G are modified in wheat. However, no alteration
in the isoelectric point of the acidic isoforms of wheat-purified p26
or p28 was observed following treatment with potato acid phosphatase,
alkaline phosphatase, -protein phosphatase, or phosphatase 2A.
Moreover, the isoelectric point of recombinant p26 or p28 was not
altered following treatment with casein kinase II (data not shown).
Although in vivo labeling of phosphoproteins with
32P also failed to establish that the acidic p26 or p28
isoforms are phosphoproteins (data not shown), wheat leaves take up
32P poorly, and consequently, the possibility that eIF-4E
and eIF-iso4E are modified by phosphorylation in wheat cannot be
excluded.
When eIF-iso4F was examined in control and heat-shocked leaves using
antibodies raised against purified eIF-iso4F, no substantial change was
observed in either eIF-iso4G (p86) or eIF-iso4E (p28) (Fig.
7, left panels). Only the acidic p28 isoform
was present in both control and heat-shocked leaves, suggesting that a
short or long heat shock does not result in the generation of
detectable levels of the basic isoform. As eIF-iso4G (p86) is a basic
protein and consequently does not resolve well, it is difficult to
conclude whether heat shock has an effect on this factor. Antibodies
raised against recombinant p28 confirmed that only the acidic isoform of eIF-iso4E (pI 6.1) was present in leaves and that the heat shock
treatment employed had no detectable effect (Fig. 7, middle panels). These same membranes were then probed with anti-p26
antibodies without stripping the anti-p28 antibodies from the membrane.
With this approach, residual p28 can be detected along with p26, and their relative pI values directly compared (Fig. 7, right
panels). Only the acidic pair of p26 isoforms (pI 6.15/6.25) was
present in leaves, and as with p28, heat shock had no impact on their isoelectric point. Note that the most acidic isoform of p26 partially overlaps with p28, making it somewhat difficult to distinguish p28 from
the most acidic p26 isoform (Fig. 7, right panels). These data suggest that, unlike mammalian eIF-4E, wheat eIF-4E and eIF-iso4E do not undergo modification following heat shock.
eIF-2
eIF-2 is a three-subunit complex that is responsible
for initiator Met-tRNA binding to the 40 S subunit. Phosphorylation of the -subunit prevents GDP/GTP exchange in eIF-2
in animal cells and yeast, resulting in its inhibition (reviewed in Refs. 15 and 58).
Phosphorylation of eIF-2
occurs following amino acid starvation in
yeast (reviewed in Refs. 27, 28, 29) or viral infection (reviewed in Ref.
28), heme deprivation, and heat shock (13) in animal cells and
therefore represents another key regulatory component of the
translational machinery.
Like its animal counterpart, wheat eIF-2 is also a three-subunit
complex composed of an -subunit (42 kDa), a
-subunit (38 kDa),
and a
-subunit (50 kDa) (59, 60, 61). Although the
-subunit of
mammalian eIF-2 is smaller than the
-subunit, the recent isolation
of the subunit cDNAs for wheat eIF-2 has clearly established the
identity of the 42-kDa polypeptide as the
-subunit and the 38-kDa
polypeptide as the
-subunit.3
eIF-2 purified from wheat embryos was displayed using two-dimensional
gel electrophoresis and stained with Coomassie Brilliant Blue (Fig.
8). Multiple (five to six) isoforms for the -subunit were observed over a pI range of 6.1-6.6 (Figs. 8 and
9B, right panel). An acidic
doublet representing the
-subunit was observed at a pI of 5.4-5.6,
and the
-subunit is a highly basic protein (Fig. 8). Western
analysis of eIF-2 from embryos using affinity-purified anti-
- or
anti-
-subunit antibodies confirmed the assignment of these two
subunits (Fig. 9). To determine the nature of the modification of the
-subunit, eIF-2 from embryos was treated with phosphatase 2A and
resolved using two-dimensional gel electrophoresis for subsequent
Western analysis. Treatment with the phosphatase substantially
dephosphorylated the eIF-2
subunit (Fig. 9, compare left
panels in A and B), resulting in a shift in
pI for this subunit that corresponded to that observed for the
-subunit present in leaves (see the following paragraph). No other
phosphatase tested (potato acid phosphatase, alkaline phosphatase, or
-protein phosphatase) resulted in the dephosphorylation of the
-subunit (data not shown). The
-subunit also underwent some
extent of dephosphorylation following treatment with phosphatase 2A
(Fig. 9, compare right panels in A and
B).
Membranes containing protein extracted from leaves that had been
resolved using two-dimensional gel electrophoresis were also probed
individually with affinity-purified anti-- and anti-
-subunit antibodies (Fig. 10, left and middle
panels). Only the most abundant
-subunit isoforms were
observed, although a long exposure of membranes (from a separate
experiment) that were probed with both anti-
- and anti-
-subunit
antibodies revealed the less abundant
-subunit isoforms (Fig. 10,
right panels). The
-subunit was observed when the
membranes were probed with anti-
-subunit antibodies (Fig. 10,
left panels), and as the anti-
-subunit antibodies contain a small amount of anti-
-subunit antibodies, a weak signal
representing the most abundant
-subunit isoform can also be seen,
which serves as a point of reference. The
-subunit can also be seen
in the panels representing a long exposure of membranes probed with
both anti-
- and anti-
-subunit antibodies (Fig. 10, right
panels). Comparison of eIF-2 from leaves and embryos revealed that
the
-subunit exists in a phosphorylated state in embryos and is
converted to a hypophosphorylated or dephosphorylated state in leaves
(compare the purified factor from embryos with the factor present in
24 °C-treated leaves; Fig. 10, left panels). There
appears to be at least two basic isoforms of eIF-2
in leaves. A heat
shock, whether short or long in duration, had little detectable impact
on the
-subunit (Fig. 10, left panels). If
phosphorylation of wheat eIF-2
results in its inactivation, as it
does in mammalian cells and yeast, then wheat eIF-2
may be
maintained in an inactive (phosphorylated) state in the seed and
shifted to an active (dephosphorylated) state upon germination. The
dephosphorylation of eIF-2
in leaves does correlate with the
activation of translation that occurs during germination. Following a
heat shock, however, the lack of phosphorylation of the
-subunit and
only minor changes in the
-subunit suggest that the response to heat
stress by the translational apparatus may differ substantially in
plants compared with yeast and animal cells.
Poly(A)-binding Protein
The cap and the poly(A) tail function
in a cooperative manner to establish an efficient level of translation
(11). This means that neither element functions well in the absence of
the other and suggests communication between these two regulatory elements. Heat shock causes not only a loss in cap function, but a loss
also in the co-dependence between the cap and the poly(A) tail (10),
which could be a consequence of a heat shock-induced inactivation of
PAB or a reduction in the communication between this protein and the
5-terminus. PAB can therefore be considered as a participant in the
translation initiation process.
PAB is present in multiple isoforms in yeast and sea urchin (62),
although the nature of the modification has not been determined. PAB
was purified from wheat embryos, and following its resolution using
two-dimensional gel electrophoresis and transfer to a membrane, it was
probed with anti-wheat PAB antibodies. As in yeast and sea urchin,
multiple isoforms were observed for wheat PAB that exhibited a pI range
of 6.9-7.5 (Fig. 11), although upon longer exposure,
additional isoforms with a pI range of 6.1-7.5 can be seen (data not
shown). No change in the distribution of the PAB isoforms was observed
in leaves or following a heat shock (Fig. 11), suggesting that the PAB
isoforms are not regulated developmentally or following heat shock.
This suggests also that the loss in co-dependence between the cap and
the poly(A) tail following a heat shock may be a consequence of the
observed heat-induced modifications of the initiation factors.
To determine the nature of the post-translational modification of PAB,
the most acidic isoforms (pI 6.1-6.5) of PAB were isolated following
the separation of purified PAB on a preparative isoelectric focusing
apparatus (Fig. 12A). Treatment of the
acidic PAB isoforms with alkaline phosphatase resulted in the
dephosphorylation of the acidic isoforms and shifted their pI to that
of the most basic isoforms (pI 7.0-7.5) (Fig. 12B). These
data suggest that PAB exists as a multiply phosphorylated protein in
wheat and is the first elucidation of a modification of PAB from any
species.
Conclusions
The changes in phosphorylation of both
transcription and translation factors following thermal stress provide
the basis for a rapid response in reprogramming gene expression (2,
63). We have determined the number of isoforms and the isoelectric states of several of the translation initiation factors in wheat and
established that several of these are modified by phosphorylation. We
have also examined whether their phosphorylation state changes during
development or following a heat shock. eIF-4A, eIF-4B, the - and
-subunits of eIF-2, and PAB can exist as phosphoproteins in
vivo depending on the developmental stage and environmental conditions. Moreover, eIF-4E (p26), eIF-iso4E (p28), and eIF-iso4G (p86) are post-translationally modified in wheat in a way that is
consistent with their phosphorylation. Although the translational machinery of plants is functionally similar to that of yeast and animals, several differences were observed that suggest that the stress
response at the translational level may differ significantly in plants.
These differences may underlie the unique challenge that environmental
stress presents to plants compared with other eukaryotes. As plants are
sessile, they cannot avoid environmental changes and use a variety of
methods, including changes in physiology and morphology, to minimize
the deleterious effects of abiotic stress. Whereas eIF-4E, eIF-4B, and
eIF-2
are modified in mammals following a heat shock, in wheat, only
eIF-4A and eIF-4B are subject to heat-induced modifications. Although
phosphorylation of eIF-4A following a prolonged stress may not account
for the rapid changes in translation observed in plants following a
short heat shock, eIF-4B does undergo rapid dephosphorylation following
thermal stress as well as during germination. Heat shock had little
impact on wheat eIF-4E, eIF-iso4E, or eIF-2
. Thus, in their response to thermal stress, plants may differ from animals in two key points: in
the regulation of eIF-4E/eIF-iso4E and of eIF-2
. It should be noted,
however, that the developmental regulation of the isoelectric state of
eIF-4E, eIF-iso4E, and eIF-2
during germination suggests that the
activity of these initiation factors may indeed be regulated developmentally in plants, but that the translational response of
plants to a heat shock may be limited to other components of the
translational machinery, such as eIF-4B and eIF-4A.
We thank Jolinda Traugh for the gift of casein kinase II.