From the Department of Chemistry and Biochemistry and the Institute
for Cellular and Molecular Biology, University of Texas, Austin, Texas
78712 and the Department of Biochemistry, University of
California, Riverside, California 92521
Received for publication, August 9, 2000, and in revised form, October 18, 2000
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ABSTRACT |
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Eukaryotic initiation factor 3 (eIF3) is a
multisubunit complex that is required for binding of mRNA to 40 S
ribosomal subunits, stabilization of ternary complex binding to 40 S
subunits, and dissociation of 40 and 60 S subunits. These functions and
the complex nature of eIF3 suggest multiple interactions with many components of the translational machinery. Recently, the subunits of
mammalian and Saccharomyces cerevisiae eIF3 were
identified, and substantial differences in the subunit composition of
mammalian and S. cerevisiae were observed. Mammalian eIF3
consists of 11 nonidentical subunits, whereas S. cerevisiae
eIF3 consists of up to eight nonidentical subunits. Only five of the
subunits of mammalian and S. cerevisiae are shared in
common, and these five subunits comprise a "core" complex in
S. cerevisiae. eIF3 from wheat consists of at least 10 subunits, but their relationship to either the mammalian or S. cerevisiae eIF3 subunits is unknown. Peptide sequences derived
from purified wheat eIF3 subunits were used to correlate each subunit
with mammalian and/or S. cerevisiae subunits. The peptide
sequences were also used to identify Arabidopsis thaliana
cDNAs for each of the eIF3 subunits. We report seven new cDNAs
for A. thaliana eIF3 subunits. A. thaliana eIF3
was purified and characterized to confirm that the subunit composition and activity of wheat and A. thaliana eIF3 were similar. We
report that plant eIF3 closely resembles the subunit composition of
mammalian eIF3, having 10 out of 11 subunits in common. Further, we
find a novel subunit in the plant eIF3 complex not present in either mammalian or S. cerevisiae eIF3. These results suggest that
plant and mammalian eIF3 evolved similarly, whereas S. cerevisiae has diverged.
Eukaryotic initiation factor 3 (eIF3)1 is the most complex
and least understood of the protein synthesis initiation factors. eIF3
has been purified from wheat germ (1-3), HeLa cells (4, 5), rabbit
reticulocytes (6-12), and Saccharomyces cerevisiae (13-16). Depending upon the organism and method of purification, there
are 6-11 nonidentical subunits present in functional eIF3 complexes
that have been isolated and characterized. Recently, the subunits from
the human and S. cerevisiae eIF3 complex have all been
identified and sequenced. Surprisingly, there are substantial differences in the subunit composition between mammalian and S. cerevisiae eIF3 complexes. The mammalian eIF3 complex composition varies depending upon the method of purification (6, 7, 9-12);
however, the mammalian eIF3 complex appears to contain 11 subunits,
p170 (17), p116 (18), p110 (19), p66 (20), p44 (21, 22), p48 (23), p47
(20), p40 (20), p36 (19), p35 (21) and
p28.2 The S. cerevisiae eIF3 complex was determined to consist of a core
complex of five subunits, Tif32p, Prt1p, Nip1p, Tif35p, and Tif34p
(13-15, 24) that correspond to mammalian subunits p170, p116, p110,
p44, and p36, respectively (see Table II). There are other
proteins associated with the S. cerevisiae eIF3 complex, TIF31p (Clu1p; Ref. 25), GCD10p (15, 26), eIF5 (15, 22), and Sui1p
(eIF1; Ref. 27), that are not present in the mammalian complex (11,
24). TIF31 is not an essential gene in S. cerevisiae, and the role of the TIF31 protein in the eIF3 complex
is not clear (25). GCD10 has recently been shown to have a role in a
nuclear complex required for maturation of initiator methionyl-tRNA
(28). eIF5 and eIF1 (Sui1), previously characterized initiation
factors, participate in subunit joining and initiation codon
recognition, respectively (15, 22, 24).
Several functions have been ascribed to eIF3 including stabilization of
ternary complex binding to the 40 S ribosome, binding of mRNA to
the 40 S ribosome, and promotion of dissociation of 40 and 60 S
ribosomal subunits (29-31); however, the exact mechanisms of these
interactions are not understood. It is known that mammalian eIF3, like
eIF4A, interacts with the central domain of mammalian eIF4G, but eIF3
appears to have a separate binding site from eIF4A (32-34). The
interaction of mammalian eIF3 with eIF4G could play a role in
positioning the mRNA and associated initiation factors on the 40 S
ribosome at the initiation codon (35). Mammalian eIF4B, another
mRNA-associated initiation factor, was shown also to interact with
mammalian eIF3 (36). Interestingly, a similar interaction of S. cerevisiae eIF4B and S. cerevisiae eIF3 could not be
demonstrated, suggesting that there may be some fundamental differences
in the way eIF3 functions in different organisms (25).
The eIF3 complex isolated from wheat germ contains nine or 10 subunits
(2, 3, 37). The expression patterns of some of the subunits of wheat
eIF3 indicated differential regulation and expression during seed
development and germination and following heat shock (38). Extensive
biochemical analysis of the wheat eIF3 complex showed that certain
subunits of the complex were protected from trypsin digestion (39),
alkylation (40), or iodination (40). These results suggest that these
subunits may be integral to the structure and therefore less
solvent-accessible. Furthermore, a group of subunits was found to be
resistant to dissociation by mild urea treatment, suggesting that these
subunits are more tightly associated with each other within the complex (40). Based on the various studies of the interactions of eIF3 subunits, a model for the interaction of some of the subunits of eIF3
has been proposed (16, 35). This preliminary model suggests that the
five subunits shared in common between S. cerevisiae and
mammalian eIF3 may have a similar architecture.
Although mammalian and S. cerevisiae eIF3 complexes have
some subunits in common, they each have subunits that are unique to
their respective complexes. It is important to know whether eIF3s from
other eukaryotes will follow a similar pattern of containing a few
common subunits but have other subunits that are unique. Higher plants
represent a distinct evolutionary path from mammals and fungi. The
composition of plant eIF3 should therefore give a good indication of
whether eIF3 complexes from eukaryotes will have substantial
differences in subunit composition. We report in this paper that the
plant eIF3 subunit composition closely resembles that of mammalian
eIF3, sharing 10 out of 11 subunits. The plant eIF3 complex contains a
unique subunit that is not present in mammalian or S. cerevisiae eIF3. These findings suggest that the eIF3 complexes
from most eukaryotes will probably have a subunit composition that is
similar to the plant and mammalian complexes and that the subunit
composition of the S. cerevisiae complex most likely
represents a divergent evolutionary path.
Preparation of Wheat eIF3 and Tryptic Peptides--
Wheat germ
used for preparation of eIF3 and other initiation factors was purchased
from Bob's Red Mill (Milwaukie, OR). Wheat eIF3 was prepared as
described previously (37). eIF3 activity was measured in an in
vitro assay system dependent upon the addition of eIF3 as
described previously (37). Purified wheat eIF3 was separated on a
10 × 15-cm-long, 1.0-mm-thick, 15% Laemmli SDS gel (15%
acrylamide, 0.55% bisacrylamide) and blotted onto polyvinylidene difluoride membrane and stained with Ponceau S. The membrane was sent
to Dr. John Leszyk (Protein Microsequencing Laboratory, University of
Massachusetts Medical School, Worcester, MA) for trypsin digestion and
peptide sequence analysis.
Arabidopsis thaliana Suspension Cell Culture--
Suspension
cultures of A. thaliana (Columbia University) were obtained
from Dr. A. N. S. Reddy (Colorado State University) and
maintained on Murashige and Skoog (MS) medium containing, per
liter, 4.3 g of MS basal salts (Sigma), 30 g of sucrose
(Sigma), 1.0 ml of 1000× MS vitamins (Sigma), and 0.5 mg of
2,4-diphenoxyacetic acid (Sigma). The medium was adjusted with KOH to a
pH of 5.7 before autoclaving. Suspension cultures were maintained by
transferring every 7 days 1 part of stationary stage culture to 4 parts
fresh MS medium in an Erlenmeyer flask that was 2.5-5 times larger
than the final volume. The flasks were rotated at 115 rpm on a gyratory shaker at 26 °C in the dark.
Preparation of A. thaliana Cell Extract--
Cells from 7-day
suspension cultures were harvested by pouring cultures through eight
layers of cheesecloth. The cells were rinsed with buffer containing 20 mM Hepes·KOH, pH 7.6, 14 mM
Mg(OAc)2, 0.1 M KOAc, and 3% sucrose. Excess
liquid was squeezed from the cells by hand and transferred to dry
cheesecloth as necessary. The wet weight of the cells was determined,
and the cells were placed in a blender with a volume of extraction
buffer (10 mM Tris·OAc, pH 8.2, 14 mM
Mg(OAc)2, 60 mM KOAc, 1 mM
dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) 2 times the wet weight of the cells. The cells and buffer were processed
for ~2 min or until smooth. The cell debris was removed by
centrifugation at 30,000 × g for 40 min at 4 °C.
The supernatant (S-30) was flash frozen in liquid nitrogen and stored
at Purification of A. thaliana eIF3--
The S150 from ~200 g of
cells was made to 40% saturation with solid ammonium sulfate, and the
precipitate was collected by centrifugation at 30,000 × g for 15 min at 4 °C. The protein pellets were
resuspended in 13 ml of buffer N1 (20 mM Hepes,
pH 7.6, 10% glycerol, 1 mM dithiothreitol, 0.1 EDTA)
containing 0.08 M KCl and dialyzed overnight against 2 liters of the same buffer. The dialyzed sample was clarified by
centrifugation at 12,000 × g for 10 min at
4 °C.
The dialyzed sample (~200 mg of protein) was made 15% in glycerol
and applied to a 20-ml column of Whatman DE-52 cellulose equilibrated
in buffer N15 (20 mM Hepes, pH 7.6, 15%
glycerol, 1 mM dithiothreitol, 0.1 EDTA) containing 0.08 M KCl. The column was washed with buffer N15
containing 0.08 KCl until the A280 was less than
0.1. The protein was eluted with a 5× linear gradient from 0.08 M KCl to 0.4 M KCl in N15 buffer.
The resulting fractions were assayed in a wheat germ translation system
(37). The eIF3 activity eluted from the column between 0.15 and 0.25 M KCl.
The pooled DEAE material was adjusted to 0.08 M KCl with
N15 buffer containing no KCl and loaded onto a 0.5-ml
column of SP-Sepharose equilibrated in the same buffer. After loading
the sample, the column was washed with N15 containing 0.08 KCl, and the eIF3 was eluted with N15 containing 0.2 M KCl. Fractions with maximum specific activity were pooled.
A. thaliana eIF-3 from two SP-Sepharose columns, ~1.3 mg
of protein, were pooled and concentrated 3-fold in a Centricon 10 microconcentrator (Amicon) according to the manufacturer's
instructions. The concentrated sample, ~400 µl, was applied to a
13.2-ml 15-40% glycerol gradient in 20 mM Hepes·KOH, pH
7.6, 0.1 mM EDTA, 1.0 mM dithiothreitol, and
0.2 M KCl. A sample of 0.74 mg in 400 µl of purified
wheat eIF3 was applied to a second gradient. The gradients were
centrifuged at 21,000 × g at 4 °C in a Beckman SW
41 Ti rotor for 18 h. Fractions of 0.3 ml were collected and
assayed for eIF3 activity and protein concentration by the method of
Bradford (41).
Electrophoresis and Mass Spectrometry of A. thaliana
eIF3--
About 50 µg of A. thaliana eIF3 or wheat eIF3
from the glycerol gradients were precipitated with methanol and
chloroform to remove the glycerol and salts (42). The precipitates were
collected in a microcentrifuge tube and air-dried. The resulting
pellets were dissolved in 40 µl of 1× Laemmli SDS gel loading buffer
and applied to a 10 × 15-cm-long, 1.0-mm-thick, 15% Laemmli SDS
gel containing 14.45% acrylamide and 0.55% bisacrylamide (43). The gel was electrophoresed at a constant current of 20 mA until the bromphenol blue tracking dye ran off the bottom. The gel was stained in
45% methanol, 10% acetic acid with 0.25% Coomassie Brilliant Blue R
dye and destained. The individual bands of A. thaliana were
cut from the gel and digested with trypsin prior to analysis by mass
spectroscopy (44). The peptides were separated by HPLC on a Varian 9000 using a 10-cm, 1-mm (inner diameter) C18 column. The mass spectrometry
was carried out on a Finnegan MAT LCQ mass spectrometer in the mass
spectrometry facility of the Department of Chemistry and Biochemistry,
University of Texas at Austin (Dr. Mehdy Moini, Director).
Sequencing and Identification of Tryptic Peptides from Wheat eIF3
Subunits--
To determine which of the wheat eIF3 subunits correlated
with the mammalian or S. cerevisiae eIF3 subunits, it was
necessary to obtain protein sequence information. Tryptic peptides were prepared from a highly purified preparation of wheat eIF3 separated by
SDS-PAGE. The peptide sequences obtained for each subunit of wheat eIF3
are shown in Table I. The peptide
sequences were compared with GenBankTM to find potential
matches. The p41 subunit of wheat eIF3 gave three peptides that
corresponded to two different human eIF3 subunits, indicating that two
polypeptides of different sequences migrate with the same mobility on
SDS-PAGE. Previous work from this laboratory showed that the wheat p41
subunit separated into two distinct isoelectric species of pH 5.3 and
7.2 (40). The combination of peptide analysis and two-dimensional gel
data shows that wheat p41 is really two distinct polypeptides with the
same SDS-PAGE mobility. Further, quantitation of the band intensities
of the wheat eIF3 subunits shown in Fig. 3 suggests that there are two proteins at this position relative to the other subunits. Except for
wheat eIF3-p56, matches to mammalian and/or S. cerevisiae eIF3 subunits were found using wheat peptides. The correlation of wheat
subunits to mammalian subunits is shown in Table I. A nomenclature is
proposed based upon the mammalian eIF3 subunits and gives a letter
designation, rather than a molecular weight designation (see Table
II). The proposed nomenclature is
flexible enough to allow for the differences in subunit composition and to accommodate any additions as more is learned about the subunits and
associated proteins from different organisms.
DNA Sequence Analysis of A. thaliana eIF3 Subunits--
The plant
A. thaliana genome and expressed sequence tag (EST) projects
have provided a wealth of easily obtained sequence information. This
resource was used to "mine" for cDNA sequences encoding the
A. thaliana eIF3 subunits using the wheat peptide sequences
shown in Table I. Three A. thaliana full-length cDNAs had already been identified and deposited for eIF3c (AF040102 (45),
eIF3f (U54561) (20), and eIF3i (U36765) (46)). For the seven remaining
subunits (eIF3a, eIF3b, eIF3d, eIF3e, eIF3g, eIF3h, and eIF3k) ESTs or
cDNAs were identified. A full-length A. thaliana EST for
the p56 subunit (eIF3l) is currently not available. A partial gene
sequence is located on a bacterial artificial chromosome derived from
A. thaliana chromosome 5, but a bacterial artificial chromosome with the complete gene sequence for this novel subunit has
not been deposited in the data base. A complete rice gene was
identified using the wheat peptides and used for sequence comparison
purposes in this report (see below).
The ESTs for eIF3a, eIF3d, eIF3e, eIF3g, eIF3h, and eIF3k were obtained
from the Arabidopsis Biological Resource Center (Ohio State
University) and completely sequenced. A cDNA for eIF3b was obtained
by screening a cDNA library with a probe from a less than
full-length EST for eIF3b. New accession numbers for the complete
cDNA sequences were obtained and are indicated in Table II along
with the original EST accession numbers.
The complete genes for all of the A. thaliana eIF3 subunits,
except eIF3l, were identified in the data base, and accession numbers
for the gene(s) are given in Table
III. A nomenclature for the genes
for the plant eIF3 subunits is proposed that uses the translation
initiation factor 3 (TIF3) designation. The species is indicated
(e.g. At for A. thaliana), the subunit letter
designation is capitalized, and the gene(s) are designated with a
number. Subunits eIF3c, eIF3d, and eIF3g each have two genes. The
remaining subunits only have one gene, although additional genes may be found when the Arabidopsis genome is completed.
Purification and Analysis of the A. thaliana eIF3 Complex and
Comparison with the Wheat eIF3 Complex--
Assignments of subunits of
A. thaliana eIF3 could be made based on deduced protein
sequences, but it is important to show that the proteins predicted to
be in the A. thaliana eIF3 complex were in fact in a
functional complex. Therefore, A. thaliana eIF3 was purified
to homogeneity, and the identity of subunits present in the complex was
confirmed by mass spectrometry.
A. thaliana eIF3 was purified from a suspension cell culture
as described under "Experimental Procedures." The final step of
purification was centrifugation through a glycerol gradient. A. thaliana eIF3 migrated in the glycerol gradient similarly to wheat
eIF3 (see Fig. 1). The ability of
A. thaliana eIF3 to support polypeptide synthesis was
compared with purified wheat eIF3 in an in vitro translation
assay. Wheat and A. thaliana eIF3 were found to have a
similar specific activity (see Fig. 2).
A. thaliana and wheat eIF3 were compared by SDS-PAGE as
shown in Fig. 3. A. thaliana
eIF3 has a similar number of subunits compared with wheat eIF3,
although a few subunits differ in apparent molecular weight. The
relative intensities of the subunits in this gel (see the legend to
Fig. 3) suggest that the subunits are present in close to stoichimetric
amounts; however, those subunits that appear to be present in less than
stoichimetric amounts are in regions of overlap, and quantitation is
not as accurate. The relative intensity of the p41 subunit supports the
presence of two polypeptides comigrating at this position. It should be
noted that the protein migrating between subunits eIF3h and eIF3i in
the A. thaliana eIF3 preparation (indicated by an
asterisk in Fig. 3) and the doublet (also indicated by an
asterisk) migrating between subunits eIF3e and eIF3h,i in
the wheat eIF3 preparation appear to be the same protein. The proteins
in the wheat doublet give the same tryptic digest pattern, suggesting
that they are the same protein, the smaller one presumably a
degradation product. The wheat peptides and the mass spectrometry of
the A. thaliana protein (asterisk) both identify
the same A. thaliana unidentified open reading frame. Data
base analysis did not yield any similar proteins from other organisms.
The appearance of the protein doublet in wheat eIF3 preparations is
variable. This protein, therefore, may be another plant-specific
subunit of eIF3 or a loosely associated protein of unknown function.
Further analysis will be necessary to distinguish between these two
possibilities.
To identify the subunits of A. thaliana eIF3 in the SDS-PAGE
gel and to correlate them to the wheat eIF3 subunits, the protein bands
were excised from the SDS-PAGE and treated with trypsin. Each subunit
digest was subjected to HPLC, and the masses of the tryptic peptides
were obtained by mass spectrometry. The masses of the tryptic peptides
were compared with the predicted peptide masses based on deduced
A. thaliana protein sequences using the Peptide Mass program
(available on the World Wide Web). In all cases, at least eight
peptides were identified that matched the predicted masses. The
A. thaliana eIF3 subunits shown in Fig. 3 are labeled
according to their identity confirmed by mass spectrometry.
Protein Sequence Comparisons of eIF3 Subunits--
The deduced
A. thaliana protein sequences for eIF3 subunits were used to
search the Caenorhabditis elegans, Schizosaccharomyces pombe, and Drosophila melanogaster genomes for
corresponding genes. The proteins were aligned using MACAW (47), and a
graphical representation is shown in Fig.
4. The substantial similarity among the
subunits (see Table IV) suggests that
many aspects of the structure and function of eIF3 are conserved
throughout eukaryotes. It should be noted that the C. elegans, S. pombe, and D. melanogaster sequences are included based solely on similarity to proteins present
in the mammalian and/or A. thaliana eIF3 complexes. eIF3 complexes from C. elegans, S. pombe, and D. melanogaster have not been purified and characterized for subunit
composition.
eIF3a (p170, TIF32p, or RPG1p)--
The A. thaliana
eIF3a cDNA sequenced in this report encodes a protein that appears
to be highly conserved, particularly at the N terminus of the protein.
The A. thaliana eIF3a is 74 and 79% similar to maize and
tobacco eIF3a, respectively, compared with 45-51% with other
eukaryotes (Table IV). Like other eIF3a subunits, the A. thaliana eIF3a contains a PCI or PINT motif. The PCI
(proteosome, COP9, initiation
factor) (48) or PINT (protesome, int-6,
Nip-1, Trip-15) (49) motif is conserved in the
middle portion of all eIF3a subunits as well as in the C termini of two other eIF3 subunits, eIF3c and eIF3e (48-50). The PCI motif is also
found in subunits of the proteosome and COP9, both of which are large
multisubunit complexes like eIF3 (48, 49). Although the function of the
PCI motif is not known, it is speculated to have a role in the assembly
of large multisubunit complexes (48, 50). Interestingly, there is a
conserved nuclear localization signal sequence identified by PSORT
(available on the World Wide Web) in the C-terminal region of
the A. thaliana eIF3a and other eukaryotic eIF3a protein
sequences. The significance, if any, of this nuclear localization
signal sequence has not been determined
eIF3b (p116, PRT1p)--
A. thaliana eIF3b contains a
conserved RNA recognition motif (RRM) located near the N terminus that
is similar to the RRM found in other eIF3b subunits. The A. thaliana eIF3b is 80% similar to tobacco eIF3b (51), compared
with 45-56% similar to nonplant eIF3b subunits.
The S. cerevisiae eIF3b gene, PRT1, was
originally isolated as a conditional lethal mutation that reduced the
stability of ternary complex binding to 40 S subunits (52, 53).
Mutations in the PRT1 gene also lead to yeast
temperature-sensitive mutants in cell cycle regulation (54, 55) and DNA
synthesis (56). The tobacco eIF3b transcript expression was shown to
accumulate in tissues with high mitotic activity and to be cell
cycle-regulated (51). These results suggest that eIF3 and protein
synthesis are central to progression of the cell cycle.
eIF3c (p110, NIP1p)--
The S. cerevisiae eIF3c gene,
NIP1, was originally isolated as an essential gene involved
in nuclear import (57). S. cerevisiae eIF3c interacts with
eIF5 and eIF1 (Sui1p) both in vivo and in vitro,
suggesting a key role in the process of initiation (15). A. thaliana eIF3c is 78% similar to a cDNA isolated from
Medicago truncatula (58), compared with 42-55%
similar to nonplant eIF3c subunits. The eIF3c subunit from A. thaliana was originally identified as a protein that copurifies
with the nuclear COP9 complex; however, eIF3c is not part of the core
COP9 complex (45). The COP9 complex plays a role in light signal
transduction and structurally resembles the 26 S proteosome, suggesting
a common evolutionary ancestor (59). Complexes similar to COP9 have
been identified in other eukaryotes, suggesting a conserved role in
development regulation (59, 60). It is intriguing that plant eIF3c
copurifies with COP9 and also shares the PCI domain in common with some
of the COP9 subunits. A regulatory role for eIF3c has been suggested (70), but the significance of the COP9
and eIF3c interaction in the cell has yet to be determined. The eIF3c
in wheat eIF3 is specifically phosphorylated by a wheat kinase
incorporating up to 6 pmol of phosphate/pmol of eIF3 complex (61). This
suggests the potential for regulation by phosphorylation, although no
effect in vitro on protein synthesis by phosphorylation was
observed (61); however, the phosphorylation may affect some other
process, such as an interaction with other protein complexes
(e.g. COP9).
The A. thaliana eIF3c gene is induced during
photomorphogenesis and shows differential tissue accumulation (45). The
eIF3c subunit from M. truncatula was identified in a screen
for genes induced during symbiosis with a plant fungal agent (58).
These observations suggest that the eIF3c mRNA and its translation
are developmentally regulated or are altered in response to changes in
the environment.
There are two genes for A. thaliana eIF3c, both on
chromosome 3; however, only AtTIF3C1 appears to be
transcriptionally active, since no ESTs corresponding to
AtTIF3C2 are present at this time in the data base. This
suggests that AtTIF3C2 is either a pseudogene, is expressed
at very low levels, or is expressed in a tissue- or
time-specific manner.
eIF3d (p66)--
The eIF3d subunit in mammalian eIF3 does not
appear to have an equivalent in the S. cerevisisae eIF3
complex; nor does there appear to be similar protein encoded in the
S. cerevisiae genome. The A. thaliana cDNA
sequenced in this report is not full-length. The missing amino-terminal
sequence was derived from gene AtTIF3D1 (see Table III). The
A. thaliana eIF3d subunit contains a basic region in the N
terminus similar to that of mammalian eIF3d. Although the amino acids
flanking this region are conserved, the sequence of the RNA binding
region itself is not conserved among eukaryotes; however, the overall
character of the region being rich in basic amino acids is maintained.
The amino acids comprising this RNA binding region in eIF3d are over
20% basic and have predicted pIs greater than 9.
There are two genes for A. thaliana eIF3d, and both appear
to be transcriptionally active, since ESTs are present for both genes
in the data bases. Furthermore, peptides from both gene products are
present and are represented in the eIF3 complex isolated from A. thaliana (see Fig. 3, d1 and d2), suggesting
that either gene product may function in eIF3.
eIF3e (p48)--
The A. thaliana eIF3e subunit, like
eIF3a and eIF3c, contains a PCI motif in the C terminus of the protein.
Like eIF3d, eIF3e is not present in the S. cerevisiae eIF3
complex. However, epitope-tagged S. pombe eIF3e was shown to
coimmunoprecipitate with S. pombe eIF3b, suggesting that it
is part of the S. pombe eIF3 complex (71,
72). Interestingly, A. thaliana eIF3e, like eIF3c, is suggested to be associated with the
COP9 complex (70). Antibodies to A. thaliana eIF3e show both
nuclear and cytoplasmic staining, and green fluorescent protein fused
to eIF3e also showed both nuclear and cytoplasmic
localization.3 This is similar to the observations with
human eIF3e (int-6), that also showed nuclear localization
(62). The int-6 gene is a frequent site for integration of
mouse mammary tumor virus and is implicated in regulating cell
proliferation (62, 63). The association of eIF3e with the COP9 complex
and its nuclear localization, in addition to its presence in the eIF3
complex, suggests that this subunit has a dual function that may be
regulatory. The A. thaliana eIF3e protein sequence is the
only other A. thaliana eIF3 subunit besides eIF3a that
contains a predicted nuclear localization signal using PSORT.
eIF3f (p47)--
eIF3f, like eIF3d and eIF3e, is not present in
the S. cerevisiae eIF3 complex. However, epitope-tagged
S. pombe eIF3f was shown to coimmunoprecipitate with
S. pombe eIF3b, suggesting that this subunit is present in
the S. pombe complex.4 A region near the N
terminus contains a conserved motif similar to the MOV34 protein family
(20). This MOV34 motif or MPN domain family has members with diverse
functions such as the 26 S proteosome, COP9, and transcription
complexes, suggesting that these proteins evolved from a common
ancestor (48, 49). The role of the MOV34 domain, like the PCI domain,
is not clear, but it may have a structural role in multiprotein
complexes. However, it is possible that these motifs allow interaction
with other large complexes such as the proteosome or COP9 that have
regulatory functions (48, 49). Interestingly, this subunit was highly
expressed throughout wheat seed development when other eIF3 subunits
were down-regulated and was present in higher amounts in expanded wheat
leaves than other eIF3 subunits, suggesting expression beyond what is
necessary for eIF3 (38).
eIF3g (p44, TIF35p)--
eIF3g contains a consensus RRM in
the C terminus that resembles an RRM found in poly(A)-binding protein
(21, 64). There is also a CCHC zinc-finger motif N-terminal to the RRM
that is conserved. Both human and S. cerevisiae eIF3g were
shown to bind nonspecifically to either 18S rRNA or mRNA (21,
22, 64).
The S. cerevisiae eIF3g gene (TIF35) was shown to
be essential, and depletion of eIF3g resulted in the loss of eIF3i from the complex (64). The S. cerevisiae eIF3g was shown to
interact specifically with S. cerevisiae eIF3b and eIF3i in
genetic experiments, in the yeast two-hybrid system, by
coimmunoprecipitation and by copurification (15, 16, 64, 65). However,
of these interactions, the stronger is with eIF3i (see below). In
addition, the S. cerevisiae eIF3g gene can suppress defects
in the S. cerevisiae eIF3i gene (65). However, the RNA
binding motif was not required for eIF3g to complement mutations in
S. cerevisiae eIF3i (65). Deletion of the RRM resulted in a
slow growth phenotype, suggesting that the RNA binding motif is
required for maximal function but is not essential (64). S. cerevisiae eIF3g was shown to interact with eIF4B and requires the
presence of the RRM for the interaction to occur (25).
Human eIF3g displays different interaction patterns than the S. cerevisiae eIF3g. It was shown to interact with human eIF3 subunits a, b, c, and h and with itself in a yeast two-hybrid analysis
but not with eIF3i (21). The strongest interactions of human eIF3g were
with eIF3a and with eIF3b (21). It was also shown that human eIF3g was
not able to complement a S. cerevisiae eIF3g knock-out (21).
Interestingly, the only monoclonal antibody to wheat eIF3 subunits that
specifically inhibits translation initiation and the binding of
mRNA to 40 S ribosomal subunits was to eIF3g, suggesting a key role
for this subunit in the binding of mRNA to 40 S ribosomes (66).
There are two A. thaliana genes for eIF3g that encode
proteins that show 79% similarity and 73% identity to each other.
Both AtTIF3G1 and AtTIF3G2 gene products are
expressed, since ESTs for these genes are found in the data base;
however, only peptides corresponding to the gene product of
AtTIF3G1 were found in the A. thaliana eIF3 complex.
eIF3h (p40)--
A. thaliana eIF3h is also a member of
the MOV34 family of proteins (11, 20). There is a gene for a similar
protein in the S. pombe genome; however, the presence of
this subunit in the S. pombe complex has not been
determined. Interestingly, a gene with sufficient similarity to eIF3h
from C. elegans could not be identified in the data base.
The C. elegans eIF3 complex will have to be isolated to
determine whether there is an eIF3h-like subunit.
eIF3i (p36, TIF34p)--
eIF3i contains 5-7 WD repeat elements,
depending upon the criteria of the motif search program used (16, 19,
46). WD elements fold into a circular structure termed a " eIF3k (p28)--
Both the A. thaliana and wheat eIF3
complexes contain a subunit of ~28,000 Da. The presence of a
28,000-Da subunit in the mammalian eIF3 complex has been
confirmed2; however, a subunit of this molecular weight is
not present in the S. cerevisiae complex.
An A. thaliana EST for eIF3k was identified based on
similarity to peptides obtained from wheat eIF3k and completely
sequenced. The deduced protein sequence is shown in Fig.
5 and compared with the protein sequences
of eIF3k from human2 and with deduced protein sequences
from C. elegans and D. melanogaster genomes. The
A. thaliana sequence is 43, 42, and 45% similar to putative
human, C. elegans, and D. melanogaster eIF3k,
respectively. No sequence similar to eIF3k was found for S. pombe, suggesting that this subunit may be unique to higher
eukaryotes. The presence of eIF3k in eIF3 complexes from other higher
eukaryotes will have to be confirmed by purification of the protein
complexes.
There are numerous ESTs for the A. thaliana eIF3k subunit,
suggesting that it is highly expressed. Examination of the eIF3k sequences using protein domain search programs, ProDom (available on the World Wide Web) or InterPro (available on the World Wide Web), did not yield any similarity to other proteins of known function or protein motifs. The predicted secondary structure from
Jpred (available on the World Wide Web) indicates that overall the protein is eIF3l (Wheat eIF3-p56)--
A subunit of ~56,000 Da is present
in the wheat eIF3 complex. It was previously thought that the 56,000 Da
subunit might correspond to the S. cerevisiae GCD10 subunit
based on protein mobility; however, the sequence analysis presented in
this paper shows that this is a novel protein. Peptides from wheat
eIF3l were used to search the data base for a corresponding protein in
A. thaliana. A full-length A. thaliana cDNA
or gene is currently not available. However, a rice gene was found that
encodes a protein with a sequence that is similar to both of the wheat
eIF3l peptides (see Fig. 3). Blast searches with rice eIF3l yielded
numerous matches to other plant ESTs, suggesting that it is a highly
expressed transcript. The mRNA for maize eIF3l appears to be
particularly abundant in a maize root cDNA library.
Comparisons with protein data bases were used to identify a human
cDNA and to identify D. melanogaster and C. elegans genes encoding proteins that are similar to eIF3l (Fig.
6). The human and D. melanogaster are more similar to the rice eIF3l, with 54 and 53%
similarity respectively, whereas the C. elegans is only 35%
similar. Overall, the C. elegans protein was less similar to
the other eIF3l-like proteins, indicating that it may be more distantly
related. There was no S. pombe or S. cerevisiae
equivalent for eIF3l, suggesting that this subunit may be unique to
higher eukaryotes. There were no obvious motifs or related proteins for eIF3l using ProDom or Interpro data base searches. The overall secondary structure predicted by Jphred appears to be
There is no equivalent of eIF3l in the mammalian eIF3 complex; however,
the presence of a mammalian cDNA that encodes a similar protein
suggests that whatever the role of eIF3l, it has been retained in the
evolution of higher eukaryotes. Further analysis will be necessary to
determine the function of the eIF3l subunit and whether it is present
in other eukaryotic eIF3 complexes or is a plant-specific subunit.
In this report, we find that the subunit composition of plant eIF3
is more similar to mammalian eIF3 than to S. cerevisiae eIF3. In plant eIF3, 10 out of 11 subunits are equivalent to mammalian eIF3 subunits based on amino acid sequence similarity. Wheat, a
monocot, and A. thaliana, a dicot, have a similar subunit
composition, suggesting that eIF3 complexes from all higher plants
share a similar composition.
The plant eIF3 complex does not appear to contain a protein similar to
mammalian eIF3j (p35); nor is there a similar protein encoded in the
genomes of S. pombe or C. elegans. A gene
containing a short region of similarity was found in the D. melanogaster genome, but this may not be a genuine eIF3j subunit.
Consequently, further analysis is necessary to determine whether eIF3j
is specific to mammalian eIF3 complexes. Similarly, the eIF3l subunit
of plant eIF3 may be specific to the plant eIF3 complex, since it is
present in both the wheat and A. thaliana eIF3 but not in
mammalian eIF3. However, proteins that are similar to plant eIF3l are
present in other eukaryotes including mammals. Further analysis of eIF3 complexes isolated from other eukaryotes will be necessary to determine
whether other species have an eIF3j-like or an eIF3l-like subunit, or
other unique subunit(s).
The analysis of deduced protein sequences from the genomes of S. pombe, D. melanogaster, and C. elegans
indicate that other eukaryotes will have an eIF3 subunit composition
more similar to mammals and plants than to S. cerevisiae.
The S. cerevisiae eIF3 complex appears to have retained five
subunits in common with other eukaryotes, but its evolution has
diverged independently of other eukaryotes. Other S. cerevisiae initiation factors, with the exception of eIF4B (69),
show significant sequence conservation (e.g. eIF4A, eIF1A,
eIF5) and similar subunit composition (e.g. eIF2 and eIF4F)
with other eukaryotes. The differences in subunit composition of eIF3
between S. cerevisiae and mammalian or plant eIF3 are the
most dramatic noted in the translational machinery so far. It is
therefore not surprising that mammalian or plant eIF3 subunits shared
in common with S. cerevisiae have lost the ability to
complement S. cerevisiae eIF3 mutants in those subunits. It
is apparent that the analysis of eIF3 function in higher eukaryotes will require another genetic model system in addition to S. cerevisiae.
Now that a clearer picture of the subunit composition and role of the
different subunits of eIF3 is beginning to emerge, further biochemical
and genetic analyses will be possible to unravel the mysteries of this
complicated initiation factor in eukaryotes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
80 °C. The supernatant was thawed and centrifuged at
150,000 × g for 3 h at 4 °C to remove ribosomes. The resulting supernatant (S-150) was flash frozen in liquid
nitrogen and stored at
80 °C.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
Sequence of tryptic peptides of wheat eIF3 subunits
Eukaryotic eIF3 subunits
A. thaliana ESTs, cDNAs, and genes for the subunits of eIF3
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Fig. 1.
Glycerol gradient analysis of wheat and
A. thaliana eIF3. Wheat eIF3 (0.75 mg in 0.4 ml)
(A) or A. thaliana eIF3 (1.3 mg in 0.4 ml)
(B) was each applied to a 34 ml 15-40% linear glycerol
gradient as described under "Experimental Procedures." After
centrifugation, fractions of 0.3 ml were collected. The protein
concentration ( ) and eIF3 activity (
) were determined.
Sedimentation is from left to right.
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Fig. 2.
Comparison of purified wheat and A. thaliana eIF3 activity. The activity of the eIF3
preparations obtained from the glycerol gradients shown in Fig. 1 was
measured as described previously (37). A fractionated assay dependent
upon the addition of eIF3 was programmed with 5 pmol of satellite
tobacco necrosis virus RNA; 8 pmol of [14C]leucine was
incorporated in the absence of any added eIF3. The indicated amounts of
wheat ( ) or A. thaliana eIF3 (
) were added to the
100-µl reaction.
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Fig. 3.
SDS-PAGE analysis of wheat and A. thaliana eIF3. The wheat and A. thaliana
eIF3 preparations obtained from the glycerol gradients shown in Fig. 1
were analyzed by SDS-PAGE. About 50 µg of purified wheat or A. thaliana eIF3 were precipitated with methanol/chloroform as
described under "Experimental Procedures" and resuspended in 40 µl of 1× SDS gel loading buffer. The samples were applied to a 15%
acrylamide gel and electrophoresed at 20 mA. The gel was stained with
Coomassie Brilliant Blue R and destained. The stained bands were
removed from the gel, treated with trypsin, separated by HPLC, and
analyzed by mass spectrometry as described under "Experimental
Procedures." The subunits are marked according to the nomenclature
proposed in Table II. The relative intensities of the subunits in this
gel were measured using ImageJ (available on the World Wide
Web). The eIF3l subunit intensity was chosen arbitrarily to be
1. The wheat eIF3 subunit intensities relative to the wheat eIF3l band
are as follows: 1.0 (a), 0.96 (b), 0.5 (c), 0.93 (d), 1.2 (e), 1.0 (f); 0.93 (g); 1.7 (h, i), 0.84 (k), and 1.1 (*; both bands). The A. thaliana
eIF3 subunit intensities relative to the A. thaliana eIF3l
band are as follows: 0.6 (a), 0.9 (b), 1.2 (c; both bands), 1.3 (d; both bands), 1.1 (e),
0.96 (f), 1.1 (g), 1.6 (h), 1.3 (i), 0.6 (k), and 0.9 (*).
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Fig. 4.
Graphical representation of the protein
sequence alignments for the subunits of A. thaliana
eIF3 with other eukaryotes. Protein alignments were carried
out using MACAW version 1 (47). The accession numbers for sequences
used are in Table IV. The blue color
indicates a higher degree of similarity in the protein. Protein motifs
or domains found in A. thaliana subunits were identified
using ProDom (available on the World Wide Web) and are indicated
below the alignment. Note that the S. pombe,
C. elegans, and D. melanogaster are included
based solely on protein similarity. The eIF3 complexes have not been
isolated and characterized from these organisms.
Comparison of A. thaliana eIF3 subunits with other eukaryotes
-propeller" (67). This circular structure is thought to provide a
scaffold that is important in protein-protein interactions and
formation of complexes (67). The presence of the WD repeats in eIF3i
suggests that the function of eIF3i may be central to the structural
integrity of the complex (16, 68). This idea is supported by the fact that S. cerevisiae temperature-sensitive eIF3i mutants have
lower levels of all of the eIF3 subunits, and the eIF3i-depleted eIF3 complex no longer binds to 40 S subunits (68). Temperature-sensitive S. cerevisiae mutants with amino acid substitutions in
certain WD repeats were more severely affected than mutants in other
regions, suggesting that these WD repeats are important for subunit
interactions and complex stability (16). The human eIF3i is identical
to the TRIP-1 protein (19). The TRIP-1 protein was found in a yeast two-hybrid screen to associate with TGF-
type II receptors (46). The
A. thaliana eIF3i was originally reported as a protein
similar to the mouse TRIP-1 gene product (46). Another EST for eIF3i was identified and sequenced in this report. It is identical to the
previously reported A. thaliana eIF3i.
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Fig. 5.
Sequence alignment of eIF3k (wheat
eIF3-p28). The protein sequences were aligned using GCG Pile-Up
(Genetics Computer Group, Inc.) and enhanced with Boxshade (available
on the World Wide Web). The peptide sequences similar to those
obtained from wheat eIF3k (see Table I) are overlined.
-helical with only a small region of
-sheet near the N terminus. Since there are no obvious motifs for eIF3k, it is
difficult to speculate on its function at this time. Biochemical analysis of the wheat eIF3 complex suggests that this subunit is
protected from the solvent, since it was not alkylated (40), was not
iodinated appreciably (40), and was resistant to trypsin digestion
(39), and since no monoclonal antibodies were obtained to this
subunit (40).
-helical with
a small portion of
-sheet near the C terminus. Biochemical analysis
of the wheat eIF3 complex indicated that eIF3l was iodinated to the
highest extent of all the subunits and was moderately alkylated, and
monoclonal antibodies were obtained (40). These observations would
suggest that eIF3l is surface-exposed in the complex; however, wheat
eIF3l was resistant to trypsin treatment (39) despite a composition of
over 10% basic amino acids.
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Fig. 6.
Sequence alignment of eIF3l (wheat
eIF3-p56). The protein sequences were aligned using GCG Pile-Up
(Genetics Computer Group, Inc.) and enhanced with Boxshade (available
on the World Wide Web). The peptide sequences similar to those
obtained from wheat eIF3l (see Table I) are overlined.
CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Arlen Johnson for critical reading of the manuscript, Dr. Farida Safadi-Chamberlain for advice on the growth of the A. thaliana suspension culture, and Daniel Mchugh for assistance in preparation of the MACAW alignments.
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Note added in proof |
---|
The complete gene sequence for A. thaliana eIF31 was located on recently deposited bacterial artificial chromosome (AC084432). The gene encodes a 60,189 Da polypeptide with 77% similarity to the rice eIF31 described in this report. The similarity to human, D. melanogaster, and C. elegans eIF31 was 60%, 59%, and 39%, respectively. The mass spectrometry analysis of A. thaliana eIF31 matched 13 peptides predicted from the gene sequence.
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FOOTNOTES |
---|
* This work was supported by National Science Foundation Grant MCB 980873, Department of Energy Grant DE-FG03-97ER20283, Welch Foundation Grant F-1339 (to K. S. B.), and United States Department of Agriculture Grant 00-35301-9086 (to D. R. G.).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) AF291711, AF285834, AF291714, AF285832, AF291712, AF285833, AF285835, and AF291713.
§ To whom correspondence should be addressed. Tel.: 512-471-4562; Fax: 512-471-8696: E-mail: kbrowning@mail.utexas.edu.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M007236200
2 G. L. Mayeur and J. W. B. Hershey, personal communication.
3 A. von Arnim and D. Chamovitz, personal communication.
4 C. Norbury, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: eIF, eukaryotic initiation factor; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; EST, expressed sequence tag; TIF3, translation initiation factor 3; RRM, RNA recognition motif.
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---|
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---|
1. | Ceglarz, E., Goumans, H., Thomas, A., and Benne, R. (1980) Biochim. Biophys. Acta 610, 181-188[Medline] [Order article via Infotrieve] |
2. |
Seal, S. N.,
Schmidt, A.,
and Marcus, A.
(1983)
J. Biol. Chem.
258,
866-871 |
3. |
Checkley, J. W.,
Cooley, L. L.,
and Ravel, J. M.
(1981)
J. Biol. Chem.
256,
1582-1586 |
4. | Brown-Luedi, M. L., Meyer, L. J., Milburn, S. C., Yau, P. M., Corbett, S., and Hershey, J. W. B. (1982) Biochemistry 21, 4204-4206 |
5. | Milburn, S. C., Duncan, R. F., and Hershey, J. W. B. (1990) Arch. Biochem. Biophys. 276, 6-11[Medline] [Order article via Infotrieve] |
6. | Schreier, M. H., Erni, B., and Staehelin, T. (1977) J. Mol. Biol. 116, 727-753[Medline] [Order article via Infotrieve] |
7. | Safer, B., Adams, S. L., Kemper, W. M., Berry, K. W., Lloyd, M., and Merrick, W. C. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2584-2588 |
8. | Merrick, W. C. (1979) Methods Enzymol. 60, 108-123[Medline] [Order article via Infotrieve] |
9. | Nygard, O., and Westermann, P. (1982) Biochim. Biophys. Acta 697, 263-269[Medline] [Order article via Infotrieve] |
10. | Meyer, L. J., Milburn, S. C., and Hershey, J. W. B. (1982) Biochemistry 21, 4206-4212[Medline] [Order article via Infotrieve] |
11. | Hershey, J. W. B., Asano, K., Naranda, T., Vornlocher, H. P., Hanachi, P., and Merrick, W. C. (1996) Biochimie (Paris) 78, 903-907[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Chaudhuri, J.,
Chakrabarti, A.,
and Maitra, U.
(1997)
J. Biol. Chem.
272,
30975-30983 |
13. |
Naranda, T.,
MacMillan, S. E.,
and Hershey, J. W. B.
(1994)
J. Biol. Chem.
269,
32286-32292 |
14. |
Danaie, P.,
Wittmer, B.,
Altmann, M.,
and Trachsel, H.
(1995)
J. Biol. Chem.
270,
4288-4292 |
15. |
Phan, L.,
Zhang, X. L.,
Asano, K.,
Anderson, J.,
Vornlocher, H. P.,
Greenberg, J. R.,
Qin, J.,
and Hinnebusch, A. G.
(1998)
Mol. Cell. Biol.
18,
4935-4946 |
16. |
Asano, K.,
Phan, L.,
Anderson, J.,
and Hinnebusch, A. G.
(1998)
J. Biol. Chem.
273,
18573-18585 |
17. |
Johnson, K. R.,
Merrick, W. C.,
Zoll, W. L.,
and Zhu, Y. X.
(1997)
J. Biol. Chem.
272,
7106-7113 |
18. |
Méthot, N.,
Rom, E.,
Olsen, H.,
and Sonenberg, N.
(1997)
J. Biol. Chem.
272,
1110-1116 |
19. |
Asano, K.,
Kinzy, T. G.,
Merrick, W. C.,
and Hershey, J. W. B.
(1997)
J. Biol. Chem.
272,
1101-1109 |
20. |
Asano, K.,
Vornlocher, H. P.,
Richter-Cook, N. J.,
Merrick, W. C.,
Hinnebusch, A. G.,
and Hershey, J. W. B.
(1997)
J. Biol. Chem.
272,
27042-27052 |
21. |
Block, K. L.,
Vornlocher, H. P.,
and Hershey, J. W. B. (1908)
(1998)
J. Biol. Chem.
273,
31901-31903 |
22. |
Bandyopadhyay, A.,
and Maitra, U.
(1999)
Nucleic Acids Res.
27,
1331-1337 |
23. |
Asano, K.,
Merrick, W. C.,
and Hershey, J. W. B.
(1997)
J. Biol. Chem.
272,
23477-23480 |
24. | Linder, P., Vornlocher, H. P., Hershey, J. W. B., and McCarthy, J. E. G. (1999) Yeast 15, 865-872[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Vornlocher, H. P.,
Hanachi, P.,
Ribeiro, S.,
and Hershey, J. W. B.
(1999)
J. Biol. Chem.
274,
16802-16812 |
26. | Garcia-Barrio, M. T., Naranda, T., Vazquez de Aldana, C. R., Cuesta, R., Hinnebusch, A. G., Hershey, J. W. B., and Tamame, M. (1995) Genes Dev. 9, 1781-1796[Abstract] |
27. | Naranda, T., MacMillan, S. E., Donahue, T. F., and Hershey, J. W. B. (1996) Mol. Cell. Biol. 16, 2307-2313[Abstract] |
28. |
Anderson, J.,
Phan, L.,
Cuesta, R.,
Carlson, B. A.,
Pak, M.,
Asano, K.,
Björk, G. R.,
Tamame, M.,
and Hinnebusch, A. G.
(1998)
Genes Dev.
12,
3650-3662 |
29. | Merrick, W. C., and Hershey, J. W. B. (1996) in Translational Control (Hershey, J. W. B. , Mathews, M. B. , and Sonenberg, N., eds) , pp. 31-70, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
30. | Pain, V. M. (1996) Eur. J. Biochem. 236, 747-771[Abstract] |
31. |
Chaudhuri, J.,
Chowdhury, D.,
and Maitra, U.
(1999)
J. Biol. Chem.
274,
17975-17980 |
32. |
Lamphear, B. J.,
Kirchweger, R.,
Skern, T.,
and Rhoads, R. E. (1983)
(1995)
J. Biol. Chem.
270,
21975-21972 |
33. | Imataka, H., and Sonenberg, N. (1997) Mol. Cell. Biol. 17, 6940-6947[Abstract] |
34. |
Morino, S.,
Imataka, H.,
Svitkin, Y. V.,
Pestova, T. V.,
and Sonenberg, N.
(2000)
Mol. Cell. Biol.
20,
468-477 |
35. | Preiss, T., and Hentze, M. W. (1999) Curr. Opin. Genet. Dev. 9, 515-521[CrossRef][Medline] [Order article via Infotrieve] |
36. | Methot, N., Song, M. S., and Sonenberg, N. (1996) Mol. Cell. Biol. 16, 5328-5334[Abstract] |
37. | Lax, S. R., Lauer, S. J., Browning, K. S., and Ravel, J. M. (1986) Methods Enzymol. 118, 109-128[Medline] [Order article via Infotrieve] |
38. | Gallie, D. R., Le, H., Tanguay, R. L., and Browning, K. S. (1998) Plant J. 14, 715-722[CrossRef] |
39. | Lauer, S. J., Burks, E. A., and Ravel, J. M. (1985) Biochemistry 24, 2924-2928[Medline] [Order article via Infotrieve] |
40. | Heufler, C., Browning, K. S., and Ravel, J. M. (1988) Biochim. Biophys. Acta 951, 182-190[Medline] [Order article via Infotrieve] |
41. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
42. | Wessel, D., and Fluegge, U. I. (1984) Anal. Biochem. 138, 141-143[Medline] [Order article via Infotrieve] |
43. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
44. | Takada, Y., Nakayama, K., Yoshida, M., and Sakairi, M. (1994) Rapid Commun. Mass Spectrom. 8, 695-697[Medline] [Order article via Infotrieve] |
45. | Karniol, B., Yahalom, A., Kwok, S., Tsuge, T., Matsui, M., Deng, X. W., and Chamovitz, D. A. (1998) FEBS Lett. 439, 173-179[CrossRef][Medline] [Order article via Infotrieve] |
46. | Chen, R. H., Meittinen, P. J., Maruoka, E. M., Choy, L., and Derynck, R. (1995) Nature 377, 548-552[CrossRef][Medline] [Order article via Infotrieve] |
47. | Schuler, G. D., Altschul, S. F., and Lipman, D. J. (1991) Proteins 9, 180-190[Medline] [Order article via Infotrieve] |
48. | Hofmann, K., and Bucher, P. (1998) Trends Biochem. Sci. 23, 204-205[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Aravind, L.,
and Ponting, C. P.
(1998)
Protein Sci.
7,
1250-1254 |
50. | Glickman, M. H., Rubin, D. M., Coux, O., Wefes, I., Pfeifer, G., Cjeka, Z., Baumeister, W., Fried, V. A., and Finley, D. (1998) Cell 94, 615-623[Medline] [Order article via Infotrieve] |
51. | Shen, W. H., and Gigot, C. (1999) Plant Sci. 143, 45-54[CrossRef] |
52. | Hartwell, L. H., and McLaughlin, C. S. (1968) J. Bacteriol. 96, 1664-1671 |
53. | Hartwell, L. H., and McLaughlin, C. S. (1968) Proc. Natl. Acad. Sci. U. S. A. 62, 468-474 |
54. |
Hanic-Joyce, P. J.
(1985)
Genetics
110,
591-607 |
55. | Hanic-Joyce, P. J., Johnston, G. C., and Singer, R. A. (1987) Exp. Cell Res. 172, 134-145[Medline] [Order article via Infotrieve] |
56. | Dumas, L. B., Lussky, J. P., McFarland, E. J., and Shampay, J. (1982) Mol. Gen. Genet. 187, 42-46[Medline] [Order article via Infotrieve] |
57. | Gu, Z., Moerschell, R. P., Sherman, F., and Goldfarb, D. S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10355-10359[Abstract] |
58. | van Buuren, M. L., Maldonado-Mendoza, I. E., Trieu, A. T., Blaylock, L. A., and Harrison, M. J. (1999) Mol. Plant-Microbe Inter. 12, 171-181[Medline] [Order article via Infotrieve] |
59. | Wei, N., and Deng, X. W. (1999) Trends Genet. 15, 98-103[CrossRef][Medline] [Order article via Infotrieve] |
60. | Deng, X. W., Dubiel, W., Wei, N., Hofmann, K., Mundt, K., Colicelli, J., Kato, J., Naumann, M., Segal, D., Seeger, M., Carr, A., Glickman, M., and Chamovitz, D. A. (2000) Trends Genet. 16, 202-203[CrossRef][Medline] [Order article via Infotrieve] |
61. | Browning, K. S., Yan, T. F. J., Lauer, S. J., Aquino, L. A., Tao, M., and Ravel, J. M. (1985) Plant Physiol. 77, 370-373 |
62. | Desbois, C., Rousset, R., Bantignies, F., and Jalinot, P. (1996) Science 273, 951-953[Abstract] |
63. | Marchetti, A., Buttitta, F., Miyazaki, S., Gallahan, D., Smith, G. H., and Callahan, R. (1995) J. Virol. 69, 1932-1938[Abstract] |
64. |
Hanachi, P.,
Hershey, J. W. B.,
and Vornlocher, H. P.
(1999)
J. Biol. Chem.
274,
8546-8553 |
65. |
Verlhac, M. H.,
Chen, R. H.,
Hanachi, P.,
Hershey, J. W. B.,
and Derynck, R.
(1997)
EMBO J.
16,
6812-6822 |
66. | Lauer, S. J., Browning, K. S., and Ravel, J. M. (1985) Biochemistry 24, 2928-2931[Medline] [Order article via Infotrieve] |
67. | Fulop, V., and Jones, D. T. (1999) Curr. Opin. Struct. Biol. 9, 715-721[CrossRef][Medline] [Order article via Infotrieve] |
68. | Naranda, T., Kainuma, M., MacMillan, S. E., and Hershey, J. W. B. (1997) Mol. Cell. Biol. 17, 145-153[Abstract] |
69. | Metz, A. M., Wong, K. C. H., Malmström, S. A., and Browning, K. S. (1999) Biochem. Biophys. Res. Commun. 266, 314-321[CrossRef][Medline] [Order article via Infotrieve] |
70. |
Yahalom, A.,
Kim, T. H.,
Winter, E.,
Karniol, B.,
von Arnim, A. G.,
and Chamovitz, D. A.
(2000)
J. Biol. Chem.
276,
334-340 |
71. |
Crane, R.,
Craig, R.,
Murray, R.,
Dunand-Sauthier, I.,
Humphrey, T.,
and Norbury, C.
(2000)
Mol Biol. Cell.
11,
3993-4003 |
72. |
Bandyopadhyay, A.,
Matsumoto, T.,
and Maitra, U.
(2000)
Mol. Biol. Cell
11,
4005-4018 |