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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 747-753
RED CELLS
Protein 4.1R binding to eIF3-p44 suggests an interaction between
the cytoskeletal network and the translation apparatus
Chia-Lung Hou,
Chieh-ju C. Tang,
Steve R. Roffler, and
Tang K. Tang
From the Institute of Life Science, National Defense Medical Center,
and the Institute of Biomedical Sciences, Academia Sinica, Taipei,
Taiwan.
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Abstract |
Erythroid protein 4.1 (4.1R) is an 80-kd cytoskeletal protein that
stabilizes the membrane-skeletal network structure underlying the lipid
bilayer. Using the carboxyl terminal domain (22/24 kd) of 4.1R as bait
in a yeast 2-hybrid screen, we isolated cDNA clones encoding a
polypeptide of eIF3-p44, which represents a subunit of a eukaryotic
translation initiation factor 3 (eIF3) complex. The eIF3 complex
consists of at least 10 subunits that play an essential role in the
pathway of protein translation initiation. Northern blot analysis
revealed that eIF3-p44 (approximately 1.35 kb) is
constitutively expressed in many tissues. The essential sequence for
this interaction was mapped to the carboxyl-terminus of 4.1R (residues
525-622) and a region (residues 54-321) of eIF3-p44. The direct
association between 4.1R and eIF3-p44 was further confirmed by in vitro
binding assays and coimmunoprecipitation studies. To characterize the
functions of eIF3-p44, we depleted eIF3-p44 from rabbit reticulocyte
lysates either by anti-eIF3-p44 antibody or by GST/4.1R-80 fusion
protein. Our results show that the eIF3-p44 depleted cell-free
translation system was unable to synthesize proteins efficiently. The
direct association between 4.1R and elF3-p44 suggests that 4.1R may act
as an anchor protein that links the cytoskeleton network to the
translation apparatus.
(Blood. 2000;96:747-753)
© 2000 by The American Society of Hematology.
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Introduction |
Erythroid protein 4.1 (4.1R) was originally identified
as an 80-kd cytoskeletal protein (4.1R-80) in red blood cells. It is believed to maintain red cell morphology and mechanical strength by
linking membrane integral proteins such as glycophorin C and band 3 to
the spectrin-actin-based cytoskeletal network. Deficiency of 4.1R in
red blood cells leads to assembly of an unstable cytoskeleton structure
that manifests as hereditary elliptocytosis, a disease characterized by
the loss of normal discoid morphology and the presence of oval or
elliptical red cells with unstable membranes.1 Furthermore,
erythrocytes from 4.1R-null mice exhibit erythroid membrane skeleton
abnormalities, which further suggests that the loss of 4.1R compromises
membrane skeleton assembly in erythroid cells.2
The protein 4.1 family encompasses a group of structure-related
proteins. The prototypical erythroid membrane skeletal protein 4.1 (4.1R) is encoded by a complexly spliced gene located on human chromosome 1p33-p34.2.3,4 Heterogeneity of 4.1R isoforms in
size and subcellular localization has been noted. These isoforms are
generated by complex alternative splicing of 4.1R pre-mRNA and
by posttranslational modification of 4.1R proteins.5-7
Western blot analysis revealed 4.1R isoforms ranging in size from 30 to 175 kd among various mammalian nonerythroid cells.8 The
erythroid 4.1R is located mainly beneath the peripheral membrane of
mature red cells. However, the immunoreactive epitopes of 4.1R in
nonerythroid cells have been identified in the cytoplasm, stress
fibers,9 nucleus,10,11
centrosomes,12 and cell-cell contact
regions.13
In addition to the prototypical erythroid 4.1R, 3 new 4.1-like genes
have been identified that reveal high sequence homology with the 30-, 10-, and 22/24-kd domains of 4.1R. These include 2 homologues that are
highly expressed in the brain and neurons (4.1B, 4.1N) and another that
is generally expressed throughout the body (4.1G).14,15
These 4.1-like genes appear to map to distinct chromosomes in humans
and mice.16
Limited chymotryptic digestion of erythroid 4.1R-80 (80 kd) generates 4 structural domains (30, 16, 10, and 22/24 kd). The 30-kd domain
mediates the attachment of 4.1R to the plasma membrane by binding to
the cytoplasmic domains of the transmembrane proteins, glycophorin
C,17-19 and band 3.20,21 It also interacts with p55,18,19 calmodulin,22 and pICln, a protein
involved in cellular volume regulation.23 The 16-kd domain
has phosphorylation sites for protein kinase C and protein kinase A
(PKA),24,25 whereas the 10-kd domain contains an exon
16-encoded peptide that is important for interaction with spectrin
and actin complexes.26-28
The physiological function of the 22/24-kd domain is less well
characterized. Recent reports showed that the carboxyl terminal domain
(22/24 kd) of the 135-kd 4.1R (4.1R-135) isoform interacts with the
nuclear mitotic apparatus protein (NuMA), suggesting that some 4.1R
isoforms may play roles in organizing the nuclear architecture and the
mitotic spindle.29 To further elucidate the possible
functions of the 22/24-kd domain in nonerythroid cells, we searched for
proteins that bind to this particular domain. We performed a yeast
2-hybrid screen using the 22/24-kd domain as bait. Several positive
clones including NuMA, eIF3-p44, Sec 14-like protein, and 26S
proteasome subunit p55 were isolated. One of these clones, encoding
eIF3-p44, a subunit of eukaryotic translation initiation factor 3 (eIF3),30,31 was further analyzed. eIF3 is a large
translation initiation complex that contains at least 10 subunits. It
plays a central role in the binding of the initiator methionyl-tRNA
and mRNA to the 40S ribosomal subunit to form the 40S initiation
complex.32 In this study, we show that 4.1R associates with
eIF3-p44 both in vitro and in vivo, which suggests possible
interactions between the cytoskeleton network and the translation apparatus.
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Materials and methods |
Yeast 2-hybrid screen
Yeast 2-hybrid screening was conducted using the yeast strain Y190,
which contains the HIS3 and lacZ reporter genes
required for GAL4-dependent transcriptional activation. The carboxyl
22/24-kd domain (residues 462-641) of 4.1R (4.1R-22) was subcloned in
frame to the GAL4 DNA binding domain (GAL4-BD) in the pAS2-1 vector (Clontech, Palo Alto, CA). This construct was used as bait to screen
human lymphocyte and testis cDNA libraries (Clontech) fused to a GAL4
activation domain (GAL4-AD) in the pACT2 vector. The 2 plasmids were
then cotransformed into Y190 yeast. The transformants were selected on
SD minimal medium containing 25 mmol/L
3-amino-1,2,4-triazole, but without the addition of leucine
( Leu), tryptophan (Trp), and histidine (His), as
described by the manufacturer (Clontech). Positive colonies (ie, Leu+,
Trp+, His+) were further tested for  -galactosidase
activity using colony-lift and liquid assays, as described by the
manufacturer (Clontech).
To identify the minimum regions of 4.1R and eIF3-p44 that bind to each
other, constructs containing various portions of 4.1R were subcloned
into the pAS2-1 vector (Figure 1A), and
different eIF3-p44 constructs (Figure 1B) were inserted into the pACT2
vector. Yeast cells (Y187) were simultaneously transformed with the 2 constructs and assayed for -galactosidase activity.
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| Fig 1.
Interaction between regions of 4.1R and eIF3-p44 in the
yeast 2-hybrid system.
The eIF3-p44 clone (p44-1) was first isolated by a yeast
2-hybrid screen from a human lymphocyte cDNA library using the 22/24-kd
domain of 4.1R (4.1R-22) as bait. (A) Schematic diagram of the various
portions of 4.1R that interact with eIF3-p44 in a yeast 2-hybrid
screen. The constructs containing various portions of 4.1R fused to the
DNA binding domain of Gal4 (Gal4-BD) were cotransformed with an
eIF3-p44/pACT2 clone (p44-1) that expressed eIF3-p44 (residues 54-321)
fused to the activation domain of Gal4 (Gal4-AD). (B) Schematic diagram
of various portions of eIF3-p44 that interact with 4.1R. A series of
eIF3-p44 deletion mutants, fused to Gal-AD in the pACT2 vector, were
cotransformed with 4.1R-22/pAS2-1 in Y187 cells. +,
expression of the lacZ reporter gene using the colony-lift
assay; , nonexpression of the reporter gene. Right-hand column
represents results of the liquid assay for -galactosidase
activity.
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Northern blot analysis and cDNA library screening
A blot filter containing 2 µg poly (A)+ RNA derived
from multiple human tissues was purchased from Clontech. The blot was
hybridized to a cDNA probe derived from the 0.9-kb fragment of
eIF3-p44 corresponding to amino acid residues 54-321 (eIF3-p44-1) under previously described conditions.5 The
same blot was stripped and reprobed with human -actin cDNA to
quantify RNA loading.
To obtain the full-length eIF3-p44 cDNA, the same probe was
used to screen a human testis cDNA library (Clontech). The conditions for screening and isolation were previously described.5
Plasmids and antibodies
eIF3-p44-1 (residues 54-321), originally identified from a yeast
2-hybrid screen (Figure 1B), and eIF3-p44 (residues 1-321), a
full-length eIF3-p44 cDNA isolated from a cDNA library, were used as
templates to generate GST/eIF3-p44-1 and GST/eIF3-p44 constructs,
respectively. The cDNA fragments spanning the coding regions of
eIF3-p44-1 and eIF3-p44 were polymerase chain reaction-amplified and
fused in frame to GST in the pGEX-2T expression vector (Pharmacia, Uppsala, Sweden). GST/eIF3-p44-1 and GST/eIF3-p44 fusion proteins were
generated and purified on glutathione-agarose beads (Sigma, St. Louis,
MO) as previously described.23 GST/eIF3-p44-1 fusion protein was used to produce polyclonal antibodies, and GST/eIF3-p44 fusion protein was used for the in vitro binding assay. Polyclonal antibodies against eIF3-p44 were raised in rabbits and in mice, as
previously described.33 Briefly, eIF3-p44-1 fusion protein was mixed with complete Freund's adjuvant (Sigma) and injected subcutaneously into New Zealand White rabbits or ICR mice. After 4 weeks, the animals were boosted with GST/eIF3-p44-1 antigen mixed with
incomplete Freund's adjuvant (Sigma). Two weeks later, sera were
collected and precipitated with ammonium sulfate. The precipitated IgG
fractions were dialyzed against 0.1 mol/L phosphate-buffered saline
(PBS), pH 8.0.
The cDNA fragments encoding the N-terminal headpiece domain (residues
55-198) of the 135-kd 4.1R and the C-terminal 22/24-kd domain (residues
462-641) of the 80-kd 4.1R isoform were individually subcloned into the
pGEX-2T vector. The recombinant proteins were expressed in E. coli and purified on glutathione-agarose beads as previously
described.23 The immunization and generation of hybridomas
(anti-N-4.1R antibody) against the N-terminal headpiece of the 135-kd
4.1R isoform were performed as previously described.33 Polyclonal anti-C-4.1R antibodies against the C-terminal 22/24-kd domain were raised in rabbits and mice as described above. Anti-FLAG monoclonal antibody (mAb) was purchased from Sigma, and anti--actin mAb was purchased from Boehringer (Indianapolis, IN).
In vitro binding assay
The cDNA encoding an 80-kd 4.1R isoform was constructed in the pSG5
vector (Stratagene, La Jolla, CA). Synthetic sense-capped mRNA was
generated from the T7 promoter within the vector. Sense mRNA was
translated in a coupled in vitro transcription/translation system (TNT
rabbit reticulocyte lysate; Promega, Madison, WI) in the presence of
35S-methionine to radiolabel newly synthesized proteins.
Equal amounts of the labeled 80-kd 4.1R protein were incubated with
affinity-purified GST or GST/eIF3-p44 fusion proteins previously
immobilized on glutathione-agarose beads as described.23 After incubation, the immobilized complexes were washed 3 times with
bead binding buffer (50 mmol/L potassium phosphate, pH 7.5, 150 mmol/L
KCl, 1 mmol/L MgCl2, 10% glycerol, 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride). Bound protein complexes were
analyzed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by
autoradiography.23
Cell culture, transfection, and Western blot analysis
Molt-4 cells (a human lymphoid cell line) were maintained in
RPMI-1640 medium, as previously described.34 SiHa cells (a human cervical carcinoma cell line) were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The cDNA encoding human eIF3-p44-1 (residues 54-321) was inserted in frame into
a cytomegalovirus promoter-driven FLAG epitope-tagged expression vector
(Kodak, New Haven, CT), designated FLAG-eIF3-p44-1. SiHa cells
(1 × 106) were transiently transfected with or
without 10 µg FLAG-eIF3-p44-1 cDNA, as previously
described.23
Twenty-four hours after transfection, cells were washed in PBS and
lysed in 1 mL ice-cold EBC buffer (50 mmol/L Tris-HCl, pH 8.0, 120 mmol/L NaCl, 0.5% NP-40, 1 µg aprotinin, 1 µg leupeptin, and 2 mmol/L phenylmethylsulfonyl fluoride) for 30 minutes on ice, and the lysate was cleared by centrifugation.
Solubilized proteins (10 µg) were separated on a 10% SDS-PAGE,
blotted onto a polyvinylidene difluoride membrane, and probed with
anti-eIF3-p44 rabbit polyclonal antibody, anti-FLAG mAb, or
anti--actin mAb. Secondary antibodies were goat antirabbit or goat
antimouse IgG conjugated with horseradish peroxidase (Promega).
Immunoreactive polypeptides were then detected by the Western
Exposure Chemiluminescent Detection System (Pierce, Rockford, IL) as
previously described.23
Coimmunoprecipitation assay
Coimmunoprecipitation of 4.1R and eIF3-p44 was performed using
Molt-4 cell extracts. Cells were lysed in EBC buffer, and the lysate
was centrifuged at 10 000g for 10 minutes at 4°C. The
supernatant (approximately 5 mg) was precleared with protein
A-Sepharose beads (Sigma), immunoprecipitated with preimmune serum
(approximately 40 µg) or anti-eIF3-p44 antibody (approximately 40 µg) overnight at 4°C, and incubated with protein A-Sepharose
beads for another 2 hours. Immunoprecipitates were collected by
centrifugation at 5000g for 5 minutes at 4°C and washed 3 times with EBC buffer. Samples were resuspended in 20 µL SDS sample
buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, 5% -mercaptoethanol,
and 10 µg/mL bromophenol blue) and were boiled at 95°C for 5 minutes. The samples were then centrifuged at 10,000g for 10 minutes at 4°C, and the supernatants were separated on 7.5%
SDS-PAGE. After transfer to a polyvinylidene difluoride membrane, the
immunoreactive proteins were detected by anti-N-4.1R or anti-C-4.1R
antibodies, as described above. For reverse immunoprecipitation, the
cell lysates were immunoprecipitated with anti-C-4.1R antibodies and
analyzed by immunoblotting with anti-eIF3-p44 antibodies.
In vitro depletion assay
To deplete eIF3-p44, rabbit reticulocyte lysates (12.5 µL) were
preincubated for 2 hours at 4°C with preimmune serum (2 µg), anti-eIF3-p44 antibody (2 µg) previously coupled to protein
A-Sepharose beads, immobilized GST (10 µg), or GST/4.1R-80 fusion
proteins (10 µg) previously conjugated to glutathione-agarose beads.
Samples were centrifuged at 5000g for 5 minutes at
4°C to remove the bound complexes, and the supernatant
was then assayed for in vitro translation activity using the
luciferase gene as the template. The
35S-methionine-labeled luciferase proteins were analyzed
on 10% SDS-PAGE and visualized by autoradiography. To test translation efficiency in the presence of GST/4.1R-80 fusion protein, the indicated
amounts (0.25 to approximately 2 µg) of soluble GST/4.1R-80 fusion
protein or GST protein (2 µg) were mixed with rabbit reticulocyte lysate for 2 hours at 4°C. Samples were examined for
in vitro translation activity as described above.
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Results |
4.1R and eIF3-p44 interact in a yeast 2-hybrid assay
We previously showed that pICln (a protein involved in cellular
volume regulation) binds to the 30-kd domain of 4.1R in a yeast
2-hybrid system.23 Using a similar approach, we set out to
identify proteins that interact with the 22/24-kd domain of 4.1R
(4.1R-22). The cDNA encoding the 22/24-kd domain, fused in frame to the
Gal4 DNA-binding domain (Gal4-BD), was used as bait to screen human
lymphocyte and testis cDNA libraries fused to the Gal4 activation
domain (Gal4-AD). We screened approximately 3 × 106
transformants, and 63 positive clones were obtained.
A search of the GenBank database revealed that at least 4 known genes
(NuMA, eIF3-p44, Sec 14-like protein, and 26S proteasome subunit p55)
were found from the screen. Among these known proteins, we further
examined NuMA and eIF3-p44. NuMA is a nuclear mitotic apparatus protein
that had previously been isolated in a yeast 2-hybrid assay using the
full-length 135-kd 4.1R as bait.29 This clone was also
independently isolated 17 times in our screen. The other known
gene that interacts with the 22/24-kd domain of 4.1R (residues 462-641;
Figure 1A) is eIF3-p44. The interaction of 4.1R and
eIF3-p44 in the yeast 2-hybrid assay appears to be specific:
eIF3-p44-1/pACT2 did not bind to the empty vector pAS2-1 containing only unfused Gal4-BD (Figure 1A). The 4.1R-22/pAS2-1 also
did not interact with the unfused Gal4-AD vector, pACT2 (Figure 1B). It
has been suggested that mRNA and the translation apparatus are
associated with the cytoskeleton.35,36 Our finding raises the interesting possibility that 4.1R may act as a linker between the
translation apparatus and the cytoskeleton.
Cloning and Northern blot analysis of eIF3-p44
Using yeast 2-hybrid screening, we isolated a cDNA clone (p44-1;
Figure 1B) encoding a eukaryotic translation initiation factor 3 subunit (eIF3-p44).30,31 Clone p44-1 represents part of the coding sequence and the 3'-untranslated region (UTR) of eIF3-p44, including the stop codon TAA followed by the polyadenylation signal AATAAA. Northern blot analysis revealed that the eIF3-p44
transcript (1.35 kb) was detectable in all tissues examined and was
predominantly expressed in testis, heart, pancreas, and skeletal
muscle (Figure 2). To obtain the cDNA
that covers the entire coding region of eIF3-p44, we screened a
human testis cDNA library using eIF3-p44-1 cDNA as a probe.
Several full-length clones were isolated, and their sequences were
confirmed on both strands.
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| Fig 2.
Northern blot analysis of eIF3-p44 in various human
tissues.
Blot filters containing 2 µg poly (A)+RNA were hybridized
with a 32P-labeled cDNA fragment (0.9 kb) encoding amino
acid residues 54-321 of human eIF3-p44. The same blot was stripped and
reprobed with -actin to quantify RNA loading. eIF3-p44 mRNA
is abundantly expressed in human tissues including testis, skeletal
muscle, pancreas, and heart.
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Interaction between 4.1R and eIF3-p44 occurs through the 22/24-kd
domain of 4.1R and amino acids 54-321 of eIF3-p44
To determine the region of 4.1R that interacts with eIF3-p44,
constructs containing different regions of 4.1R in the pAS2-1 vector
and eIF3-p44-1 in the pACT2 vector were cotransformed into the yeast
Y187 strain and plated on SD medium lacking tryptophan and leucine.
After incubation for 2 days, the colonies were subjected to a liquid
assay for -galactosidase activity. Figure 1, panel A shows that
eIF3-p44-1 interacted with 4.1R-80 (80 kd), 4.1R-32 (10 kd + 22/24 kd),
4.1R-22 (22/24 kd), and 4.1R-2 (residues 525-622), but not with 4.1R-30
(30 kd), 4.1R-1 (residues 462-531), 4.1R-3 (residues 608-641), or the
pAS2-1 vector alone. Similar results were obtained using the
colony-lift assay (data not shown) to examine for positive
interactions. Thus, the 98 amino acids of 4.1R derived from the
22/24-kd domain (4.1R-2) are required and sufficient for interaction
with eIF3-p44.
To map the region of eIF3-p44 that binds to 4.1R, we expressed
different domains of eIF3-p44/pACT2 clones and cotransformed them into
Y187 with 4.1R-22/pAS2-1. As shown in Figure 1B, only p44 (encoding the
entire coding region of eIF3-p44) and p44-1 (residues 54-321) showed
substantial binding to 4.1R-22, whereas p44-2 (residues 54-185)
revealed a weak interaction. In contrast, peptide segments of eIF3-p44
encompassing amino acids 185-321 (p44-3) or 231-321 (p44-4), which
contain the RNA recognition motif, did not interact with 4.1R-22.
Therefore, we believe that the entire structure of eIF3-p44,
particularly the region spanning amino acids 54-321, are necessary and
important for the maintenance of the correct conformation for eIF3-p44
binding to 4.1R. Taken together, these results indicate that the
interacting regions of these 2 proteins are restricted to the amino
acids residues 525-622 of 4.1R and 54-321 of eIF3-p44.
4.1R interacts with eIF3-p44 in vitro
The direct interaction between 4.1R and eIF3-p44 was further
analyzed using an in vitro binding assay. The full-length
eIF3-p44 cDNA was constructed in frame into the pGEX2T vector.
The GST/eIF3-p44 fusion protein was expressed and purified with
glutathione-agarose beads (Figure 3A, lane
3). Immobilized GST/eIF3-p44 protein was incubated with
35S-methione-labeled 4.1R (80-kd isoform), and retention on
the beads was analyzed by SDS-PAGE. As shown in Figure 3B, GST/eIF3-p44 fusion protein interacted with the labeled 80-kd 4.1R (Figure 3B, lane
3). In contrast, the control GST fusion protein failed to bind to
labeled 4.1R (Figure 3B, lane 2). Consistent with our results from the
yeast 2-hybrid assays, these results further confirm that eIF3-p44
specifically interacts with 4.1R in vitro.
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| Fig 3.
In vitro binding assay indicates that eIF3-p44 binds to
4.1R.
The eIF3-p44 cDNA was subcloned into the pGEX-2T vector
and expressed as a GST/eIF3-p44 fusion protein in Escherichia
coli. (A) Purified GST (lane 2) and GST/eIF3-p44 fusion protein
(lane 3) were visualized by Coomassie blue staining. (B) Binding of
GST/eIF3-p44 to 4.1R-80 (80-kd isoform) in vitro.
35S-methionine-labeled 4.1R-80 (lane 1) was
incubated with affinity-purified GST (lane 2) or GST/eIF3-p44 fusion
protein (lane 3) previously coupled to glutathione-agarose beads.
After incubation, the bound complexes were analyzed by SDS-PAGE and
autoradiography. Radiolabeled 4.1R-80 bound to GST/eIF3-p44 fusion
protein (lane 3) but not GST alone (lane 2).
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4.1R interacts with eIF3-p44 in vivo
Polyclonal antibodies against eIF3-p44 were raised to further
characterize the interaction between 4.1R and eIF3-p44. To test the
specificity of this antibody, we transiently transfected
FLAG-eIF3-p44-1 into SiHa cells. The cell extract was prepared
24 hours after transfection and analyzed by anti-FLAG mAb or
anti-eIF3-p44 polyclonal antibodies. Figure
4 shows that FLAG-eIF3-p44-1
(residues 54-321) protein was recognized by both anti-FLAG
(Figure 4A, lane 1) and anti-eIF3-p44 (Figure 4B, lane 1) antibodies,
whereas, the endogenous eIF3-p44 in both transfected (Figure 4B, lane
1) and untransfected (Figure 4B, lane 2) SiHa and Molt-4 (Figure 4B,
lane 3) cells was only detected by anti-eIF3-p44 antibody.
Because the SiHa cells were transfected with an N-terminal-truncated
clone, FLAG-eIF3-p44-1 (residues 54 to 321), a low molecular
weight band was detected in transfected cells (Figure 4B, lane 1).
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| Fig 4.
Characterization of anti-eIF3-p44 antibody.
The production of antibody against eIF3-p44 is described in
"Materials and methods." SiHa cells were transiently transfected
with a FLAG-tagged eIF3-p44-1 plasmid. Cell lysates (10 µg)
prepared from transfected cells (lane 1), untransfected cells (lane 2),
and Molt-4 cells (lane 3) were separated by SDS-PAGE, transferred to a
membrane, and immunoblotted with anti-FLAG antibody (A) or
anti-eIF3-p44 antibody (B). The same blot was reprobed with an antibody
against -actin as a control. Positions of the molecular weight
markers are shown on the left.
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To examine whether endogenous 4.1R is associated with eIF3-p44 in vivo,
we performed a co-immunoprecipitation assay. Cell lysates prepared from
Molt-4 cells were first immunoprecipitated with either preimmune serum
or anti-eIF3-p44 antibodies. Coprecipitated proteins were then detected
by anti-N-4.1R (against the headpiece 209 amino acids of the 135-kd
isoform) (Figure 5A) or anti-C-4.1R (against the C-terminal 22/24-kd domain) (Figure 5B) antibodies. As
shown in Figure 5, panels A and B (lane 2), no endogenous 135-kd or
80-kd 4.1R was detected when preimmune serum was used for the immunoprecipitation experiment. In contrast, the 135-kd isoform of 4.1R
was coprecipitated with anti-eIF3-p44 antibodies and was detected by
anti-N-4.1R antibody (Figure 5A, lane 3). Similarly, both 135-kd and
80-kd 4.1R isoforms were coprecipitated with anti-eIF3-p44 and
immunoreacted with anti-C-4.1R antibody (Figure 5B, lane 3). The in
vivo association of 4.1R and eIF3-p44 was further confirmed by reverse
immunoprecipitation (Figure 5C). Cell lysates were first
immunoprecipitated with either preimmune serum (Figure 5C, lane 2) or
anti-C-4.1R antibody (Figure 5C, lane 3), and this was followed by
immunoblotting of co-precipitated proteins with anti-eIF3-p44 antibody.
Because the heavy chains of the immunoglobulins comigrate with eIF3-p44
under reducing conditions on SDS-PAGE, immunoprecipitates were
dissolved in sample buffer lacking -mercaptoethanol to maintain the
disulfide bonds between the light and heavy chains of the
immunoglobulins. As shown in Figure 5, panel C, eIF3-p44 was pulled
down by anti-C-4.1R antibody (lane 3) but not by pre-immune serum (lane
2). Taken together, these results indicate that eIF3-p44 interacts with
4.1R in vivo.
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| Fig 5.
4.1R interacts with eIF3-p44 in vivo.
Cell lysates prepared from Molt-4 cells were immunoprecipitated with
preimmune serum (lane 2) or anti-eIF3-p44 antibody (lane 3). Bound
protein complexes were analyzed by immunoblotting with antibodies
against the N-terminal portion of 135-kd 4.1R (A, anti-N-4.1R) or
against the C-terminal 22/24-kd domain of 4.1R (B, anti-C-4.1R
antibody). Lane 1 was loaded with Molt-4 cell lysates (10 µg) and
immunoblotted with anti-N-4.1R (A), anti-C-4.1R (B), or anti-eIF3-p44
antibody (C). The 135-kd 4.1R isoform was specifically detected by
anti-N-4.1R antibody (A, lane 3), whereas 2 alternative splicing
isoforms of 4.1R (135-kd and 80-kd) were recognized by anti-C-4.1R
antibody (B, lane 3). (C) Reverse immunoprecipitation. Cell lysates
prepared from Molt-4 cells were immunoprecipitated with preimmune serum
(lane 2) or anti-C-4.1R antibody (lane 3) and analyzed by
immunoblotting with anti-eIF3-p44 antibody. Lane 1 represents a
positive control in which Molt-4 cell extracts were immunoblotted with
anti-eIF3-p44 antibody. Samples were dissolved in SDS sample buffer
containing -mercaptoethanol (A, B) or lacking -mercaptoethanol
(C, lanes 2 and 3).
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Cell-free translation system deficient in eIF3-p44 was
unable to synthesize proteins efficiently
To verify that eIF3-p44 plays a role in protein
biosynthesis,30,31 we attempted to deplete eIF3-p44
activity in reticulocyte lysates using anti-eIF3-p44 antibodies (Figure
6) or GST/4.1R-80 fusion protein (Figure
7). As seen in Figure 6, eIF3-p44 was
completely removed from the reticulocyte lysates using immobilized
antibodies against eIF3-p44 (Figure 6A, lane 4), and immunodepletion of
eIF3-p44 from reticulocyte lysates resulted in the loss of template
mRNA (luciferase) translation (Figure 6B, lane 4). The
preimmune serum, though it partially depleted eIF3-p44 from the
reticulocyte lysates (Figure 6A, lane 3), did not significantly inhibit
mRNA translation (Figure 6B, lane 3).
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| Fig 6.
Immunodepletion of eIF3-p44 by anti-eIF3-p44 antibody
results in the loss of mRNA translation.
Rabbit reticulocyte lysates were incubated without (lane 1)
or with protein A-Sepharose beads that had been preincubated with PBS
buffer (lane 2), preimmune serum (lane 3), or anti-eIF3-p44 antibody
(lane 4) as described in "Materials and methods." (A) After
centrifugation, the proteins in supernatants were separated by SDS-PAGE
and immunoblotted with anti-eIF3-p44 or anti--actin antibody.
-Actin was used as an internal control. (B) Treated lysates were
tested for in vitro translation activity using the luciferase
gene as a template. 35S-methionine-labeled luciferase was
analyzed by SDS-PAGE and autoradiography.
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| Fig 7.
Depletion of eIF3-p44 using immobilized GST/4.1R-80
fusion protein.
(A) Purified, bacterially produced GST (lane 2) and GST/4.1R-80 fusion
protein (lane 3) were visualized by Coomassie blue staining.
(B) Rabbit reticulocyte lysates were incubated without GST (lane 1),
with GST (lane 2), or with GST/4.1R-80 fusion protein (lane 3) coupled
to glutathione-agarose beads as described in "Materials and
methods." After centrifugation, the supernatants were subjected to
SDS-PAGE, and blots were probed with anti-eIF3-p44 or anti--actin
antibodies. (C) Lysates without treatment (lane 1) or pretreated with
affinity-purified GST (lane 2) or GST/4.1R-80 (lane 3) beads were
tested for in vitro translation activity as described in Figure 6B.
|
|
Similar effects were also observed when immobilized GST/4.1R-80 fusion
protein was used to deplete eIF3-p44. As seen in Figure 7, the eIF3-p44
in reticulocyte lysate was significantly removed by the addition of 10 µg GST/4.1R-80 fusion protein (Figure 7B, lane 3) immobilized on
glutathione-agarose beads, and the eIF3-p44-depleted lysate produced
substantially decreased luciferase mRNA translation (Figure 7C,
lane 3). In contrast, immobilized GST alone did not significantly
remove eIF3-p44 (Figure 7B, lane 2), and the GST-treated lysate was not
defective in mRNA translation (Figure 7C, lane 2). Furthermore, the
addition of increasing amounts (0.25 to approximately 2 µg) of
soluble GST/4.1R-80 fusion protein (Figure
8A), but not GST protein (2 µg; Figure
8C, lane 2), resulted in increasing loss of the protein translation
efficiency of luciferase mRNA (Figure 8B). Taken together,
these results suggest that eIF3-p44 can be depleted by either
anti-eIF3-p44 antibodies or its interaction protein (4.1R) and that
eIF3-p44-depleted or GST/4.1R-80 treated lysates do not efficiently
synthesize proteins.
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| Fig 8.
GST/4.1R-80-treated lysates exhibit reduced in vitro
translation activity in a dose-dependent manner.
(A) Affinity-purified GST/4.1R-80 fusion protein (0.25 to approximately
2.0 µg) was visualized by Coomassie blue staining. (B) Rabbit
reticulocyte lysates were incubated with the indicated amounts of
soluble GST/4.1R-80 fusion protein for 2 hours at 4°C
and tested for in vitro translation activity, as described in Figure
6B. (C) Rabbit reticulocyte lysates were incubated with (lane 2) or
without (lane 1) affinity-purified GST protein (2 µg) and tested
for in vitro translation activity. In vitro translation
activity was not inhibited by GST (C, lane 2) but was inhibited by the
addition of GST/4.1R-80 fusion protein in a dose-dependent manner
(B).
|
|
| |
Discussion |
We and others5,6,34 have shown that 4.1R is composed of
multiple isoforms that are heterogeneous in size and subcellular localization.10-12 Many of these isoforms are present in
nonerythroid cells3,5 or in erythroid cells at different
developmental stages.6 Their biologic significance,
however, has not yet been well characterized. In the current study we
identified eIF3-p44, a subunit of the eukaryotic initiation factor 3 (eIF3) complex, as a binding target of 4.1R. The interaction between
the carboxyl terminal domain (22/24 kd) of 4.1R and eIF3-p44 was first
discovered using the yeast 2-hybrid system and then confirmed by in
vitro binding, in vitro depletion, and coimmunoprecipitation studies.
The finding that eIF3-p44 directly interacts with a cytoskeletal
protein (4.1R) was informative. The interaction between eukaryotic translation components during the early phase of protein synthesis has
been well characterized.37 Eukaryotic translation
initiation begins with the assembly of a preinitiation complex,
including a small ribosome subunit (40S), eIF1A, eIF3, and
eIF2-GTP-tRNA. Next this complex binds to mRNA in a reaction requiring
adenosine triphosphate and the mRNA m7G-cap binding protein
complex (eIF4A, eIF4B, eIF4E, and eIF4G) and then scans toward the
3' end until it forms a stable complex at the first AUG
initiation codon. eIF4G serves as a scaffold protein for the assembly
of eIF4E and eIF4A and recruits Mnk1 to phosphorylate
eIF4E.38
eIF3 is a large, multi-subunit complex (approximately 600 kd) that
plays a central role in the initiation of translation. It was
originally isolated from rabbit reticulocyte lysates, and it contains
at least 10 different protein subunits ranging from 35 to 170 kd.32 Among these protein subunits, eIF3-p44 binds specifically to eIF3-p170 and contains a consensus RNA recognition motif near its carboxyl terminus.30 eIF3 binds to 40S
ribosomal subunits, resulting in the dissociation of 80S ribosomes.
eIF3 also stabilizes initiator methionyl-tRNA binding to 40S subunits and participates in mRNA binding through its interaction with eIF4G.32 Interestingly, 4.1R was reported to be an
actin-associated protein that binds to the spectrin-actin complex
through its 10-kd domain.26-28 Purified 4.1R also interacts
with tubulin39 and myosin.40 In the current
study, we show that eIF3-p44 binds to both 80-kd and 135-kd 4.1R
isoforms (Figure 5) and that this interaction is through the carboxyl
terminal 22/24-kd (residues 525-622) domain of 4.1R and amino acids
residues 54-321 of eIF3-p44 (Figure 1). These findings suggest that
4.1R may serve as a bridging molecule that attaches the translational
apparatus to the cytoskeletal scaffold.
Recent studies have shown that mRNA and polysomes are associated with
the cytoskeleton and that this association may influence the transport,
anchoring, and translation of mRNA.35,36 For example, the
elongation factor 1 alpha (EF-1 ) is abundantly expressed and
constitutes 1% to 2% of the total proteins in normal growing cells.
It catalyzes the GTP-dependent binding of aminoacyl-tRNA to ribosomes,
and it regulates the faithfulness and rate of polypeptide elongation
during translation.41 EF-1 is an actin-binding
protein,42 and the involvement of EF-1 in translation is
possibly regulated by its ability to dissociate from actin in response
to local environmental changes in pH. The release of EF-1 from actin
filaments would then facilitate polypeptide elongation by the
F-actin-associated translational apparatus.43
Unlike EF-1, whose involvement in translation is regulated in a
pH-dependent manner, the association of 4.1R to the cytoskeleton and
translational apparatus may be regulated by phosphorylation. It has
been reported that 4.1R promotes a high-affinity association between
spectrin and F-actin and that this association appears to be regulated
by phosphorylation. 4.1R can be phosphorylated by various kinases,
including protein kinase C and PKA.44 The phosphorylation
of 4.1R by these kinases decreases the ability of 4.1R to bind to
spectrin and inhibits the formation of the spectrin-actin-4.1R
complex.45 We observed a significant inhibition of the
protein translation activity when the reticulocyte lysates were
incubated with 4.1R previously phosphorylated by PKA (unpublished data). This observation raises the possibility that the involvement of
4.1R in the cytoskeleton and translational apparatus may be regulated
by phosphorylation. The PKA-phosphorylated amino acids of 4.1R have
recently been identified as Ser-331 in the 16-kd domain and Ser-467 in
the 10-kd spectrin-actin-binding domain.25 Awareness
of such interaction may in turn lead to understanding the
molecular mechanism of how phosphorylation regulates the
4.1R-linked cytoskeleton to the translational apparatus.
Depletion of eIF3-p44 from rabbit reticulocyte lysates using
anti-eIF3-p44 antibodies (Figure 6) or immobilized GST/4.1R-80 fusion
protein (Figure 7) resulted in a reduction of the lysates' ability to
synthesize proteins efficiently. This is because eIF3-p44-depleted lysate may lose the entire eIF3 complex or the factors essential for
protein translation. However, the preincubation of reticulocyte lysates
with affinity-purified GST/4.1R-80 fusion protein without removing
eIF3-p44 from the lysates caused a dose-dependent inhibition of mRNA
translation (Figure 8). eIF3-p44 contains an RNA recognition motif near
its C-terminus that can bind to both 18S rRNA and -globin mRNA and
that appears to be a nonspecific RNA binding protein.30 In
the current study, we found that the 22/24-kd domain (residues 525-622)
of 4.1R directly binds to eIF3-p44 (residues 54-321; Figure 1B). This
finding suggests that the direct interaction of 4.1R with eIF3-p44 may
sterically hinder the binding of eIF3-p44 to RNA. Therefore, the
addition of increasing amounts of soluble GST/4.1R-80 fusion protein to
lysates may result in a reduction of mRNA translation activity (Figure
8).
Although the functional interactions of the 30-kd and 10-kd domains of
4.1R have been well documented, the roles of the 22/24-kd domain and
the headpiece (209 amino acids) of the 135-kd 4.1R isoform remain
unclear. Recently, several 4.1R- or 4.1G-associated proteins, including
pICln,23 NuMA,29 P4.1-CAP (CPAP),46 and FKBP13,15 have been isolated using yeast 2-hybrid
screens. The different subcellular locations and uncommon features of
these 4.1R-associated proteins implies the functional diversity and complexity of 4.1R. For example, the 30-kd domain of 4.1R mediates its
binding to pICln, a protein involved in cellular volume
regulation,23 whereas the 22/24-kd domain and the headpiece
(209 amino acids) of the 135-kd isoform interact with a nuclear mitotic
apparatus protein (NuMA)29 and a centrosome protein
(CPAP),46 respectively. In the current study, we also show
that the 22/24-kd domain of 4.1R interacts with eIF3-p44, a subunit of
mammalian translation initiation factor 3. Further study of the
physiologic implications of the interaction between 4.1R and associated
proteins will eventually provide a more complete understanding of the
various functional roles of 4.1R in nonerythroid cells.
| |
Footnotes |
Submitted October 25, 1999; accepted March 9, 2000.
Supported by grant NSC88-2314-B001-013 from the National
Science Council, Republic of China, and an institutional grant from Academia Sinica, Taiwan, Republic of China.
Reprints: Tang K. Tang, Institute of Biomedical
Sciences, Academia Sinica, 128 Yen-Chiu-Yuan Road, Sec. 2, Taipei 115, Taiwan; e-mail: tktang{at}ibms.sinica.edu.tw.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction