The j-Subunit of Human Translation Initiation Factor eIF3 Is
Required for the Stable Binding of eIF3 and Its Subcomplexes
to 40 S Ribosomal Subunits in Vitro*
Received for publication, November 21, 2003, and in revised form, December 18, 2003
Published, JBC Papers in Press, December 19, 2003, DOI 10.1074/jbc.M312745200
Christopher S. Fraser‡, Jennifer Y. Lee, Greg L. Mayeur, Martin Bushell§, Jennifer A. Doudna¶,
and John W. B. Hershey?
From the Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616
Eukaryotic initiation factor 3 (eIF3) is a 12-subunit
protein complex that plays a central role in binding of
initiator methionyl-tRNA and mRNA to the 40 S riboso-
mal subunit to form the 40 S initiation complex. The
molecular mechanisms by which eIF3 exerts these func-
tions are poorly understood. To learn more about the
structure and function of eIF3 we have expressed and
purified individual human eIF3 subunits or complexes
of eIF3 subunits using baculovirus-infected Sf9 cells.
The results indicate that the subunits of human eIF3
that have homologs in Saccharomyces cerevisiae form
subcomplexes that reflect the subunit interactions seen
in the yeast eIF3 core complex. In addition, we have
used an in vitro 40 S ribosomal subunit binding assay to
investigate subunit requirements for efficient associa-
tion of the eIF3 subcomplexes to the 40 S ribosomal
subunit. eIF3j alone binds to the 40 S ribosomal subunit,
and its presence is required for stable 40 S binding of an
eIF3bgi subcomplex. Furthermore, purified eIF3 lack-
ing eIF3j binds 40 S ribosomal subunits weakly, but
binds tightly when eIF3j is added. Cleavage of a 16-
residue C-terminal peptide from eIF3j by caspase-3 sig-
nificantly reduces the affinity of eIF3j for the 40 S ribo-
substantially less stabilization of purified eIF3–40S
complexes. These results indicate that eIF3j, and espe-
cially its C terminus, play an important role in the re-
cruitment of eIF3 to the 40 S ribosomal subunit.
Eukaryotic initiation factor 3 (eIF3)1was first isolated and
purified as a high molecular weight complex from rabbit reticu-
locytes (1–3). The mammalian factor possesses a molecular
mass of about 600 kDa and contains at least 12 nonidentical
protein subunits, named in order of decreasing molecular
weight as recommended (4): eIF3a, eIF3b, eIF3c, eIF3d, eIF3l,
eIF3e, eIF3f, eIF3g, eIF3h, eIF3i, eIF3j, and eIF3k (5, 6). Spe-
cific functions for mammalian eIF3 have been identified by a
variety of in vitro experiments. It binds directly to 40 S ribo-
somal subunits in the absence of other initiation components
(1), and affects the association/dissociation of ribosomes (7–10).
It promotes the binding of Met-tRNAiand mRNA to the 40 S
ribosomal subunit (5), and binds directly to eIF1 (11), eIF4B
(12), eIF4G (13, 14), and eIF5 (15). Clearly, eIF3 plays a central
role in the initiation pathway, perhaps structurally organizing
other translational components on the surface of the 40 S
An eIF3 complex was first identified and isolated from Sac-
charomyces cerevisiae by employing either of two assay sys-
tems: stimulation of methionyl-puromycin synthesis based on
mammalian assay components (16) and stimulation of protein
synthesis in a heat-inactivated yeast lysate derived from a
conditional mutant of eIF3b (17). Purification of eIF3 using an
oligohistidine-tagged eIF3b identified a core of five subunits
associated with eIF5 (18). The five core subunits, eIF3a, eIF3b,
eIF3c, eIF3g, and eIF3i, are all essential for yeast growth and
are conserved in mammalian eIF3. Recently, eIF3j has been
shown to associate loosely with the core eIF3 complex in yeast
(19) and has been suggested to augment the stability of eIF3
and possibly play a role in the 40 S ribosomal subunit assembly
Given the similarities between other yeast and mammalian
initiation factors, the structural differences observed with eIF3
are somewhat surprising. These discrepancies may be due in
part to subtle differences in the strengths of various protein-
protein interactions. It is likely that the true subunit composi-
tion of eIF3 will not be resolved until a functional protein
complex is reconstituted from separated subunits. To further
understand the structure and function of mammalian eIF3, we
have utilized the baculovirus expression system to prepare
human eIF3 subunits that have orthologs in S. cerevisiae. In
this study, we overexpressed different combinations of eIF3
subunits, including FLAG-tagged subunits, and affinity-puri-
fied the subcomplexes by anti-FLAG affinity beads. This ap-
proach allows assembly of human eIF3 components in vivo,
recovery of stable subcomplexes and determination of their
functions in in vitro assays for initiation. It also has the poten-
tial to generate structural information about subunit-subunit
interactions and to identify specific functions of individual sub-
units. We focus here on the function of eIF3j in promoting the
binding of core subcomplexes and purified eIF3 to 40 S riboso-
mal subunits and its reduced activity following cleavage by
* This work was supported by Grant GM-22135 from the National
Institutes of Health. The costs of publication of this article were de-
frayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
‡ Present address: Dept. of Molecular and Cell Biology, and Howard
Hughes Medical Institute, University of California, Berkeley, CA
§ Supported by Grant 063233/B/00/Z from the Wellcome Trust while
in the laboratory of Dr. P. Sarnow. Present address: Dept. of Microbi-
ology and Immunology, Stanford University School of Medicine, 299
Campus Dr., Stanford, CA 94305.
¶ Present address: Dept. of Molecular and Cell Biology, and Howard
Hughes Medical Institute, University of California, Berkeley, CA
? To whom correspondence should be addressed: Dept. of Biological
Chemistry, School of Medicine, University of California, Davis, CA
95616. Tel.: 530-752-3235; Fax: 530-752-3516; E-mail: jwhershey@
1The abbreviation used is: eIF3, eukaryotic initiation factor 3.
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 279, No. 10, Issue of March 5, pp. 8946–8956, 2004
Printed in U.S.A.
This paper is available on line at http://www.jbc.org
by guest on November 18, 2015
Chemicals and Biochemicals—Materials for tissue culture and DNA
oligonucleotides are from Invitrogen; [35S]methionine is from ICN;
DNA-modifying enzymes are from New England BioLabs; anti-FLAG
affinity beads are from Sigma. Unless otherwise stated, all other chem-
icals are from Sigma.
Cell Culture—Spodoptera frugiperda 9 (Sf9) cells were grown in
100-ml spinner flasks at 27 °C in Sf-900 II serum-free medium (Invitro-
gen). For experiments, cells were seeded at 1 ? 107cells in 100-mm
dishes prior to infection with the recombinant viruses described in
individual figure legends.
Construction of Recombinant Baculoviruses—Baculoviruses allowing
the expression of single subunits of human eIF3 were constructed in
derivatives of the pFASTBAC1 vector (Invitrogen). DNA oligonucleo-
tides were designed, annealed, and ligated into pFASTBAC1 to produce
FLAG-FASTBAC1. This vector includes an NcoI site containing an
AUG codon upstream of the FLAG-tag sequence followed by an in-frame
AUG within an NdeI site. In addition, pET-28c (Novagen) was digested
with BglII and XhoI and ligated into pFASTBAC1 digested with BamHI
and XhoI to produce pFASTpET.
Untagged eIF3 subunit expression constructs were created as fol-
lows. The cDNA for full-length eIF3i (GenBankTMnucleotide accession
number U39067) (21) was excised from an untagged construct in pET-
28c by digesting with XbaI and XhoI and ligating into the equivalent
sites of pFASTpET. Similarly, the eIF3g sequence (accession no.
U96074) was released from pET-T7p44 (22) by digesting with XbaI and
XhoI and inserted into pFASTpET. The full-length cDNA of eIF3b
(accession no. U62583), a kind gift from Nahum Sonenberg (McGill
University), was modified by PCR to include NcoI and NdeI sites at the
5?-end of the construct by using the primer 5?-CCATGGGGCATATG-
CAGGACGCGGAGAACGT-3?. This allowed the creation of an un-
tagged eIF3b coding sequence when ligated between NcoI and XhoI in
FLAG-FASTBAC1. Full-length eIF3a (accession no. D50929) was mod-
ified by PCR so that the full-length coding sequence could be released by
digestion with NdeI and SalI. The digestion of FLAG-FASTBAC1 con-
taining untagged eIF3b with NdeI and XhoI allowed the ligation of
eIF3a that had been digested with NdeI and SalI to result in untagged
To create FLAG fusion proteins, constructs were generated as fol-
lows. eIF3g and eIF3j were excised from pET-NHp44 (22) and pET-
NHp35 (22) respectively, by digestion with NdeI and XhoI and ligated
into FLAG-FASTBAC1 at the equivalent sites. eIF3i was subcloned
from pGEXp36 (21) into pET-28c by digesting with NdeI and EcoRI and
the resulting construct digested with NdeI and XhoI and ligated into
FLAG-FASTBAC1 at the equivalent sites. The PCR-modified eIF3b
sequence was digested with NdeI and XhoI and inserted into the same
restriction sites of FLAG-FASTBAC1, while the PCR-modified eIF3a
was digested with NdeI and SalI and inserted into the NdeI and XhoI
sites of FLAG-FASTBAC1.
The recombinant FASTBAC vectors above were recombined with
baculovirus DNA using DH10BAC E. coli (Invitrogen) and the high
molecular weight DNA (“bacmid”) purified according to the manufac-
turer’s guidelines. Sf9 cells were transfected with bacmid DNA by using
the calcium phosphate method (Promega) and viral stocks were pre-
pared by three-step growth amplification according to the manufactur-
er’s guidelines. eIF3b-HMK-FLAG, eIF3j-HMK-FLAG, and His6-eIF3c-
Myc viruses were kind gifts from Hiroaki Imataka, Shigenobu Morino,
and Nahum Sonenberg (McGill University).
Expression of eIF3 Subunits and Preparation of Cell Extracts—Sf9
cells (1 ? 107) were infected with baculoviruses expressing a single
FLAG-tagged subunit of eIF3 and/or untagged subunits of eIF3, as
indicated in each experiment. The cells were grown for 24 h and then
supplemented with 0.5 mCi of [35S]methionine for an additional 36 h.
Cells were harvested after placing on ice, by washing once with phos-
phate-buffered saline (50 mM sodium phosphate, pH 7.0, 150 mM NaCl)
and scraping in 1 ml of Buffer A (20 mM Tris-HCl, pH 7.5, 120 mM KCl,
10 mM 2-mercaptoethanol, 1% (v/v) Triton X-100, 10% glycerol). Follow-
ing a 5-min incubation on ice with occasional vortexing, extracts were
centrifuged for 10 min at 12,000 ? g in a cooled microcentrifuge. The
supernatant was either used immediately or frozen in liquid nitrogen
and stored at ?70 °C.
Immunoprecipitations and Western Blots—For isolation of FLAG-
tagged proteins and associated proteins, Sf9 cell extracts were sub-
jected to affinity purification on anti-FLAG beads (Sigma) as recom-
mended by the manufacturer. Briefly, cell extracts were incubated with
anti-FLAG beads at 4 °C with gentle agitation for 30 min. The resin was
washed four times with Buffer A, and protein was eluted by incubation
at 4 °C for 45 min with FLAG peptide (100 ?g/ml) in Buffer B (20 mM
Tris-HCl, pH 7.5, 70 mM KCl, 1 mM dithiothreitol, 2 mM Mg(OAc)2, 10%
glycerol). A fraction of the recovered proteins was subjected to SDS-
PAGE and the gel was analyzed either by Coomassie Blue staining,
exposure to x-ray film overnight to detect radioactive bands, or transfer
of proteins to a polyvinylidene difluoride membrane (Millipore) for
Western blotting. eIF3 subunits were detected with polyclonal goat
anti-eIF3 antiserum (1:2000), whereas eIF3c-Myc was probed with
monoclonal anti-Myc antibodies (1:2000, Santa Cruz Biotechnology).
Protein bands were revealed by incubation with the appropriate alka-
line phosphatase-conjugated secondary antibody.
Purification of eIF3 and 40 S Ribosomal Subunits—eIF3 was puri-
fied from HeLa cells as described previously (23), with some modifica-
tions. Briefly, HeLa cell lysate from 200 g of cell pellet was passed
through Q Sepharose Fast Flow ion exchange media and eluted using a
potassium chloride gradient. Fractions containing eIF3 were precipi-
tated using ammonium sulfate and then passed through a Superdex
200 gel filtration column. Fractions were then diluted and purified
using cation exchange. The purity of eIF3 was determined by SDS-
PAGE and Coomassie Blue staining.
Ribosomal subunits were isolated from HeLa cells as described (24).
Purity of the ribosomal subunits was assessed by sucrose gradient
centrifugation; quality was demonstrated by their efficient formation of
80 S ribosomes in 5 mM Mg(OAc)2buffer (10, 24).
Assembly and Analysis of 40 S Ribosomal Complexes—40 S com-
plexes were assembled by incubating purified 40 S ribosomal subunits
(17 pmol) with either purified HeLa eIF3 (17 pmol), radiolabeled re-
combinant subcomplexes, or individual recombinant subunits isolated,
and purified from insect cells. Following incubation for 3 min at 37 °C
in Buffer B lacking glycerol, the reactions were chilled for 5 min on ice,
layered over 10–40% (w/v) linear sucrose gradients containing buffer B,
and centrifuged in a Beckman SW-40 rotor at 38,000 rpm for 3.5 h at
4 °C. After centrifugation, each gradient was fractionated using an
ISCO gradient fractionator, and the absorbance profile at 254 nm was
monitored. Fractions were collected, precipitated with methanol, and
the presence of eIF3 subunits determined by SDS-PAGE and autora-
diography and/or Coomassie Blue staining. Alternatively, the total ra-
dioactivity in each gradient fraction was determined by measuring
trichloroacetic acid-precipitable radioactivity in a scintillation counter.
Expression of Human eIF3 Subunits in Sf9 Cells and Puri-
fication of the Recombinant Proteins—Construction and pro-
duction of baculoviruses expressing each subunit of human
eIF3 that has a homolog in S. cerevisiae (subunits a, b, c, g, i,
and j) were performed as described under “Experimental Pro-
cedures.” Initially, all but the eIF3c subunit was tagged at the
N terminus with a FLAG peptide, allowing for more efficient
purification. Extracts prepared from Sf9 cells infected with
individual recombinant baculovirus strains were subjected to
affinity purification by using anti-FLAG beads and proteins
were eluted with the FLAG peptide as described under “Exper-
imental Procedures.” Each of the purified FLAG-tagged eIF3b,
eIF3g, eIF3i, and eIF3j preparations exhibits a single major
protein band, indicating that these proteins are stable as iso-
lated subunits in insect cells (Fig. 1A). Each of the proteins has
an apparent molecular weight equal or very close to the corre-
sponding subunit derived from eIF3 purified from HeLa cells.
eIF3a also was expressed in Sf9 cells but does not accumu-
late to a high level in the soluble fraction of cell extracts (Fig.
1B, lane 1). Instead, a large amount of eIF3a was found in the
insoluble fraction (data not shown), which may be due to de-
naturation or an association of eIF3a with components of the
cytoskeleton (25–27). Therefore, we asked if coinfection of cells
with other subunits of eIF3 might promote the solubility of
eIF3a in cell extracts. Previously, both eIF3b and eIF3c have
been shown to bind to eIF3a in mammalian (28) and yeast (29)
eIF3. While eIF3c did not affect the solubility of eIF3a in Sf9
cells (data not shown), when cells were coinfected with viruses
expressing eIF3a and eIF3b (Fig. 1B, lane 2) a significant
amount of eIF3a became soluble in these cell extracts. This
presumably reflects an association of the two proteins in vivo.
Human eIF3j Stabilizes eIF3–40S Subunit Association
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Human eIF3j Stabilizes eIF3–40S Subunit Association
by guest on November 18, 2015
Doudna and John W. B. Hershey
2004, 279:8946-8956. J. Biol. Chem.
L. Mayeur, Martin Bushell, Jennifer A.
Christopher S. Fraser, Jennifer Y. Lee, Greg
Subcomplexes to 40 S Ribosomal Subunits
Stable Binding of eIF3 and Its
Initiation Factor eIF3 Is Required for the
The j-Subunit of Human Translation
Modification, and Degradation:
Protein Synthesis, Post-Translation
doi: 10.1074/jbc.M312745200 originally published online December 19, 2003
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