From the Department of Plant Sciences, Tel
Aviv University, Tel Aviv 69978, Israel and § Department
of Botany, the University of Tennessee,
Knoxville, Tennessee 37996-1100
Received for publication, July 27, 2000, and in revised form, September 25, 2000
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ABSTRACT |
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The Arabidopsis COP9 signalosome is a
multisubunit repressor of photomorphogenesis that is conserved among
eukaryotes. This complex may have a general role in development. As a
step in dissecting the biochemical mode of action of the COP9
signalosome, we determined the sequence of proteins that copurify with
this complex. Here we describe the association between components of
the COP9 signalosome (CSN1, CSN7, and CSN8) and two subunits of
eukaryotic translation initiation factor 3 (eIF3), eIF3e (p48, known
also as INT-6) and eIF3c (p105). To obtain a biochemical marker for
Arabidopsis eIF3, we cloned the Arabidopsis
ortholog of the eIF3 subunit eIF3b (PRT1). eIF3e coimmunoprecipitated
with CSN7, and eIF3c coimmunoprecipitated with eIF3e, eIF3b, CSN8, and
CSN1. eIF3e directly interacted with CSN7 and eIF3c. However, eIF3e and
eIF3b cofractionated by gel filtration chromatography in a complex that
was larger than the COP9 signalosome. Whereas eIF3, as detected through
eIF3b, localized solely to the cytoplasm, eIF3e, like CSN7, was also
found in the nucleus. This suggests that eIF3e and eIF3c are probably
components of multiple complexes and that eIF3e and eIF3c associate
with subunits of the COP9 signalosome, even though they are not
components of the COP9 signalosome core complex. This interaction may
allow for translational control by the COP9 signalosome.
Light is the major environmental signal regulating plant
development. Its effect on plant development is dramatically seen in
seedlings of dicotyledonous plants such as Arabidopsis.
Dark-grown seedlings, which undergo skotomorphogenesis, differ vastly
from light-grown seedlings, which go through photomorphogenesis, both at the morphological and molecular levels. Illumination of dark-grown seedlings induces photomorphogenesis. The transition from
skotomorphogenic growth to photomorphogenic growth involves changes in
transcription, translation (1), and protein modifications such as
ubiquitination and protein degradation (2).
Multisubunit protein complexes play central roles in the regulation of
all three processes and thus in the light-controlled regulation of
plant development. The COP9 signalosome (previously referred to as the
COP9 complex (30)) is a recently discovered protein complex of at least
eight subunits (renamed CSN1-CSN8 (4)) with a key role in regulating
development in plants and animals (reviewed in Ref. 5). The COP9
signalosome has been found primarily in the nucleus and has been
implicated in regulating protein kinase pathways (6-8).
Four Arabidopsis mutants that lack the COP9 signalosome
(cop9, fus6, fus5, and
cop8) exhibit a constitutive
photomorphogenic (cop) phenotype, both at the
morphological and molecular levels, in the absence of light. Therefore,
the COP9 signalosome acts as negative regulator of photomorphogenic
development in the dark. However, as in other eukaryotes, the
biochemical activity of the COP9 signalosome remains to be elucidated.
The pleiotropic nature of the mutants implies that the COP9 signalosome
acts at the nexus between multiple photoreceptors and a variety of
downstream regulatory events controlling specific aspects of cellular
differentiation, presumably via transcription, translation, and/or
post-translational modification. However, given that all mutations
known to disrupt the COP9 signalosome are seedling-lethal and begin to
show defects during late embryogenesis, i.e. before the
decision between the photomorphogenic and skotomorphogenic pathways is
made, it is likely that the COP9 signalosome is involved in more than
just the dark repression of photomorphogenesis (5, 9).
The COP9 signalosome is similar to two other multisubunit protein
complexes, eukaryotic translation initiation factor 3 (eIF3)1 and the regulatory
lid of the 19 S component of the proteasome (9, 10). Multiple subunits
of all three complexes share a common motif, termed the proteasome-COP9
signalosome-initiation factor 3 (11) or proteasome-Int6-Nip1-Trip15
(12) domain. The conservation between the COP9 signalosome and
proteasome lid is especially striking, where all eight subunits of each
complex have a corresponding similar protein (~20% amino acid
identity) in the other complex. Therefore, the three complexes may
share an overall architecture and a common evolutionary ancestor.
The role of eIF3 includes stabilizing the ternary complex between eIF2,
GTP, and tRNAiMet and promoting mRNA
binding to the 40 S ribosomal subunit (13, 14). Various reports have
placed the size of eIF3 between 550 and 700 kDa (17-23). Its subunit
composition has been a matter of debate, with the exact composition
often being dependent on the method of purification. Mammalian eIF3 now
appears to contain 11 subunits as follows: p170, p116 (PRT1), p110,
p66, p48 (INT-6), p47 (mov34), p44, p40, p36, p35, and p25, whose
SDS-PAGE patterns fit reasonably well with the wheat eIF3 pattern
(15-20). Recently, it has been reported that plant eIF3 closely
resembles the subunit composition of mammalian eIF3 having 10 out of 11 subunits in common and also contains a novel subunit not present in
either mammals or Saccharomyces cerevisiae (21). The
S. cerevisiae eIF3 core complex is smaller and consists of
only five subunits that are homologous to human, p170, p116, p110, p44,
and p36 (22), although other purifications have identified additional
proteins, which may bind the core complex with lower affinities
(23-25). A unified eIF3 nomenclature has recently been proposed, with
the largest subunit as eIF3a and the smallest eIF3k (21).
Interestingly, two of the eIF3 subunits have been independently
identified. The S. cerevisiae subunit corresponding to human
eIF3c (p110) was originally isolated in a screen for mutants with
defects in nuclear targeting (26), whereas the mammalian eIF3e (p48)
subunit, also known as INT-6, was found in nuclear bodies and has been
implicated in carcinogenesis (27).
Recent findings suggested interactions between the COP9 signalosome,
the regulatory 19 S proteasome subcomplex, and eIF3. The CSN1 subunit
of the Arabidopsis COP9 signalosome interacts with the AtS9
subunit of the proteasome lid in a yeast two-hybrid assay (28).
Analysis of proteins that copurify with the COP9 signalosome from
cauliflower identified another component of the proteasome, p43/RPN7,
and two putative subunits of eIF3, eIF3c (p105) and eIF3e (p48). We
have previously shown that Arabidopsis eIF3c is highly
conserved with the human eIF3c subunit and that eIF3c associates in
yeast with the COP9 signalosome components CSN1 and CSN8, although
probably not as a core component of the COP9 signalosome (29).
To clarify the relationship between the COP9 signalosome and eIF3, we
have now analyzed the eIF3e protein that copurifies with the COP9
signalosome. We show that eIF3e is likely the
Arabidopsis ortholog of the human eIF3e. Our data indicate
that eIF3e and eIF3c are subunits of Arabidopsis eIF3 that
also interact with components of the Arabidopsis COP9 signalosome.
Plant Materials and Growth Conditions--
Wild type plants are
in the Arabidopsis thaliana Columbia background. The
cop9-1 mutant is in the Wassilewskija background (30). Plant
germination and growth conditions in darkness and white light were as
described previously (31). Day/night cycle conditions consisted of
16 h of white light at 75 µmol m Isolation and Cloning of Arabidopsis eIF3e and
eIF3b--
Internal peptide sequences of the cauliflower COP9
signalosome-associated protein eIF3e were described previously (29). Based on amino acid sequence data from four peptides, a cDNA (EST number 142E10T7) for eIF3e was identified through the
Arabidopsis genome sequencing effort, sequenced on both
strands, and deposited under GenBankTM accession
number AF255679.
A search of the Arabidopsis data base with the human eIF3b
identified an expressed sequence tag (EST clone 195A21T7) of 578 base
pairs. This EST was used as a probe in a screen of a Antibody Production and Affinity Purification--
The complete
coding sequence of eIF3e was cloned into the pET29b vector (Novagen,
Madison, WI) and a pGEX-3C vector (Amersham Pharmacia Biotech). Because
of poor solubility of the His6-tagged eIF3e, the protein
was solubilized with SDS sample buffer and run on an SDS-PAGE gel. The
fusion protein was cut from the gel and used to immunize rabbits
(AniLab, Rehovot, Israel). For affinity purification of the antibodies,
the glutathione S-transferase (GST)-eIF3e fusion was
immobilized on an N-hydroxysuccinimide Hi-Trap column
(Amersham Pharmacia Biotech). Antibodies bound to the GST-eIF3e protein
were eluted with low pH buffer (2 M glycine, pH 2.5). The
resulting affinity purified anti-eIF3e antibodies were neutralized by
addition of 1 M Tris-HCl, pH 8.8.
The sequence encoding amino acids 58-538 of eIF3b was cloned into
pGEX-4T1 and pET28a. GST-eIF3b fusion protein was over-produced in
Escherichia coli strain BL21 and was used to immunize
rabbits (AniLab, Rehovot, Israel). The His6-eIF3b was
purified on a nickel column, and eIF3b antibodies were
affinity-purified over HIS6-eIF3b coupled to an
N-hydroxysuccinimide Hi-Trap column (Amersham Pharmacia Biotech) as described above.
Protein Extraction and Immunoblot
Analysis--
Arabidopsis or cauliflower, as indicated, was
ground in liquid nitrogen, and the powder was suspended in a phosphate
buffer, pH 7.0, containing 10% glycerol, 10 mM NaCl, 10 mM MgCl2, and 5 mM EDTA with
freshly added proteinase inhibitors including 0.5 mM
phenylmethylsulfonyl fluoride and Complete Protease Inhibitor Mixture
Tablets (Roche Molecular Biochemicals). Protein concentrations were
determined by the BCA protein assay (Pierce). Proteins separated by
SDS-PAGE were transferred to Immobilon-P membranes (Millipore, Bedford,
MA) and probed with 1:5000 dilution of affinity-purified rabbit
polyclonal antibodies. Bound antibody was detected with alkaline
phosphatase-coupled secondary antibodies, nitro blue tetrazolium, and
5-bromo-4-chloro-3-indolyl phosphate as substrates.
Yeast Two-hybrid Assay--
The complete coding region of eIF3e
was cloned into the EcoRI-NotI site of pJG4-5
with partial EcoRI digestion to make an in-frame fusion with
the transcription activation domain. The complete coding sequence of
eIF3b was cloned into the EcoRI site of pEG202 to make an
in-frame fusion with LexA. CSN1, CSN7, CSN8, and eIF3c yeast constructs
have been described previously (8, 29). The plasmids were transformed
into yeast EGY48. Selection for interaction was as described (33).
Gel Filtration Chromatography--
Total homogenates were
prepared as described above. The homogenate was spun in a
microcentrifuge for 30 min at 22,000 × g at 4 °C,
and the supernatant was filtered through a 0.45-µm filter (Sartorius
AG, Goettingen, Germany). Approximately 100 µg of total soluble
protein was fractionated at 4 °C through a Superose 6 HR column
(Amersham Pharmacia Biotech), with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4,
1.4 mM KH2PO4) containing 5 mM MgCl2 and 10% glycerol, at a flow rate of
0.3 ml/min. Fractions of 0.5 ml each were collected and concentrated by
Strata Clean Resin Beads (Stratagene, La Jolla, CA). The protein
standards for the gel fractionation were as follows: thyroglobulin (669 kDa), apoferritin (443 kDa), catalase (232 kDa), aldolase (158 kDa),
and bovine serum albumin (66 kDa).
Immunoprecipitation and Pull-down Assay--
Total plant extract
was cleared of aggregates by centrifugation for 30 min at 4 °C at
22,000 × g. 100 µg of total protein was incubated
with antibodies in PBS buffer and 1% Tween for 4 h at 4 °C.
Following incubation on an Orbiton rotator (Boekel Industries Inc.),
the samples were centrifuged for 15 min at 22,000 × g,
and the supernatant was transferred to new tubes containing 20 µl of
protein A-agarose beads (Sigma). The tubes were incubated for an
additional hour at 4 °C. The tubes were spun at 22,000 × g for 1 min, and protein A beads were washed 5 times with 1 ml of RIPA buffer (150 mM NaCl, 50 mM Tris-HCl,
pH 8.0, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS). Proteins
were eluted by boiling for 5 min in SDS sample buffer, separated by
SDS-PAGE, and blotted for antibody detection.
For the pull-down assay with CSN7, constant amounts (140 µg) of total
soluble proteins from E. coli expressing GST-eIF3e were incubated for 1 h at room temperature in PBS buffer containing 1%
Triton X-100, with increasing amounts of total soluble proteins from
E. coli expressing either His6-CSN7 or control
empty pET28a vector (4 µg/µl each). Following incubation on a
rotator, the tubes were centrifuged for 15 min at 22,000 × g, and the supernatant was transferred to new tubes
containing 20 µl of Ni-NTA-agarose (Qiagen, Germany). The tubes were
incubated for an additional hour. The tubes were spun at 22,000 × g for 1 min. As the Ni-NTA agarose absorbed many nonspecific
proteins, the Ni-NTA beads were washed 4 times with PBS containing 1%
Triton X-100 and once with buffer containing 8 M urea, 0.1 M sodium phosphate, and 0.01 M Tris-HCl, pH
8.0. For pull-down assay with eIF3c, constant amounts of total soluble
protein (16 µg) from E. coli expressing
His6-eIF3c were incubated for 1 h at room temperature
in PBS buffer containing 1% Triton X-100 and 5 µg of bovine serum
albumin, with increasing amounts of total soluble proteins from
E. coli expressing either GST-eIF3e or control empty pEGX-3C
vector (1 µg/µl each). Following incubation and centrifugation as
above, the supernatant was transferred to new tubes containing 20 µl
of 50% glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) for
additional hour. The tubes were spun at 22,000 × g for
1 min and washed 5 times with 1% Triton X-100 in PBS. Proteins were
eluted from both Ni-NTA and glutathione beads by boiling for 5 min in
SDS sample buffer, run on an SDS-PAGE gel, and blotted for antibody detection.
Immunofluorescence and Transient Expression of GFP Fusion
Proteins--
The procedure for protoplast isolation and
immunofluorescence assay was similar to a previously published
procedure (34). Root or leaf protoplasts were isolated from 8-day-old
wild type or
For the transient assay, GFP fusion proteins were expressed from the
GFP cloning vector pAVA319 (35) in onion epidermal cells by using
particle bombardment. The relative nuclear and cytoplasmic localization
was determined by quantitative fluorescence measurement (35). The
location of the nucleus was determined under bright-field illumination.
At least 15 cells were analyzed for each GFP fusion protein.
p48 Is Similar to the Mammalian eIF3e--
To elucidate the
composition of the COP9 signalosome, the complex was originally
purified from cauliflower, and the peptides from individual proteins
were microsequenced (29, 36). Based on amino acid sequence data from
four cauliflower peptides, an Arabidopsis cDNA for p48
was identified. This cDNA has an open reading frame of 1326 base
pairs that encodes 441 amino acids with a predicted mass of 51,808 Da
and a pI of 5.92. The Arabidopsis protein is highly similar
to cauliflower, with 68 of the 75 amino acids from the cauliflower
peptides being identical to the Arabidopsis protein. The
cDNA encodes a protein highly similar to the eIF3e subunit
of mammalian eIF3 and its likely homologs (Table
I). We therefore refer to this protein as
Arabidopsis eIF3e. eIF3e is found on chromosome 3, BAC
F2809. No protein similar to eIF3e was found in S. cerevisiae. Like mammalian eIF3e and other subunits of eIF3 and
the COP9 signalosome, eIF3e contains a C-terminal PCI domain.
Sequence data base searches indicate that eIF3e is most likely a single
gene in Arabidopsis.
Although the human eIF3e is postulated to be a subunit of eIF3 (16), it
is also found in the nucleus (27), has recently been shown to interact
with the interferon signaling pathway (37), and is similar to the S2
subunit of the mammalian COP9 signalosome (9). To analyze further
eIF3e, and its possible interaction with either the COP9 signalosome or
eIF3, we needed a control protein that is thought to be solely a
component of eIF3. eIF3b is a known subunit of eIF3 in yeast and humans
(19, 24, 25). eIF3b is found exclusively in a high molecular weight
complex (22), and in yeast mutants, eIF3b was unstable outside of the eIF3 complex (38). In addition, eIF3b did not copurify with the COP9
signalosome (29, 36). We identified a cDNA encoding a putative
homolog of eIF3b in Arabidopsis. The cDNA encodes a protein with a predicted molecular mass of 82,125 Da that is
highly similar over its entire length to the eIF3b subunit of mammalian and yeast eIF3 and its likely homologs (Table
II). As for other eIF3b proteins, the
Arabidopsis protein contains a domain common to a number of
RNA-binding proteins between amino acids 75 and 132.
Antibodies were generated against eIF3e and eIF3b, and
affinity-purified as described under "Experimental Procedures."
eIF3e and eIF3c Interact with COP9 Signalosome Subunits--
The
similarity of Arabidopsis eIF3e, eIF3c, and eIF3b with their
animal counterparts, which are components of eIF3, suggests that these
three proteins also interact in Arabidopsis in the same
complex. If the three proteins are components of the same complex, we
reasoned that antibodies against one should immunoprecipitate the
others. On the other hand, if one or more of the proteins interact with
the COP9 signalosome or its subunits, then we expect that these
antibodies will precipitate subunits of the COP9 signalosome. To test
this hypothesis, total protein extracts from Arabidopsis were incubated with antibodies against eIF3e, eIF3b, CSN1, or CSN8, and
the immunoprecipitate was analyzed by protein blot with antibodies
against eIF3c or CSN7. As seen in Fig.
1A, eIF3c coimmunoprecipitated with eIF3e and eIF3b, indicating that these three proteins are components of the same protein complex, presumably eIF3. eIF3c also
coimmunoprecipitated with CSN8 and CSN1 (Fig. 1A),
confirming our earlier yeast two-hybrid analysis. Since CSN8 and CSN1
proteins are found solely as components of the COP9 signalosome and are unstable as monomers (30), these results suggest that the interaction is between eIF3c and these proteins in the COP9 signalosome. eIF3e coimmunoprecipitated a core component of the COP9 signalosome, CSN7, in
extracts from both wild type and cop9 mutants, whereas antibodies against eIF3b did not coimmunoprecipitate CSN7 (Fig. 1B). These results suggest that eIF3e and eIF3c interact
with both complexes, eIF3 and the COP9 signalosome, whereas eIF3b has no connection to the COP9 signalosome. It must be pointed out that all
immunoprecipitates were washed with RIPA buffer, conditions that would
not allow identification of weak interactions.
eIF3e Interacts Directly with CSN7 and eIF3c--
CSN7 exists both
as a component of the COP9 signalosome and in a COP9
signalosome-independent form (8). The coimmunoprecipitation assays
suggested that CSN7 and eIF3e reside in the same protein complex.
Because Arabidopsis cop9 mutants lack the COP9
signalosome, and CSN7 exists as a monomer or lower molecular weight
form in this mutant, the interaction of eIF3e with CSN7 in
cop9 mutants (Fig. 1B) suggests that eIF3e
interacts directly with the CSN7 monomer. To test this hypothesis, we
examined the interaction of eIF3e with CSN7 in a heterologous system in
the absence of other Arabidopsis proteins or recognizable
homologs of the COP9 signalosome. To this end, we have used the yeast
two-hybrid assay (39). As shown in Fig.
2A, eIF3e did not interact
with the LexA domain by itself. The interaction of eIF3e with CSN7
clearly activated
As the immunoprecipitation results suggested that eIF3b interacts with
eIF3c, but not the COP9 signalosome, we monitored eIF3b further for
direct protein-protein interactions in yeast. As seen in Fig.
2B, eIF3b results in variable levels of reporter gene expression by itself. Despite this, the results clearly show a direct
interaction between eIF3b and eIF3c. eIF3b did not interact with either
CSN1 or CSN8. eIF3b also appears not to interact with eIF3e and CSN7,
although the high variability of the eIF3b-LexA-carrying strains is
problematic in this analysis. In summary, the yeast two-hybrid assay
has substantiated the hypothesis that interaction of the signalosome
subunits is specific for the e subunit but not the b subunit of
eIF3.
To substantiate further the direct interaction between eIF3e and CSN7
and eIF3c, an in vitro pull-down assay was used to determine whether these proteins interact in the absence of other plant or yeast
proteins. As shown in Fig. 3A
the amount of GST-eIF3e protein that was "pulled down" increased as
the amount of His6-CSN7 protein increased, whereas the
control failed to pull down GST-eIF3e. Similar results were obtained in
a pull-down experiment using equal amounts of His6-eIF3c
and increasing amounts of GST-eIF3e on glutathione-Sepharose beads
(Fig. 3B).
The two-hybrid results are consistent with the immunoprecipitation
analysis. Taken together with the in vitro pull-down assay, these results strongly indicate that eIF3e, but not eIF3b, interacts directly with CSN7 and eIF3c.
Subcellular Localization of eIF3e and eIF3b--
To address the
potential function of the COP9 signalosome interactive protein eIF3e,
we examined its subcellular localization in relation to that of eIF3b
and CSN7. If eIF3e and eIF3b both functioned solely as subunits of
eIF3, then we expect that they will show identical localization
patterns, most likely exclusively cytoplasmic localization. As a first
test, eIF3e was expressed transiently as a green fluorescent protein
(GFP) fusion protein in onion epidermal cells, where it was found in
both the cytoplasm and the nucleus (Fig.
4, B and F). The
distribution of eIF3e-GFP was similar to that of three core subunits of
the COP9-signalosome, namely CSN1, CSN8 (34), and CSN7 (data not
shown). GFP-eIF3e had a higher level of nuclear localization than a GFP
dimer (Fig. 4, A and E), even though its
molecular mass (80 kDa) is larger than that of the GFP dimer (55 kDa).
Therefore, even though the nuclear level did not approach that of a
nuclear control protein, GFP-NIa (Fig. 4, D and
H), eIF3e appears to have an intrinsic propensity for
nuclear uptake. In contrast, a GFP-eIF3b fusion showed exclusively
cytoplasmic localization (Fig. 4, C and G), as
expected for a subunit of eIF3.
Subsequently, an immunocytochemical assay was employed to determine the
localization of endogenous eIF3e as well as of eIF3b and CSN7 in
Arabidopsis protoplasts that were counterstained with DAPI.
Endogenous eIF3e was primarily cytoplasmic in protoplasts derived from
seedling leaf tissue (Fig. 5,
D and H). However, protoplasts from roots often
revealed both cytoplasmic as well as nuclear and/or perinuclear
staining of eIF3e (Fig. 5, C and G). By
comparison, as expected,
In summary, the subcellular localization of eIF3e was clearly distinct
from that of eIF3b and similar to CSN7. These results are consistent
with the interpretation that eIF3b functions only as a subunit of a
cytoplasmic complex (eIF3), whereas eIF3e functions in multiple
complexes both in the cytoplasm as well as in the nucleus. The
subcellular localization data are also consistent with a direct
interaction between eIF3e and the CSN7. Moreover, the nuclear
localization of eIF3e may be regulated by cell type-specific factors.
eIF3e and eIF3b Are Part of a Protein Complex Larger Than the COP9
Signalosome--
The high level of amino acid identity between eIF3e
and eIF3b and their mammalian orthologs suggested that they are
components of the eIF3 complex. On the other hand, the interactions
presented above indicate that eIF3e is also part of the COP9
signalosome. To determine if eIF3e and eIF3b are found in a large
molecular weight complex, total soluble proteins from cauliflower buds
and Arabidopsis roots were separated by gel filtration
chromatography, and the fractions were subjected to Western blot
analysis with A necessary step in elucidating the role of the COP9 signalosome
in the control of plant development is determining its exact subunit
composition. Approximately 10 individual proteins were identified in
the initial purification of the COP9 signalosome (29, 36). Most of
these proteins were subsequently shown to be orthologs of the subunits
of the mammalian COP9 signalosome, although the p105- and
p48-copurifying proteins showed similarities to components of eIF3, and
p43 was similar to a subunit of the regulatory lid of the proteasome
(29). Whereas eIF3, the lid of the proteasome, and the COP9 signalosome
are similar in size and contain subunits that may be evolutionarily
related, each complex has unique biochemical properties and different
proposed biological functions. Therefore, the association of eIF3e
(p48) and eIF3c (p105) with the COP9 signalosome raises some
interesting possibilities, leading us to one of three hypotheses. 1)
eIF3c and eIF3e are subunits of the COP9 signalosome. 2) They are
subunits of eIF3 that also interact with the COP9 signalosome or its
subunits, either through eIF3 or independent of eIF3. 3) Copurification of eIF3c and eIF3e with the COP9 signalosome may be due to similar fractionation properties of eIF3 and the COP9 signalosome.
Several lines of evidence indicate that eIF3e is a subunit of
Arabidopsis eIF3. First, eIF3e is highly conserved with
Int-6, the mammalian eIF3e (16). Second, like eIF3c, eIF3e is a
component of a large complex, in the range of eIF3 in mammals. Third,
eIF3e coimmunoprecipitates with eIF3c and interacts in yeast and
in vitro with eIF3c, indicating that they are components of
the same protein complex. Furthermore, eIF3e was recently identified in the purified Arabidopsis eIF3 (21). On the other hand,
additional evidence suggests that eIF3e as well as eIF3c are associated
with the COP9 signalosome. First, eIF3e and eIF3c copurified with the COP9 signalosome. Second, both eIF3e and eIF3c bound specific signalosome subunits by coimmunoprecipitation, and eIF3e interacted with the CSN7 subunit in vitro. These results are supported
by the two-hybrid assay (see Ref. 29 and Fig. 2). Third, if eIF3e and
eIF3c were simply components of a copurifying eIF3, one would expect to
find most subunits of eIF3 in our COP9 signalosome preparation. However, after sequencing peptides from all copurifying proteins, eIF3e, eIF3c, and possibly an eIF3f ortholog are the only eIF3-related proteins that copurify with the COP9 signalosome (29, 36). Furthermore,
eIF3c and eIF3e were present in equimolar amounts with other COP9
subunits (36). Fourth, whereas eIF3b was localized exclusively in the
cytoplasm, as expected for an eIF3 subunit, eIF3e was also found in the
nucleus in a fraction of cells, further suggesting that it plays a role
outside of eIF3. Taken together these results suggest that in
Arabidopsis, eIF3e and eIF3c interact with both eIF3 and the
COP9 signalosome.
Subcellular localization data indicate that although eIF3b is
cytoplasmic, eIF3e and CSN7 are both nuclear and cytoplasmic. When
using protoplasts from green leaves, the immunolocalization results
were identical to those with roots, with the following exception; we
were unable to detect nuclear-localized eIF3e protein. Therefore, it is
possible that eIF3e is subject to a nuclear exclusion mechanism in
photosynthetically active leaf cells. Onion cells may not show this,
either because they lack chloroplasts, as do protoplasts from roots, or
because the necessary partner protein for nuclear exclusion is present
in insufficient amounts in the onion cell.
The possible cell type specificity for the subcellular localization of
eIF3e is intriguing, although not understood. The nuclear localization
of eIF3e was reminiscent of the predominantly nuclear localization of
its ortholog Int-6 in certain mammalians cells (27, 40), although we
have not observed an association of eIF3e with nuclear bodies.
Coexpression of the human T-cell lymphotrophic virus, type I retroviral
protein, Tax, with Int-6 causes the redistribution of Int-6 from the
nuclear bodies to the cytoplasm (27), suggesting that the
nucleocytoplasmic distribution of Int-6 may be regulated. Recent data
have confirmed signals for both nuclear import and nuclear exclusion
within mammalian Int-6 and have resulted in a reappraisal of the
association of Int-6 with nuclear bodies (37). Given that the COP9
signalosome is thought to be nuclear-localized (36), even though a
cytoplasmic or perinuclear localization has not been ruled out (7, 41),
it seems most likely that eIF3e interacts with the COP9 signalosome in
the nucleus rather than in the cytoplasm. However, the signalosome
subunit CSN7 was detected in both nucleus and cytoplasm. It is
conceivable that CSN7, by virtue of binding to eIF3e, may modulate the
subcellular localization of eIF3e.
This hypothesis may be the key to understanding the significance of the
CSN-eIF3e interaction. The role of eIF3e is unclear, and indeed eIF3e
was absent from several mammalian eIF3 preparations (13, 42, 43), and
eIF3e is not a subunit of eIF3 in S. cerevisiae (22). This
suggests that eIF3e is not necessary for basic translation initiation
but rather controls eIF3 activity, providing an additional translational control mechanism in higher organisms. We propose a model
in which the COP9 signalosome, in addition to its role in
transcriptional repression, also regulates translation by
differentially sequestering eIF3 subunits (Fig.
7). As no COP9 signalosome was found
during the isolation of plant eIF3 (21), this suggests that eIF3e and
eIF3c interact with COP9 signalosome distinct from eIF3, probably in
the nucleus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
s
1 and 8 h of darkness. Brassica
oleracea (cauliflower) was purchased at the supermarket.
ZAPII size-fractionated cDNA library from Arabidopsis (32).
The library was screened as described for eIF3c (29). Phages from
positive clones were excised in vivo according to standard
procedure. The longest cDNA clone found was sequenced, and the
sequence was confirmed through comparison with the genomic eIF3b
sequence (accession number AC005405) and deposited under
GenBankTM accession number AF255680.
-Galactosidase quantitation was done as described (29).
-glucuronidase-transgenic seedlings. After fixation to
microscope slides, affinity-purified primary antibodies against eIF3e,
eIF3b, or CSN7 (FUS5) (8) were applied at a dilution of 1:100. The anti-
-glucuronidase antibody (Molecular Probes) was used at 1:500 dilution. The secondary antibody was a goat anti-rabbit immunoglobulin antibody conjugated with fluorescein, which was used at a 1:200 dilution. Protoplasts were mounted with SlowFade anti-fade reagent (Molecular Probes) containing 1 µg/ml of
4',6-diamidino-2-phenylindole (DAPI).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Comparison of Arabidopsis eIF3e and eIF3e from human, Drosophila, C. elegans, and S. pombe
Comparison of Arabidopsis eIF3b and eIF3b from human, Drosophila, S. pombe, and S. cerevisiae
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Fig. 1.
eIF3e and eIF3c coimmunoprecipitate with each
other and with components of the COP9 signalosome. Total soluble
protein extracts from Arabidopsis wild type (wt)
and cop9 mutants were immunoprecipitated with the antibodies
as indicated above each lane. Following washes with RIPA
buffer the proteins bound to the protein A beads were separated on an
8% SDS gel (A) or a 12.5% SDS gel (B),
transferred to PVDF membrane, and probed with anti-eIF3c antibodies
(A) or anti-CSN7 antibodies (B). The band seen in
B around 100 kDa is nonspecific.
-galactosidase activity indicating a direct
interaction between eIF3e and CSN7 and further supporting the
immunoprecipitation data. eIF3e also interacted with eIF3c, suggesting
that the immunoprecipitation of eIF3c and eIF3e is a result of direct
interaction between these proteins.
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Fig. 2.
Two-hybrid interactions of eIF3e and
eIF3b. A, eIF3e interacts directly with CSN7 and eIF3c.
B, eIF3b interacts with eIF3c but not CSN proteins. The
relative lacZ reporter gene activity in yeast cells for
different combinations of plasmids is shown. LacZ activity in the
negative controls (top three bars in A and
top bar in B) represents the background levels.
Five to ten individual transformants were used to measure relative
lacZ activity for each strain. Error bars
represent S.D.
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Fig. 3.
In vitro interaction of eIF3e with
CSN7 (A) and eIF3c
(B). Increasing amounts of crude soluble proteins
from E. coli expressing either His6-CSN7 or
control proteins (A) or GST-eIF3e or control proteins
(B) were incubated with constant amounts of crude soluble
protein from E. coli expressing GST-eIF3e (A) or
His6-eIF3c (B) and collected on nickel beads
(A) or glutathione-Sepharose (B) as detailed
under "Experimental Procedures." The beads were extracted, and the
proteins were separated on a 12.5% SDS gel, transferred to a PVDF
membrane, and probed with eIF3e (A) or eIF3c (B)
antibodies. The right lane shows the control GST-eIF3e
(A) and His6-eIF3c (B).
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Fig. 4.
Subcellular localization of eIF3e and eIF3b
proteins fused to GFP after transient transformation of onion epidermal
cells. Constructs encoding the GFP reporter fused to 35S promoter
and either the full coding length eIF3e (B) or eIF3b
(C) cDNA sequences were introduced into onion epidermal
cells by particle bombardment. A GFP dimer (A) and the
nuclear GFP-NIa protein (D) (35) served as controls. The
positions of the nuclei in A-D (arrows) were
determined under bright-field illumination as shown immediately below
in E-H. The white bars represent 50 µm. The
bar in A refers to A-C and
E G, and the bar in D refers to
D and H.
-glucuronidase was always cytoplasmic in
protoplasts from roots (Fig. 5, A and E). On the
other hand, the majority of Arabidopsis eIF3b was
cytoplasmic in protoplasts isolated from seedling roots and leaves
(Fig. 5, I and M, and J and
N). The localization of eIF3e overlapped that of the CSN7 protein, which was found in both nucleus and cytoplasm of root and leaf
protoplasts (Fig. 5, K and O, and L
and P).
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Fig. 5.
Immunofluorescence of eIF3e, eIF3b, and CSN7
proteins in Arabidopsis protoplasts.
A-D and I-L (1st and 3rd
row), antibody treatments. E-H and M-P
(2nd and 4th row), DAPI staining. For
A and E, protoplasts were isolated from roots of
GUS-transgenic Arabidopsis. Protoplasts in C, G,
I, K, M, and O were from wild type roots.
B, D, F, H, J, L, N, and P protoplasts were from
wild type leaves. A and E, GUS antibody.
B and F, no first antibody. C, D, G,
and H, eIF3e antibody. I, J, M, and N,
eIF3b antibody. K, L, O, and P, CSN7 antibody.
The white bar in A represents 40 µm.
-eIF3e and
-eIF3b antibodies. In both tissues, the
peak elutions of eIF3e and eIF3b were in a large molecular weight
species, which is larger than the COP9 signalosome, as shown by the
elution profile of CSN7 (Fig. 6).
However, as we have shown for eIF3c, both eIF3e and eIF3b were also
present in the 500-kDa fractions. In roots, the elution profile of CSN7
extended into the higher molecular weight fractions, overlapping the
elution profile of eIF3e and eIF3b (Fig. 6). This gel filtration
shoulder in the high molecular weight fractions is similar to the
elution profile of the COP9 signalosome subunit CSN8 from dark-grown
Arabidopsis seedlings (30), suggesting a heterogeneous pool
of complexes containing the COP9 signalosome in roots. In summary,
although eIF3e and eIF3b are primarily found in a complex larger than
the COP9 signalosome, a larger COP9 signalosome overlapping eIF3 was
detected in roots, the same tissue, where eIF3e colocalizes with the
CSN.
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Fig. 6.
eIF3e and eIF3b are part of a protein complex
larger than the COP9 signalosome. Total soluble proteins from
cauliflower (top panel) or Arabidopsis roots
(bottom panel) were separated by Superose 6 gel filtration
chromatography. Equal volumes from each fraction were separated on
12.5% polyacrylamide gel, transferred to PVDF membrane, and probed
with antibodies against eIF3e followed by re-probing of the membrane
with anti-eIF3b antibodies and anti-CSN7 antibodies. Fraction numbers
from the start of the void volume are indicated, as well as the elution
point of thyroglobulin and apoferritin (669 and 440 kDa, respectively).
The molecular weight markers were SeeBlue (NOVEX) (top
panel) and ProSieve (FMC) (lower panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Suggested model for the regulation of eIF3
activity by the COP9 signalosome. We propose that the function of
eIF3 is regulated through the binding of specific regulatory subunits
such as eIF3e and/or eIF3c by the nuclear-localized COP9 signalosome.
The regulation is achieved by either sequestering (A) or
releasing (B) eIF3e and/or eIF3c from the COP9 signalosome.
The activity of eIF3 in A and B is qualitatively
different, as shown by different arrows toward translation.
eIF3c or eIF3e may bind both complexes simultaneously. eIF3e may
interact with eIF3 through eIF3c.
The regulation of protein synthesis is an essential means of
controlling gene expression, and the translational machinery is the
target of global regulatory mechanisms (44). As initiation is the
rate-limiting step in mRNA translation, regulatory mechanisms often
target this event. In plants, translation initiation factors including
eIF3 are differentially regulated during development and following heat
shock (45). In wheat, specifically, initiation factors are expressed in
a coordinated fashion during seed germination; however, during seed
development or after heat shock treatment uncoupling of these
coordinated expression patterns is observed (45). The activity of the
initiation factors may be modulated by additional interacting proteins.
Accordingly, eIF3 may be a protein complex consisting of core subunits
and additional regulatory subunits that associate with the core as
required during development. The eIF3e protein might be such a
regulator of eIF3.
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ACKNOWLEDGEMENT |
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We are grateful to Xing-Wang Deng of Yale University for support at early stages of this project, for providing anti-CSN1 and CSN8 antibodies.
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FOOTNOTES |
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* This work was supported by Grant 96-00258 from the United States-Israel Binational Science Foundation (to D. A. C. and A. G. v. A.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF255679 and AF255680.
¶ To whom correspondence should be addressed. Tel.: 972-3-6408989; Fax: 972-3-6409380; E-mail: chamd@post.tau.ac.il.
Published, JBC Papers in Press, October 11, 2000, DOI 10.1074/jbc.M006721200
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ABBREVIATIONS |
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The abbreviations used are: eIF3, eukaryotic translation initiation factor 3; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; GST, glutathione S-transferase; EST, expressed sequence tag; Ni-NTA, nickel-nitrilotriacetic acid; DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein.
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