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INTRODUCTION |
The three stages of protein synthesis are catalyzed by three
groups of proteins: initiation, elongation, and termination factors (1). Initiation is characterized by formation of a series of initiation
complexes, each catalyzed by a different subset of initiation factors.
Recruitment of mRNA to the 43 S initiation complex to form the 48 S
initiation complex involves
eIF3,1 PABP, and the eIF4
proteins. eIF3 is a 520-kDa multimer that is required for both
Met-tRNAi and mRNA binding. PABP is a 70-kDa protein
that specifically binds poly(A) and homo-oligomerizes. The eIF4 factors
consist of: eIF4A, a 46-kDa RNA helicase; eIF4B, a 70-kDa RNA-binding
and -annealing protein that stimulates eIF4A; eIF4E, a 25-kDa
cap-binding protein; and eIF4G, a group of proteins of 154-180 kDa
that form specific complexes with all of the other proteins known to be
involved in mRNA recruitment.
Proteins sharing eIF4G homology represent the products of at least
three different genes in mammalian cells (2-4). One of these has been
mapped to the chromosomal location 3q27-qter (5). In accordance with
the nomenclature system proposed for translation factors (6), the
protein product of the gene at 3q27-qter is referred to as eIF4G-1,
whereas its two homologues are eIF4G-2 (also known as p97, eIF4G2,
DAP5, and NAT1; Refs. 3, 7, and 8) and eIF4G-3 (also know as eIF4G3,
and eIF4GII; Ref. 4). Most studies reported to date have concerned
eIF4G-1.
eIF4G appears to serve as a nucleation site for co-localization of an
unusually large number of proteins involved in mRNA metabolism.
Combining data from yeast, plant, and mammalian eIF4Gs, these include
(in approximate order of binding sites on eIF4G from N to C termini):
the influenza protein NS1 (9), the cytoplasmic poly(A)-binding protein
PABP (10, 11), the rotavirus protein NSP3 (12), the decapping protein
Dcp1 (13), the cytoplasmic cap-binding protein eIF4E (14-16), the
nuclear cap-binding protein CBP80 (17, 18), the initiation factor eIF3
(14, 19), the RNA helicase eIF4A at two distinct sites (14, 20, 21),
RNA itself (22-24), the heat shock proteins hsp27 (25) and hsp70 (26),
and the eIF4E kinase Mnk1 (27-29). In some cases it has been shown
that eIF4G-1 not only binds proteins but also affects their activities
or binding of other proteins (19, 21, 30).
The mRNA recruitment step is rate-limiting for initiation under
normal cellular conditions and appears to be highly regulated (31, 32).
The best studied regulatory mechanism involving eIF4G is its cleavage
by 2A protease of entero- and rhinoviruses and L protease of
foot-and-mouth-disease virus. Upon infection of mammalian cells with
these picornaviruses, most host protein synthesis is shut off
coincident with the appearance of viral proteins (33). This is thought
to be mediated by a switch from cap-dependent to
cap-independent translation. Complexes containing eIF4G restore
cap-dependent translation in lysates of poliovirus-infected cells (34-36). eIF4G was discovered as a result of its proteolysis coincident with the loss of cap-dependent initiation during
poliovirus infection (37). eIF4G is separated into two functional
domains, an N-terminal cleavage product (cpN) that binds
eIF4E and PABP, and a C-terminal cleavage product (cpC)
that binds eIF4A and eIF3 (14, 38). Initiation of picornaviral mRNA
translation is via an internal ribosome entry site (39, 40). Cleavage
of eIF4G drastically inhibits translation of capped mRNAs in
vitro, whereas internal initiation and initiation of uncapped
mRNAs are either unaffected or even stimulated (41-43).
Multiple isoforms of eIF4G-1 are observed by SDS-PAGE, but the origin
of these is not known. However, an analysis using 2A protease cleavage,
SDS-PAGE, and antibodies directed against different domains of eIF4G
suggested that the heterogeneity was attributable to the ~50-kDa
cpN domain (38). Digestion with L protease further delimited the source of heterogeneity to the N-terminal ~30 kDa cpN1 domain (14). Multiple cDNAs for human eIF4G-1 have
also been reported. The initial cloning, based on two overlapping
cDNAs from fetal and adult brain, predicted a protein of 154 kDa
(2). Subsequent cloning revealed a cDNA corresponding to a protein with an additional 156 aa at the N terminus (44). An initiation codon
was proposed based on alignment with eIF4G-3 and lack of any further
upstream cDNA sequence. Further cloning revealed a cDNA that
was 42 nt longer (45), indicating either that multiple mRNAs exist
or that Imataka et al. (44) had not reached the 5'-end of
the same mRNA. Finally, a fourth and fifth cDNA were reported
(46). One of these independently confirmed some of the sequence
reported by Johannes and Sarnow (45) but did not extend the cDNA.
The other was collinear with the other cDNAs up to a point,
upstream of which it deviated, suggesting a splice variant. None of
these cDNAs provided a new in-frame AUG. Thus, the most upstream
AUG codon for all five eIF4G-1 cDNAs reported to date is that
originally proposed by Imataka et al. (44).
The present study was motivated by the fact that the relationship
between the multiplicity of electrophoretic forms of the protein and
the multiplicity of cDNAs is not known. Importantly, despite
speculation based on cDNA sequences, the N terminus has not been
established for any eIF4G-1 isoform. It is important to establish the
actual protein structures, since eIF4G-1 isoforms differing at the N
terminus may contain different binding sites for proteins involved in
translational control. This study identifies an EST in the public
databases as corresponding to a longer form of eIF4G-1 mRNA.
Because it predicts a new upstream, in-frame AUG, it could encode an
even longer isoform of eIF4G-1 than those predicted from previous
cDNA sequences. Mass spectrometric analysis confirmed that this was
the case and also established the structure of two other eIF4G-1 isoforms.
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EXPERIMENTAL PROCEDURES |
Materials--
Porcine trypsin was obtained from Promega
(catalog number V5111). Endoproteinase Lys-C (catalog number P3428) and
bovine 
-chymotrypsinogen A (catalog number C4879) were obtained from
Sigma. Arg-C from Clostridium histolyticum was purchased
from Worthington (catalog number LS001641). Asp-N (catalog number
1420488) and Complete, Mini, Protease Inhibitor Cocktail Tablets
(catalog number 1836153) were obtained from Roche Molecular
Biochemicals. m7GTP-Sepharose 4B was purchased from
Amersham Biosciences.
Identification of a New EST for eIF4G-1--
As noted above,
Bushell et al. (46) reported a novel cDNA related to
eIF4G-1, GenBankTM nucleotide accession number AF002815.
The hypothetical protein encoded by this cDNA was assigned
GenBankTM protein accession number AAC78442. We compared
the cDNA sequence corresponding to aa residues 
24 to +49 of this
protein2 to the est_human
database using the program blastn found under the NCBI BLAST suite of
programs. We obtained a new EST derived from an adult melanoma:
GenBankTM nucleotide accession number AL120751. The 215-nt
segment from nt 25-240 of AL120751 exactly matched the 215-nt segment
from nt 4-219 of AF002815.
The sequence reported for AL120751 covered only the most 5'-terminal
627 nt, even though the complete eIF4G-1 cDNA is predicted to
contain 5510 nt (2, 44, 45). We obtained the plasmid corresponding to
this EST, termed Homo sapiens cDNA clone DKFZp762O191, from the German Genome Project and determined additional DNA sequence information using the DNA sequencing facility at Iowa State University. The 3'-end of AL120751 (nt 4647-5306, using a composite numbering system)3 was determined using
an oligo(dT) 3'-A primer. A 3'-terminal poly(A) tract of 111 nt was
observed using a primer corresponding to the SP6 promoter in the
vector. A sequence covering nt 439-1155 was obtained using the sense
primer 5'-AACACGCCTTCTCAGCCCCGC-3'. This corrected an entry of "N"
at nt 456 to G in the GenBank record of AL120751. A sequence covering
nt 710-1399 was obtained using the antisense primer
5'-GGGGCAAGCTGGGGGAGGAGC-3'. This sequence exactly matched nt 328-1018
of cDNA AF104913, which encodes protein AAC82471. Finally,
sequences covering nt 1-300 and 97-793 were obtained using the sense
primer 5'-CGCCACGGCCGAAGCAGCTAG-3' and antisense primer
5'-AACACGCCTTCTCAGCCCCGC-3', respectively. Overall, this extended the
sequence information for cDNA clone DKFZp762O191 by 1544 nt.
Generation of K562 Cell Lysates--
Human K562 cells were grown
in RPMI medium (Invitrogen) containing 10% fetal bovine serum
(Atlanta Biologicals, Norcross, GA). Cultures were grown to confluence
in 175-cm2 tissue culture flasks maintained in a
humidified, 5% CO2 environment at 37 °C. A standard
preparation of lysate was derived from 3.2 × 109
cells (16 T-175 flasks). Cells were resuspended on ice for 30 min in an
equal volume of Buffer A (1% Triton X-100, 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 tablet of
protease inhibitor mixture per 20 ml buffer) and then
centrifuged at 25,000 × g for 20 min. The supernatant
was frozen in liquid N2 and stored at 80 °C.
Immunological Procedures--
Two antibodies against different
regions of eIF4G-1 were
used.4 Anti-Peptide 7 antibodies were obtained as described previously (2). Anti-Peptide 10 antibodies were produced and affinity purified using the peptide
CRAQPPSSAASR, which corresponds to aa residues 55-65 of the consensus
eIF4G-1 sequence2 with an added N-terminal Cys residue, as
described previously (2). Immunoblotting was carried out as described
previously (47). Incubation with the anti-Peptide 7 antibodies (1:1000) was carried out at room temperature for 1 h. Incubation with
affinity-purified anti-Peptide 10 antibodies (1:50) was at 4 °C
overnight. Both antibodies were visualized with alkaline
phosphatase-conjugated goat anti-rabbit IgG antibodies (Vector
Laboratories, Burlingame, CA) at 1:1000.
Purification of cpN--
Recombinant 2A protease
from Coxsackievirus serotype B4 was prepared as described previously
(41). K562 cell lysates were cleared after thawing by spinning at
25,000 × g for 20 min at 4 °C. The supernatant was
incubated with recombinant 2A protease at a final concentration of
50-100 µg/ml for 30 min on ice. (As 2A protease preparations
differed somewhat in activity, the amount was adjusted to produce
complete cleavage of eIF4G-1, based on western blotting with
anti-Peptide 7 antibodies.) The 2A protease-treated lysate was then
subjected to m7GTP-Sepharose affinity chromatography as
described previously (48) but with the following modifications. One
standard batch of K562 lysate was gently rotated with 2 ml of
m7GTP-Sepharose for 1 h at 4 °C. The slurry was
then transferred to a column and the flow-through fraction was
reapplied to the column. The column was washed first with 12 volumes of
Buffer B55 (20 mM MOPS, pH 7.6, 10% (w/v)
glycerol, 0.5 mM EDTA, 0.25 mM dithiothreitol,
25 mM NaF, 55 mM KCl) and then with 3 volumes of 100 µM GTP in Buffer B55. Proteins were
eluted with 4 volumes of 200 µM m7GTP in
Buffer B55. The eluate was frozen in liquid N2
and stored at 80 °C.
Electrophoretic Separation of cpN
Isoforms--
Linear polyacrylamide was prepared (49) and added to
each cpN sample at a final concentration of 120 µg/ml as
carrier. Ice-cold 100% trichloroacetic acid was added to a final
concentration of 10% and the sample allowed to stand on ice for 30 min. The precipitate was collected by centrifugation at 25,000 × g for 20 min, washed four times with ice-cold 80% aqueous
acetone, and dissolved in 1× SDS-loading buffer (adjusted to pH 8.2;
Ref. 50). Alkylation of Cys residues was performed as described
previously (50). The sample was then adjusted to pH 6.8 with HCl, and
the cpN isoforms were separated by electrophoresis on a
16 × 20-cm gradient gel (8-15%;
acrylamide:N,N'-bisacrylamide, 30:0.8) with a 4%
stacking gel. Electrophoresis was carried out a constant current of 16 mA for 4 h followed by 24 mA for 16-20 h. Protein bands were
stained with Coomassie Blue, excised, and stored at 80 °C.
Protease Digestion--
Gel pieces were minced and further
destained with three washes (400 µl each) of 50% acetonitrile, 25 mM ammonium bicarbonate, pH 8.0. The polyacrylamide was
dehydrated for 5 min with 100% acetonitrile followed by vacuum
centrifugation in a Savant Speedvac for 30 min. In-gel digestion with
trypsin was performed overnight at 37 °C by the addition of 15 µl
of enzyme (10 µg/ml) in 25 mM NH4HCO3, pH 8.0, to each gel piece. For Lys-C
digestion, a stock enzyme solution of 50 µg/ml was made in 0.1 M Tris-HCl, pH. 8.0, and 15 µl were added to each gel
piece. Digestion was performed overnight at 37 °C. For Arg-C
digestion, the enzyme was reconstituted at 2 mg/ml in 1 mM
CaCl2, 2.5 mM dithiothreitol. In-gel digestion was carried out at room temperature overnight in 15 µl of 50 mM sodium phosphate, pH 7.6, 2.5 mM
dithiothreitol by the addition of 3 µg enzyme per gel slice. Finally,
for Asp-N digestion, the enzyme was reconstituted in H2O to
give a concentration of 40 µg/ml in 10 mM Tris, pH 7.5. In-gel digestion was carried out overnight at 37 °C in 15 µl of 50 mM sodium phosphate, pH 8.0, by the addition of 4 µl of
enzyme per gel slice.
Mass Spectrometry--
Mass spectrometric analysis was performed
at both the LSUHSC-S Research Core Facility or the Laboratory
for Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller
University. At LSUHSC-S, MALDI-TOF-MS was performed on a PerSeptive
Biosystems Voyager-DE PRO Biospectrometry work station. At Rockefeller
University, two instruments were used. Peptide mapping by MALDI-TOF-MS
was performed on a PerSeptive Biosystems Voyager-DE STR Biospectrometry
Work station. Sequence information was obtained by LC-ESI-MS/MS on a
Finnigan LCQ-DECA ion trap mass spectrometer.
Peptides were prepared for MALDI-TOF-MS after proteolytic digestion by
extraction from gel pieces twice with 50-µl portions of 50%
acetonitrile, 5.0% trifluoroacetic acid. Peptides in the extract were dried, dissolved in 15 µl of 0.1% trifluoroacetic acid,
and purified on a ZipTip (Millipore). They were eluted with 2 µl of
the appropriate organic acid (matrix) dissolved in 50% acetonitrile,
0.1% trifluoroacetic acid, and spotted on a MALDI plate. For masses in
the 800-6000 Da range, the matrix was a 0.01 mg/ml solution of
-cyano-4-hydroxy-trans-cinnamic acid (Sigma, catalog
number C-2020). For masses in the 6000-26,000 Da range, the matrix was
a 0.01 mg/ml solution of sinapinic acid
(3,5-dimethoxy-4-hydroxy-trans-cinnamic acid; Aldrich,
catalog number D13,460-0). When internal calibration was used, the
eluting solution also included peptide mass standards. Data were summed
over 50-100 acquisitions in delayed extraction mode.
At LSUHSC-S, data analysis was performed using the Data Explorer
software, version 3.5-4.0. At Rockefeller University, analysis was
performed using the software program M over Z from Proteometrics, LLC.
In both cases, spectra were subjected to algorithms for base-line correction and noise removal at two S.D. values. Throughout the current
report, monoisotopic masses are reported for <2500-Da peptides,
whereas average masses are reported for >2500-Da peptides. Theoretical
masses were determined using the Peptide Mass tool at the ExPASy
Proteomics website (ca.expasy.org/tools/). Peaks seen in both
the sample of interest and also in a blank gel treated identically were
eliminated from further consideration. For automatic matching of
observed to predicted peptide masses, we used Auto-MS Fit, version
1.2.18 (PerSeptive Biosystems). For manual matching, we considered that
peptides matched if their masses were within 1 Da for the range
800-10,000 Da. Because of unresolved microheterogeneity, caused by Met
oxidation for example, peptides with molecular masses above 10,000 Da
were considered to be matches if the experimentally determined masses
were within 0.6% of the calculated values.
LC-ESI-MS/MS analysis was carried out using a Smart System (Amersham
Biosciences) equipped with 10-ml syringe pumps and a pre-column
flow splitter (Michrom Bioresources, Auburn, CA). The chromatographic
eluate was monitored by on-line mass spectrometry using an electrospray
ion trap mass spectrometer, model LCQ-DECA (Finnigan
ThermoQuest, San Jose, CA). Peptide mixtures were diluted 10-fold with
0.01% trifluoroacetic acid (v/v) in water/methanol/acetic acid
(949:50:1, v/v/v) and loaded on a reverse phase Magic C18 column
(50 × 0.2 mm inside diameter; pore size, 100 Å; particle size, 5 µm) from Michrom Bioresources (Auburn, CA). Peptide
separation was performed at room temperature with a fast-rising
methanol gradient (in 5 min) at a flow rate of 2.8 µl/min. The eluate
was transferred through a 50-µm inside diameter fused silica
capillary from the column to the ion source of the mass spectrometer
and electrosprayed at 2.8-3.2 kV. The transport capillary in the mass spectrometer was kept at 130-150 °C in order to assist desolvation.
| |
RESULTS |
Identification of a New EST for eIF4G-1--
As noted above,
several cDNAs for eIF4G-1 have been reported (2, 44-46). Four of
the polypeptides theoretically encoded, NP_004944, AAC82471, AAC78443,
and eIF4G-Iext, are collinear, but one of them, AAC78442,
deviates from the others upstream of aa 50 (Fig.
1). In some cases, the polypeptide
sequences deposited in GenBankTM represent those encoded by
the longest open reading frame following an AUG codon
(NP_004944, AAC82471), but in other cases, the polypeptide sequence
continues in the N-terminal direction despite the absence of an AUG
codon (AAC78442, AAC78443, eIF4G-Iext). We used sequence
information from the cDNA with the longest open reading frame
(GenBankTM nucleotide accession number AF002815, which
encodes GenBankTM protein accession number AAC78442; see
Fig. 1) to search for additional ESTs. The result was a new EST entered
in GenBankTM under nucleotide accession number AL120751
(Fig. 1). The EST sequence reported (627 nt) was not previously
identified as corresponding to eIF4G-1 mRNA, but a comparison with
other eIF4G-1 cDNAs indicated an identical sequence over 215 nt.
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Fig. 1.
Alignment of polypeptide sequences derived by
theoretical translation of cDNAs and an EST corresponding to
eIF4G-1. Protein ID generally refers to the GenBankTM
protein accession numbers. One exception is AL120751, which is the
GenBankTM nucleotide accession number for the sequence of
an EST. The sequence shown is the conceptual translation product in the
+2 reading frame. The other exception is eIF4G-Iext, which
refers to the sequence published in Ref. 45. Met residues are
shaded. The underlined aa residues (55-65)
indicate the epitope used to generate anti-Peptide 10 antibodies. Names
for hypothetical forms of eIF4G-1 appear below potential N-terminal Met
residues. AAC78442, AAC78443, and AL120751 do not continue toward the C
terminus, because they are derived from only partial cDNA
sequences. The aa residue 61 shown for AL120751 differs from the
GenBankTM entry based on sequencing reported in the present
work (i.e., ANT  AGT, encoding Ser). The aa residue 214 shown for NP_004944 differs from the GenBankTM record
because of resequencing (52, 67) of the cDNA (AGT GGT, encoding
Gly instead of Ser).
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We obtained the plasmid corresponding to this GenBankTM
entry from the German Genome Project. Sequencing portions of the
plasmid insert from both the 5' and 3' termini revealed: (i) agreement over the entire EST sequence for AL120751 except for a single nucleotide sequencing ambiguity at nt 456; (ii) preservation of reading
frame between the 3'-end of the EST and other eIF4G-1 cDNA
sequences; (iii) the presence of a 111-nt poly(A) tract; (iv)
collinearity with the cDNAs encoding AAC82471, AAC78443, and
eIF4G-Iext (Fig. 1); and (v) partial collinearity with the cDNAs encoding NP_004944 and AAC78442 (see "Experimental Procedures"). If the new cDNA is collinear with these cDNAs
throughout its entire length, it corresponds to an mRNA of 5510 nt.3
Surprisingly, the polypeptide encoded by AL120751 deviates from the
AAC78442 polypeptide upstream of aa 50 (Fig. 1), even though sequences
from the cDNA encoding it, AF002815, were used to find AL120751.
Further comparison of AL120751, AF002815, and the corresponding human
genomic sequence (GenBankTM nucleotide accession number
AC078797.8; gi number 15887175; nt 122674-133235) indicates that
AF002815 lacks two exons that are present in AL120751 (data not shown).
The most 5' exon, however, is common to AF002815 and AL120751. The
absence of these two internal exons from AF002815 results in a
different reading frame upstream of aa 50. On this basis, we suggest
that AF002815 represents a splice variant that lacks internal exons present in the AL120751 cDNA.
The new polypeptide encoded by AL120751 is collinear with AAC82471,
AAC78443, eIF4G-Iext, and NP_004944, except for the Arg in
the first position of AAC78443 (aa 30 using the common numbering
system2), which is Pro in eIF4G-Iext and
AL120751 (Fig. 1). We assume this difference is due to an incomplete
codon at the junction between the AAC78443 cDNA and the vector or a
sequencing error. AL120751 predicts the longest eIF4G-1 protein
reported to date, adding 22 aa residues to the eIF4G-Iext
sequence. Furthermore, it provides a new potential translational
initiation site. Upstream of the AUG that encodes Met-1 of AL120751 are
both in-frame and out-of-frame stop codons. These features reduce the
likelihood that a start codon even further upstream could be utilized.
Immunological Analysis of eIF4G-1 Electrophoretic Variants--
To
determine whether any of the multiple bands of eIF4G-1 typically seen
by SDS-PAGE contain N-terminal sequences predicted by these cDNAs,
we developed an anti-peptide antibody (anti-Peptide 10) against aa
55-65 (underlined in Fig. 1).2,4 Both
full-length eIF4G and cpN were separated by analytical
gradient gel SDS-PAGE (Fig. 2). By silver
staining, three partially resolved, major bands are observed at
apparent molecular mass 
220 kDa in full-length eIF4G-1, as
well as numerous minor bands (lane 2). Three major and
several minor bands are also present in cpN (lane
1). Based on prior studies using various antibodies (14, 38, 51,
52), these major bands in the cpN preparation are the
N-terminal domains of the major proteins in the eIF4G preparation.
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Fig. 2.
Immunological analysis of full-length eIF4G-1
and cpN. Total protein from K562 cells was subjected
to m7GTP-Sepharose affinity chromatography followed by
analytical gradient gel SDS-PAGE. Prior to chromatography the lysate
was either untreated (lanes 2, 4, and
6) or treated with 2A protease (lanes 1,
3, and 5). Protein was detected by silver
staining (lanes 1 and 2) immunoblotting with
anti-peptide 7 (lanes 3 and 4) or anti-peptide 10 (lanes 5 and 6) antibodies.
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Some of the major bands in full-length eIF4G-1 detected by silver
staining also reacted with the highly sensitive anti-Peptide 7 antibodies (lane 4), which were developed against eIF4G-1 aa residues 523-538 (2). As with the silver-stained preparation, the
~220-kDa cluster of proteins (lane 4) is not present in
the cpN preparation (lane 3). Instead,
cpN contains three major and several minor bands that are
reactive with anti-Peptide 7 antibodies and migrate the same as Bands
1-3 of the silver-stained gel (cf. lanes 3 and
1). Most of the bands not reacting with this
antibody are identified below by mass spectrometry. By contrast, with
the new anti-Peptide 10 antibody, only the two slowest migrating forms were detected, whether in full-length eIF4G (lane 6) or
cpN (lane 5). This suggests that only the
two slowest forms contain aa 55-65 (Fig. 1).
Separation of cpN Isoforms--
The yield of eIF4G on
m7GTP-Sepharose affinity chromatography, as measured with a
monoclonal antibody, is higher if the eIF4G is first cleaved by
poliovirus infection (36). Furthermore, as shown in Fig. 2,
cpN isoforms were separated better by electrophoresis than
isoforms of full-length eIF4G. We therefore chose to separate isoforms
of cpN rather than intact eIF4G. We attempted
two-dimensional gel electrophoresis of cpN, but the bands
were extremely broad and failed to focus in the first dimension. This
behavior has been previously observed (53). Instead, we obtained
optimal resolution of cpN by one-dimensional SDS-PAGE on
preparative (16 × 20 cm) gradient gels.
Strategy for Assigning eIF4G-1 Isoforms to Bands--
Application
of anti-Peptide 7 and anti-Peptide 10 antibodies provided only limited
information about the structures of the polypeptides making up the
various electrophoretic bands. Initially, we attempted to identify them
by N-terminal sequence analysis. The various cpN isoforms
were separated by SDS-PAGE, blotted onto a polyvinylidene difluoride
membrane, and analyzed by Edman degradation at the Macromolecular
Structure Analysis Facility of the University of Kentucky. However,
this failed to yield interpretable data, presumably due to blocked N termini.
We therefore turned to sequence information provided by cDNAs and
ESTs. Five of the cDNA sequences (AAC82471, AL120751, AAC78443,
eIF4G-Iext, and NP_004944) are theoretically translated
into collinear polypeptide sequences. Due to the existence of multiple
AUG codons, six eIF4G-1 isoforms can be predicted that correspond to
alternative translational initation sites (Fig. 1). We termed these
hypothetical isoforms eIF4G-1a, -1b, -1c, -1d, -1e, and -1f (Table
I). The proteolytic peptides of
each eIF4G-1 isoform can be predicted from the composite cDNA
sequence (Fig. 3). It is also possible to
determine the masses of peptides actually present in proteolytic
digests from the various electrophoretic bands by MALDI-TOF-MS (54).
Putting information on theoretical and actual peptides together, we can
deduce which hypothetical isoforms are consistent with the peptide
pattern of each electrophoretic band. The masses of "diagnostic"
peptides, i.e. those that are unique to the various
hypothetical eIF4G-1 isoforms, are shown in Table
II.
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Fig. 3.
Predicted peptides from digestion of
hypothetical eIF4G-1 isoforms with trypsin, Arg-C, Lys-C, and
Asp-N. Peptides shown cover the cpN region of eIF4G-1
only. Tryptic peptides are numbered from N to C terminus using normal
Arabic numerals (Peptides 1, 2, etc.). Lys-C peptide names
are preceded by "k" (Peptides k1, k2, etc.). Arg-C
peptide names are preceded by "r" (Peptide r1, r2,
etc.). Asp-N peptide names are preceded by "d" (Peptide
d1, d2, etc.). Peptides that form the N terminus of one of
the hypothetical eIF4G-1 isoforms (Table I) are shaded. The
suffixes C, C1, C2, etc. denote truncated N-terminal
peptides (e.g. Peptide 2C is the C-terminal portion of
Peptide 2). The initial aa sequences of predicted N-terminal peptides,
prior to any posttranslational modification, are shown above the
corresponding tryptic peptides (MNK, MNT, etc.).
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Initial Characterization of Protein Bands in cpN
Preparations by MALDI-TOF-MS--
To determine which bands present in
cpN preparations were actually related to eIF4G-1, we
subjected a preparation of cpN to electrophoresis and
staining with Coomassie Blue (Fig. 4).
This pattern differs from that of Fig. 2, lane 1, for two
reasons. First, higher resolution was achieved on the preparative gel
(Fig. 4) than the analytical gel (Fig. 2). Second, staining with
Coomassie Blue (Fig. 4) is roughly proportional to protein quantity,
but staining with silver (Fig. 2) is disproportionately strong for cpN polypeptides (38), perhaps because of several
polyglutamic acid stretches in the sequence. Protein bands were
excised, digested with endoproteases, and the resulting peptides
analyzed by MALDI-TOF-MS (see "Experimental Procedures"). eIF4G-1
peptides were found in band b, band c plus
band d (partially resolved), band e, and
band g only, which correspond to Bands 1,
2, 3, and 4 in Fig. 2, respectively. These assignments, based on observed eIF4G-1 peptides, also agree with
immunoblotting results (Fig. 2 as well as Refs. 14, 38, 51, and
52).
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Fig. 4.
Identification of eIF4G-1 and and other
affinity-purified polypeptides by matching peptide masses to data
bases. 2A protease-treated K562 lysate was subjected to
m7GTP-Sepharose affinity chromatography as described under
"Experimental Procedures." Bound proteins were then resolved by
preparative gradient gel SDS-PAGE and stained with Coomassie Blue.
Protein bands (arbitrarily labeled a thru n) were
excised, digested with trypsin, and subjected to MALDI-TOF-MS. The
spectra were automatically matched to the NCBI protein data base by the
Auto-MS Fit program, as described under "Experimental Procedures."
Identities and confidence scores (MOWSE; Ref. 68) were generated for
each band. The human proteins that best matched the indicated bands
are: band a, hypothetical protein, GenBankTM
protein accession number T17345, MOWSE = 2.84 × 103; band b (same as Band 1 of Fig. 2), eIF4G-1,
GenBankTM AAC78443, MOWSE = 6.85 × 102; band c (same as Band 2 of Fig. 2), LINE1
reverse transcriptase homologue, GenBankTM P08547,
MOWSE = 3.19 × 102; subsequent analysis of this
band revealed eIF4G-1 peptides as well; band d, p110 subunit
of eIF3, GenBankTM NP_003743, MOWSE = 2.61 × 105; band e (same as Band 3 of Fig. 2), no
protein matched by Auto-MS Fit, but subsequent analysis revealed
eIF4G-1 peptides; band f, no protein matched by Auto-MS Fit;
band g (same as Band 4 of Fig. 2), no protein matched by
Auto-MS Fit, but subsequent analysis revealed eIF4G-1 peptides;
band h, no protein matched by Auto-MS Fit; band
i, BiP, GenBankTM AAF13605, MOWSE = 1.09 × 104; band j, PABP, GenBankTM
NP_002559, MOWSE = 8.29 × 103; band
k, DEAD-box protein p72, GenBankTM NP _006377,
MOWSE = 1.63 × 103; Band l, HSP70,
GenBankTM NP_005336, MOWSE = 3.22 × 105; band m, RNA helicase p68,
GenBankTM NP_004387, MOWSE = 5.94 × 103; band n, p36 subunit of eIF3,
GenBankTM NP_003748, MOWSE = 7.82 × 104.
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Some non-eIF4G-1 proteins present in the cpN preparation
were potentially of interest. In addition to eIF4G-1, band c
contained peptides that matched a LINE1 reverse transcriptase
homologue. Bands d and n matched the p110 and p36
subunits of eIF3, respectively (55). As noted above, several heat-shock
proteins bind to eIF4G, so it is interesting to note that band
i is the endoplasmic reticulum-resident chaperone BiP, and
Band l is hsp70-1. Band j is PABP, known to associate with eIF4G (10, 11). Finally, whereas the 46-kDa DEAD-box
helicase eIF4A is known to bind eIF4G-1 at two sites in cpC
(14, 20, 21), it is interesting to note that band k is a
72-kDa DEAD-box protein, and band m is a 68-kDa RNA helicase.
Band 1 Corresponds to eIF4G-1f--
To correlate hypothetical
eIF4G-1 isoforms (Table I) with electrophoretic bands (Fig. 2), we
examined the eIF4G-1-containing bands in more detail with four
different proteolytic enzymes over several mass ranges. A typical
spectrum for Band 1 (which corresponds to band b in Fig. 4)
in the range m/z = 500-6000 is shown in Fig. 5. Of the tryptic peptides predicted to
arise from eIF4G-1 (Fig. 3), those with masses similar to peaks
observed in Fig. 5 are listed in Table
III. The average deviation of observed
versus theoretical masses was 0.19 Da. The major peaks
(other than calibrants) matched predicted eIF4G-1 peptides, indicating
that the sample was not grossly contaminated. Peptides below 800 Da
were excluded from further consideration because of high noise levels
and paucity of significant sequence information. In addition to the
tryptic peptides listed in Table III, we detected in other spectra
Peptides 11·12 (missed cleavage), 13·14, 16, 29·30·31, 30·31,
and 37·38, some of which were either singly or doubly oxidized in Met
residues (see below).
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Fig. 5.
MALDI-TOF-MS analysis of tryptic peptides
from Band 1 of a cpN preparation.
Band 1 (see Fig. 2) from a preparative gradient gel (same as
band b in Fig. 4) was subjected to trypsin digestion and
MALDI-TOF-MS as described under "Experimental Procedures." Peaks
attributable to eIF4G-1 peptides are indicated using the numbering
system in Fig. 3. Composite peptides resulting from missed tryptic
cleavages are shown with a center dot, e.g.
Peptide 11·12·13. Peptides in which Met residues are oxidized have
the suffixes ox, ox1, and ox2 (see Table III). Peaks resulting from
peptide standards used for internal calibration are indicated as cal1,
cal2, etc. For each peak, m/z values are compared
with predicted mass values in Table III.
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Diagnostic tryptic peptides for Band 1 are shown in Fig.
6A (upper panel).
Band 1 has peaks at m/z = 4534.7 and 4550.8, which are
within 0.5 Da of the calculated masses of Peptide 2 (Table II) and the
Met-oxidized form of Peptide 2 (+16.0 Da). This "one-Met signature"
agrees with the presence of one Met residue in Peptide 2 (Fig. 3 and
Table II) and contributes to the confidence of this assignment. Other
peaks in Fig. 6A for Band 1 at m/z = 4629.7, 4645.9, and 4662.1 are within 0.6 Da of the predicted masses of Peptide
5 (Table II) and its derivatives containing either one or two oxidized
Met residues, respectively. This "two-Met signature" agrees with
the presence of two Met residues in Peptide 5 (Fig. 3 and Table II).
Finally, the peak at m/z = 4728.6 matches Peptide 28·29 (Fig. 3 and Table III). In contrast, the spectrum of Band 2 contains peaks corresponding to Peptide 5 but not Peptide 2 (Fig.
6A, lower panel), nor were peaks corresponding to
Peptides 2 or 5 observed in Bands 3 or 4 (not shown). Peptide 5 can
come from eIF4G-1e or eIF4G-1f, but not from eIF4G-1a, -1b, -1c, or -1d
(Fig. 3 and Table II). However, Peptide 2 can only come from eIF4G-1f (or an undescribed, longer form not predicted from the cDNA sequence). Thus, the presence of Peptide 2 in Band 1 but not
in Bands 2, 3, or 4 is consistent with eIF4G-1f being present only in
Band 1.
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Fig. 6.
Identification of diagnostic peptides in
Bands 1 and 2. Peptides produced from Bands 1 and 2 (Fig. 2) with four endoproteases were analyzed by
MALDI-TOF-MS. Peaks marked with an asterisk arise from MALDI
matrix adduction (69). A, peaks for tryptic peptides from
Bands 1 and 2. Closed arrows (upper panel)
indicate unmodified and Met-oxidized forms of Peptide 2. Open
arrows (upper and lower panels) indicate
unmodified and Met-oxidized forms of Peptide 5. B, peaks for
Lys-C peptides from Bands 1 and 2. The closed arrow
(upper panel) indicates Peptide k2. C, peaks for
Arg-C peptides from Band 1. The arrows denote the
N -acetylated, des-Met derivative of Peptide
r1 and the Met-oxidized form. D, Asp-N peptides from Bands 1 and 2. Peptide d1 is present only in Band 1 (upper panel),
while Peptide d1C1 is present only in Band 2 (lower panel).
An internal calibrant peak for the protein standard horse apomyoglobin
(16,952 Da) is present in both upper and lower
panels of B and D.
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Confirmation that eIF4G-1f is in Band 1 was obtained with endoprotease
Lys-C. When this enzyme was used with Band 1, we detected Peptides k2,
k4, k7, k12, k13, k15, and k21 (Fig. 3). A diagnostic peak at
m/z = 17,770 was observed in Band 1 but not Band 2 (Fig. 6B). Peptide k2 has a predicted, unmodified mass of
17,695 Da (Table II), which is 75 Da less than the peak observed in
Fig. 6B. Since Peptides 2 and 5 exist primarily in the
oxidized forms (Fig. 6A), it is likely that the peak at
17,770 consists of a mixture of Peptide k2 and derivatives oxidized at
one to four Met residues (calculated masses: 17,711, 17,727, 17,743, and 17,759 Da). The observed peak is within 0.06% of the latter
calculated mass. These results indicate that Band 1 contains Peptide
k2, which can only arise from eIF4G-1f (or a hypothetical larger form), whereas Bands 2, 3, and 4 do not contain Peptide k2.
Unfortunately, the tryptic and Lys-C peptide predicted to represent the
extreme N terminus of eIF4G-1f, MNK, is too small to be unambiguously
identified by our approach. Thus, we cannot conclude from the data in
Fig. 6, A and B, whether Peptides 2 and k2 were
contributed by eIF4G-1f or a hypothetical larger form. We therefore
turned to a different enzyme, Arg-C, which produced a diagnostic peak
at m/z = 4819.3 (Fig. 6C). The mass of
Peptide r1, which is predicted to result from Arg-C digestion of
eIF4G-1f, is 4908.6 Da (Table II). However, the mass of a derivative of Peptide r1 in which the N-terminal Met is removed and the resulting N-terminal Asp is N-acetylated (+42.0 Da) is
4819.4, within 0.1 Da of the observed peak. This agrees with our
observation that the same band was refractory to analysis by Edman
degradation (see above). A second peak at 4835.6 matches the mass of
Met-oxidized, N-acetylated Peptide r1 in
which the N-terminal Met is removed. This one-Met signature is
consistent with the postulated removal of the N-terminal Met-1 and
oxidation of Met-41 (Fig. 1). The peaks at m/z = 4819.3 and 4835.6 were observed in Band 1 but not Bands 2-4 (data not shown),
which agrees with the hypothesis that eIF4G-1f is in Band 1 but not in
the other bands.
To obtain additional evidence for eIF4G-1f in Band 1, we digested with
Asp-N. A major peak with m/z = 19,371 was observed in
Band 1 (Fig. 6D, upper panel) but not Band 2 (Fig. 6D, lower panel), Band 3, or Band 4 (data
not shown). This is similar to the mass of Peptide d1, which is 19,278 Da (Table II). Peptide d1 contains five Met residues, although based on
the data with Arg-C (Fig. 6C), the N-terminal Met may have
been removed on at least some of the polypeptides. Subtracting one Met
residue, adding an acetyl group, and oxidizing four Met residues gives
a mass of 19,253. Although this does not agree exactly with the
m/z of the observed peak, it is within 0.6% of this value.
As noted above, exact matching of predicted and observed peptide masses
can be compromised by microheterogeneity (partial removal of N-terminal Met, acetylation, oxidation). Nonetheless, the peak at
m/z = 19,371 is at least consistent with the presence
of Peptide d1 in Band 1, which is, in turn, indicative of eIF4G-1f.
Band 2 Represents a Mixture of Proteins, Including
eIF4G-1e--
Three lines of evidence suggest that Band 2 is a mixture
of proteins. First, the band is broader than Band 1, Band 3, or other non-eIF4G bands in Coomassie Blue-stained gels (Fig. 4, band
c + band d), suggesting that proteins are unresolved.
Second, Band 2 is, in fact, partially resolved into a doublet or
triplet in some gels (data not shown). Third, the Auto MS-Fit program
identified a LINE1 reverse transcriptase homologue and an eIF3 subunit
in bands c and d (Fig. 4), despite the fact that
they also contain numerous eIF4G-1 peptides (see below). Nonetheless,
we were able to obtain structural information about the isoform of
eIF4G-1 present in Band 2.
Of the tryptic peptides in cpN with masses >800 Da, we
detected Peptides 3, 4, 5, 13, 30, and 38 in Band 2, either as single peptides or composite peptides resulting from missed cleavages. A set
of peaks with m/z = 4630.0, 4646.3, and 4662.1 was
observed for Band 2 (Fig. 6A, lower panel),
corresponding to the two-Met signature of Peptide 5 (Table II). The
presence of Peptide 5 is diagnostic of a protein being initiated with a
Met upstream of eIF4G-1d, i.e. either eIF4G-1e or eIF4G-1f
(Fig. 3). We also detected Peptide 13, which indicates the eIF4G-1
isoform present is initiated upstream of eIF4G-1a (data not shown). Yet
Band 2 lacks Peptide 2 (Fig. 6A, lower panel),
indicating it does not contain eIF4G-1f. It is clear from the results
with Band 1 that Peptide 2 would have been detected if present (Fig.
6A, upper panel). The presence of Peptide 5 but
absence of Peptide 2 indicates that Band 2 contains eIF4G-1e.
With Lys-C, we detected Peptides k3, k4, k5, k7, k8, k9, k10, k13, k14,
k15, k16, k18, k19, k20, and k21 as either single or composite peptides
(Fig. 3). Unfortunately, none of these peptides gave insight into the
form of eIF4G-1 present, other than it was upstream of eIF4G-1a.
Specifically, no evidence for the diagnostic peptide k2C1 was seen.
Digestion with Asp-N revealed a peak at m/z = 15,387 in
Band 2 (Fig. 6D, lower panel). This
m/z value is similar to that of Peptide d1C1 (15,300), which
represents the N terminus of eIF4G-1e (Table II). Peptide d1C1 contains
four Met residues. If each Met were oxidized, it would add 64 Da to the
mass of this peptide, bringing the m/z value to 15,364 Da.
The resulting difference between the observed and calculated values is
0.1%, which is considerably less than the difference for any other
peptide predicted from the six hypothetical isoforms of eIF4G-1. This
peak was not observed in Band 1 (Fig. 6D, upper
panel), Band 3, or Band 4 (data not shown), providing additional
evidence that eIF4G-1e is present only in Band 2.
Band 3 Contains eIF4G-1c--
The only tryptic peptides with
masses >800 Da detected in Band 3 were Peptides 5C2, 12, 14, and 15. This is considerably fewer than for Band 1, presumably due to the lower
amount of protein typically seen in this band (Fig. 2). A diagnostic
peak was found in Band 3 at m/z = 2426.20 that was not
present in Band 1 (Fig. 7A,
lower panel). This is 42.03 Da more that the mass of Peptide 5C2 (Table II), which is the postulated N terminus of eIF4G-1c. This
peak is therefore consistent with an
N-acetylated form of Peptide 5C2, which would
agree with our inability to obtain information for this band from Edman
degradation (see above). A second peak was detected for Band 3 at
m/z = 2442.23 (Fig. 7A, lower
panel). This one-Met signature agrees with the presence of one Met
residue in Peptide 5C2 (Table II) and suggests that, unlike eIF4G-1f,
the N-terminal Met is not removed before acetylation of
eIF4G-1c. Alternatively, the nascent polypeptide may have been eIF4G-1d
from which Met-88 was removed, followed by
N-acetylation of Met-89 (Fig. 1).
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Fig. 7.
Identification of diagnostic peptides in Band
3. A, peaks for tryptic peptides from Bands 1 and 3. Arrows (lower panel) indicate
N-acetylated Peptide 5C2 and its Met-oxidized
derivative. A calibrant peak (ACTH clip 18-39, calculated mass 2465.20 Da) is present in both samples. B, peaks for Lys-C peptides
from Bands 1 and 3. Arrows (lower panel) denote
Met-oxidized derivatives of N-acetylated
Peptide k2C3, which are unique to Band 3. C, peaks for Asp-N
peptides from Band 3. The group of peaks match masses of Met-oxidized
and unoxidized forms of N-acetylated Peptide
d1C3.
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Additional evidence for diagnostic peptides in Band 3 was sought using
Lys-C. Peptides k2C3, k3, k4, k5, k7, k13, k14, k15, k16, k17, k18,
k19, k20, and k21 were detected as either single or composite peptides.
Diagnostic peptides were found at m/z = 9124.1 and
9139.5 (Fig. 7B, lower panel). These are within 1 Da of the predicted masses for the
N-acetylated form of Peptide k2C3 containing
either one (9123.3 Da) or two (9139.3 Da) oxidized Met residues. This
two Met signature is consistent with the presence of two Met residues
in Peptide k2C3 (Table II). The peak at m/z = 8883.3 of
Band 1 corresponds to doubly charged Peptide k2 (predicted
m/z = 8847.9 for the unoxidized peptide; see Fig.
6B). Its breadth is presumably due to microheterogeneity from multiple oxidized Met residues.
Finally, we analyzed the Asp-N peptides of Band 3 (Fig. 7C).
A unique peptide at m/z = 10,317 matched the predicted
mass for N-acetylated Peptide d1C3 (10,317 Da). Similarly, the observed peaks at m/z = 10,333 and
10,349 matched the masses of the singly and doubly oxidized forms of
this acetylated peptide (predicted at 10,333 and 10,349 Da). These
peptides provide additional evidence for the presence of eIF4G-1c in
Band 3.
In a separate experiment, MS fragmentation data (Fig.
8) confirmed our assignment of the
tryptic peptide detected at m/z = 2426.20 in Fig.
7A as N-acetylated Peptide 5C2
(cDNA-derived sequence given in Fig. 1).
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Fig. 8.
LC-ESI/MS spectrum of Peptide 5C2 in Band
3. Fragmentation analysis of
N-acetylated Peptide 5C2 shows ions of the b
(N-terminal) and y (C-terminal) series. The y fragments (in the range
y3+ to y20+) all match
their predicted masses, whereas the detected b fragments give values
that are shifted by +42 Da, indicating an acetylated Met residue at
position 1.
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Band 4 Represents a Mixture of Proteins That May Include
eIF4G-1a--
The amounts of eIF4G-related material in Band 4 were
insufficient to make a definitive assignment. However, peaks were
detected that suggest the presence of eIF4G-1a. Specifically, we
detected a peak in tryptic digests at 538.24 Da that is within 1 Da of Peptide 13C with one Met oxidized (537.25 Da). We also detected a peak
in tryptic digests at 580.25 that is within 1 Da of the N-acetylated, Met-oxidized form of Peptide
13C (579.26 Da). In Asp-N digests, we detected a peak at
m/z = 3574.1 that is within 1 Da of Peptide d3C (3575.0 Da; Table II). We also detected a peak at m/z = 3616.1 that is similar to N-acetylated Peptide d3C
(3617.0 Da). All of these were only slightly above the background,
making assignment of structure uncertain. Band 4 often occurs as a
diffuse doublet, indicating heterogeneity, and immunoblotting
demonstrates that the eIF4G-1 isoforms in Band 4 are much less abundant
than in Bands 1-3 (Fig. 2).
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DISCUSSION |
The data presented here permit the identification of several
eIF4G-1 primary structures and their assignment to electrophoretic bands. Band 1 consists of a novel isoform, here termed eIF4G-1f. This
is also the most abundant form, as measured by Coomassie Blue staining
and immunoreactivity to both anti-Peptide 7 and anti-Peptide 10 antibodies. Using trypsin or Lys-C, we observed Peptides 2 and k2,
which can only arise from eIF4G-1f or a hypothetical protein initiated
upstream of it. With Arg-C we detected the N-terminal peptide, Peptide
r1, and its mass indicated that it was modified in two ways: the
N-terminal Met was removed and the resulting Asn was
N-acetylated. Asp-N produced Peptide d1, in
agreement with the assignment of eIF4G-1f to Band 1, but it was too
large (19,371 Da) to allow determination of the exact N-terminal
structure. Thus, all of the data from Band 1 are consistent with its
identity as eIF4G-1f. This isoform of eIF4G-1 was not predicted from
any of the previously reported cDNA sequences. It is the longest
isoform, with 22 additional aa residues at the N terminus compared with eIF4G-Iext (Fig. 1), the longest isoform previously postulated.
Several observations point to Band 2 being a mixture of proteins.
Nonetheless, three types of data demonstrate that Band 2 contains
eIF4G-1e. First, it reacts with anti-Peptide 10 antibodies, which are directed against an epitope in only eIF4G-1e and
eIF4G-1f. Second, tryptic digests contain Peptide 5 but not
Peptide 2, despite the fact that Peptide 2 is readily
detectable in Band 1. Third, Asp-N digests contain Peptide
d1C1 in Band 2 but not Band 1. Due to the size and microheterogeneity
of Peptide d1C1, however, we were unable to determine the exact
structure at the N terminus. eIF4G-1e is the isoform predicted from
several of the cDNAs published before the present report (44-46),
although its existence had not actually been demonstrated. Comparison
of eIF4G-1e to eIF4G-1f by immunoreactivity with two different
antibodies indicates that eIF4G-1e is less abundant (Fig. 2 and data
not shown).
Both peptide matching and fragmentation data support the assignment of
Band 3 as a N-acetylated form of eIF4G-1c. In
digests with trypsin, Lys-C, and Asp-N, we detected the predicted
N-terminal peptides and their Met-oxidation products, viz.
N-acetylated Peptide 5C2,
N-acetylated Peptide k2C3, and
N-acetylated Peptide d4C3, respectively.
These peptides were found in Band 3 but not other bands. Finally, the
sequence of N-acetylated Peptide 5C2 was
established by MS fragmentation. Based on the agreement between
Coomassie Blue staining and immunoreactivity in comparison with Band 1, Band 3 appears to be homogeneous for eIF4G-1c. This form of eIF4G-1 is
clearly present in K562 cells but is less abundant than eIF4G-1f and
-1e (Fig. 2). eIF4G-1c is another novel isoform, not previously
predicted from cDNA sequences.
Additional forms of eIF4G-1 may exist but were too low in abundance to
be identified in the present study. Several peptides unique to eIF4G-1a
were detected in Band 4, but we do not judge the evidence to be
compelling. It is interesting to note that many bands
besides Bands 1-4 react with anti-Peptide 7 antibodies (Fig. 2, lane 4). One might consider them to represent
nonspecific binding of the antibodies to non-eIF4G-1 proteins except
for one additional feature: they are not present in a preparation
pretreated with 2A protease (Fig. 2, lane 3). The 2A
protease of entero- and rhinoviruses is quite specific for a consensus
aa sequence (38, 52, 56, 57). A few cellular proteins other than eIF4G are cleaved by 2A proteases (58-60), but two-dimensional
electrophoresis reveals that the overwhelming majority of cellular
proteins are unaffected by picornavirus infection (61). The likelihood
that a non-eIF4G-1 protein would both react with anti-Peptide 7 antibodies and also be a substrate for 2A protease is quite remote.
Thus, the numerous weak bands detected in anti-Peptide 7 immunoblots (Fig. 2, lane 4) may represent additional eIF4G-1 isoforms.
While there may well be additional isoforms of eIF4G-1, it
is unlikely that there are any larger than eIF4G-1f. Even if longer cDNAs are found, three considerations make it unlikely that they will encode proteins containing an extension of the eIF4G-1f
polypeptide sequence. First, we have shown that the structure of the
major form (eIF4G-1f) is initiated from the AUG that is furthest
upstream of the known cDNAs. Second, there are termination codons,
both in-frame and out of frame, upstream of this AUG. It is possible that other forms of eIF4G-1 may be encoded by mRNAs arising by alternative splicing that contain different N-terminal aa sequences, e.g. AAC78442 (Fig. 1). However, we detected no peaks
corresponding to either eIF4G-3 (4) or AAC78442. Third, eIF4G-1f is the
principle constituent of Band 1, which is the slowest migrating
immunoreactive band (Fig. 2). Drawing conclusions about eIF4G structure
from electrophoretic mobility is complicated by the fact that the
migration of eIF4G-1 on SDS-PAGE is aberrantly slow. The slowest
eIF4G-1 isoform migrates at 220 kDa and the fastest, at 205 kDa (62), yet the calculated molecular masses range from 176 to 155 kDa (Table
I). Thus, something besides the additional aa sequence reported here
must account for the disparity. Despite the aberrant mobility, we
observed that the order of bands follows the molecular mass
of the isoforms, e.g. eIF4G-1f is both the largest form
(Table I) and the slowest eIF4G band (Fig. 2), etc. Larger
forms of eIF4G-1 may exist but not be purified by the method used in
this study; it is worth noting that our method of affinity purification on m7GTP-Sepharose would exclude any hypothetical eIF4G-1
isoforms that lack an eIF4E-binding site such as the eIF4G homologue
eIF4G-2 (3, 7, 8).
Although it is clear that the various isoforms of eIF4G-1 contain
different N termini, the origin of these proteins cannot be determined
from the results presented. Several formal possibilities can be
considered. The first is alternative translation initiation of the same
mRNA by leaky scanning. The optimal consensus sequence for
initiation in animals is A/GXXAUGG (63). A "strong"
initiation codon is considered to be one containing either the purine
at 4, the G at +1, or both. The corresponding sequences for the various hypothetical eIF4G-1 isoforms are: eIF4G-1f, AAAAUGA; eIF4G-1e, CAAAUGA; eIF4G-1d, GUAAUGA; eIF4G-1c, AUGAUGA; eIF4G-1b, UUGAUGA; and eIF4G-1a, AUCAUGU. Thus, the strong initiation codons are
in mRNAs for eIF4F-1f, -1d, -1c, and -1a, perhaps explaining the
absence of eIF4G-1b. The second possibility for the multiplicity of
eIF4G-1 isoforms is internal initiation of translation. Two separate
sequences derived from the 5'-portion of eIF4G-1 mRNA can function
as internal ribosome entry sites in cultured cells (45, 64, 65). A
single mRNA corresponding to the longest cDNA reported to date
(this report; 5510 nt) could be initiated from the 5'-end, giving rise
to eIF4G-1f, and internally, giving rise to eIF4G-1c. The nucleotide
sequence representing the 5'-untranslated region of an mRNA that
would encode eIF4G-1d has already been shown to direct internal
initiation of translation (45). This may provide a mechanism for
eIF4G-1c expression, allowing for the N-terminal processing of the
nascent polypeptide (removal of Met-88 and
N-acetylation of Met-89). Finally there may
be several mRNAs differing in their 5'-terminal sequences that
encode different eIF4G-1 isoforms, some of which exclude upstream AUG
codons. These could arise from alternative splicing (e.g.
NP_004944 and AAC78442) or alternative promoter usage. Resolution of
this question will require an investigation of structure of eIF4G-1
mRNA(s) present in the cell and their translational properties. The
fact that we never observed peptides diagnostic for slower bands in
faster bands argues against the faster bands representing proteolytic
breakdown products, or polypeptides missing internal exons. The same
conclusion can be drawn from the absence of any bands running faster
than Bands 1 and 2 that bind anti-Peptide 10 antibodies (Fig.
2).
We found a surprisingly large number of ligands co-purifying with
cpN (Fig. 4). This type of analysis does not indicate
whether these ligands are bound to the cpN fragment of all
eIF4G-1 isoforms or to only a subset. These ligands include PABP and
hsp70, which is consistent with previous studies showing these to be
eIF4G-binding proteins (10, 11, 26). Less expected is the presence of the p110 and p36 subunits of eIF3. It is known that 11-subunit eIF3 has
a high affinity for eIF4G, but the binding site has been localized in
cpC, not cpN (14, 19). The association of eIF4G with the p110 and p36 subunits may mean that there are additional points of attachment in cpN. The presence of a putative
DEAD-box protein and an RNA helicase in cpN is intriguing,
since eIF4A, a different member of the DEAD-box RNA helicase family, is
well established to bind eIF4G-1 at two distinct sites in
cpC (14, 20, 21).
At present we have not determined whether there are differences in the
biochemical properties of the different eIF4G-1 isoforms. Since eIF4G
is a protein that binds an unusually large number of protein and RNA
ligands, the presence of different N-terminal sequences may direct the
binding of isoform-specific ligands. The various eIF4G isoforms may
have different affinities for ribosomes or initiation factors that link
them to ribosomes (e.g. eIF3), opening the possibility that
eIF4G-1 isoforms participate in mRNA recruitment to different
extents. The only study explicitly comparing eIF4G-1 isoforms showed
that transfection of cDNAs expressing eIF4G-1e and eIF4G-1a gave
similar growth efficiencies in soft agar, an assay for malignant
transformation (66). Yet no in vivo expression studies have
been performed to date on eIF4G-1f, the longest and most abundant form
in K562 cells.
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932. Tel.:
318-675-5161; Fax: 318-675-5180; E-mail:
rrhoad@lsuhsc.edu.
