From the Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616
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ABSTRACT |
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Translation initiation factor eIF3 is a
multisubunit protein complex required for initiation of protein
biosynthesis in eukaryotic cells. The complex promotes ribosome
dissociation, the binding of the initiator methionyl-tRNA to the 40 S
ribosomal subunit, and mRNA recruitment to the ribosome. In the
yeast Saccharomyces cerevisiae eIF3 comprises up to 8 subunits. Using partial peptide sequences generated from proteins in
purified eIF3, we cloned the TIF31 and TIF32
genes encoding 135- (p135) and 110-kDa (p110) proteins.
Deletion/disruption of TIF31 results in no change in growth
rate, whereas deletion of TIF32 is lethal. Depletion of p110 causes a severe reduction in cell growth and protein synthesis rates as well as runoff of ribosomes from polysomes, indicative of
inhibition of the initiation phase. In addition, p110 depletion leads
to p90 co-depletion, whereas other eIF3 subunit levels are not
affected. Immunoprecipitation or nickel affinity chromatography from
strains expressing (His)6-tagged p110 or p33 results in the co-purification of the well characterized p39 and p90 subunits of eIF3
as well as p110 and p33. This establishes p110 as an authentic subunit
of eIF3. In similar experiments, p135 and other eIF3 subunits sometimes, but not always, co-purify, making assignment of p135 as an
eIF3 subunit uncertain. Far Western blotting and two-hybrid analyses
detect a direct interaction of p110 with p90, p135 with p33, and p33
with eIF4B. Our results, together with those from other laboratories,
complete the cloning and characterization of all of the yeast eIF3 subunits.
The initiation phase of protein synthesis in eukaryotes is
promoted by 10 or more proteins called eukaryotic initiation factors (eIFs)1 (for reviews, see
Refs. 1 and 2). The largest and most complex of these, eIF3, is a
600-kDa factor with 8 or more subunit proteins. Based on in
vitro biochemical studies of the mammalian system, eIF3 is
implicated in a large number of reactions in the initiation pathway.
eIF3 alone among the initiation factors binds stably to 40 S ribosomal
subunits (3). The factor promotes the dissociation of 80 S ribosomes
into 40 S and 60 S subunits, affects the stability of the ternary
complex comprising methionyl-tRNAi·eIF2·GTP in the
absence of ribosomes but in the presence of mRNA, and stabilizes methionyl-tRNAi binding to 40 S subunits (1). It also is
absolutely required for mRNA binding to ribosomes, where eIF3
already bound to the 40 S ribosome interacts with a region of eIF4G, a
component of the mRNA m7G-cap binding complex, eIF4F
(4). Thus eIF3 acts as a bridge between the 40 S ribosome and the
mRNA·eIF4F complex. It is apparent that eIF3 plays a central role
in initiation by interacting with numerous other translational components.
To better understand the function of eIF3, the cDNAs encoding 11 human eIF3 subunits have been cloned and characterized: p170 (5), p116
(6), p110 (7), p66 (8), p48 (9), p47 (8), p44 (10), p40 (8), p36 (7),
p35 (10), and p28.2 Knowledge
of the primary sequences of these proteins sheds light on their
possible functions. Three of the human subunits contain RNA-binding
motifs (p116, p66, and p44), and the gene for p48 (Int-6) is
a frequent site of integration by the mouse mammary tumor virus,
possibly implicating p48 in the regulation of eIF3 activity (9). Thus,
the cloned cDNAs provide insights into subunit functions and tools
for the study of the structure of eIF3 and its interactions with other
translational components.
To elucidate the function of eIF3 during the initiation phase, we
turned our attention to the budding yeast, Saccharomyces cerevisiae, where genetic approaches and gene replacement
strategies are well developed. The other initiation factors from
mammalian and yeast cells are quite strongly conserved, ranging from 17 to 72% sequence identity (1). Thus information obtained about yeast
eIF3 may be applicable to understanding the structure/function of human
eIF3. A yeast homolog of mammalian eIF3 was isolated on the basis of
its activity in an in vitro methionyl-puromycin synthesis
assay constructed with purified mammalian components of initiation
(11). The yeast eIF3 preparation contains proteins with masses of 135, 90, 62, 39, 33, 29, 21, and 16 kDa. The genes encoding p90
(PRT1), p62 (GCD10), and p16 (SUI1)
had been identified previously, and were subsequently shown to encode
eIF3 subunits (11-13). The genes encoding the p39 (TIF34)
and p33 (TIF35) subunits have been cloned and characterized
more recently (14, 15). The p29 subunit was shown to be a degradation
product of p39 (14), whereas a protein of 93 kDa has been identified as
an eIF3 subunit (p93) encoded by NIP1 (16, 17). Nip1p (p93)
is not present in our preparation described above, apparently due to
proteolysis during purification. In this report, we describe the
cloning of TIF31 encoding p135 and show that the 21-kDa
protein is derived from a 110-kDa subunit encoded by TIF32.
Thus, 8 proteins associated with yeast eIF3 have been cloned and
characterized: p135, p110, p93, p90, p62, p39, p33, and p16.
Interactions of p135, p110, and eIF4B with other eIF3 subunits also are
defined. The report thereby completes the detailed characterization of
yeast genes encoding known eIF3 components and employs the genes in
studies designed to define the subunit composition and structure of eIF3.
Strains, Cell Growth, and General Procedures--
The strains of
S. cerevisiae employed in this study are listed in Table
I. Yeast cells were grown at 30 °C in
yeast-peptone (YP) medium or in synthetic complete (SC) medium lacking
the indicated amino acids and were supplemented with 2% glucose (D) or
galactose (G) (18). Cell growth was monitored as optical density at 600 nm (OD600). Yeast cells were sporulated on 2% agar plates
with 0.3% potassium acetate, 0.02% raffinose, and 10 µg/ml of each amino acid as sporulation medium, and tetrad analyses were carried out
as described (19). Yeast chromosomal DNA and RNA were prepared and
transformations performed according to Rose et al. (18). Escherichia coli XL-1 blue was used for all plasmid cloning
procedures and the recombinant TRX-p110 fusion protein was expressed in
E. coli AD494(DE3) (Novagen). Plasmid preparations,
recombinant DNA techniques, and Northern and Southern blotting were
performed as described (20, 21).
Cloning and Chromosomal Disruption of TIF31 and TIF32--
eIF3
was purified as described previously (11) and was fractionated by
SDS-PAGE. Internal peptide sequences for the p135 and p21 subunits were
determined in the Protein Structure Laboratory (University of
California, Davis) as described (22). The 5 peptide sequences from p135
and the two from p21 are reported in Table II and were used to perform a TblastN
search of the GenBank data base.
The five p135 peptide sequences match 51 out of 55 residues with a
putative protein encoded by cosmid clone c8270 (American Type Cell
Culture). The 7.2-kb ApaI and XhoI fragment
(GenBank accession number AF004911) was ligated into the same sites of
pBluescript KSII (Stratagene) to yield pKSCos-TIF31. For disruption of
the open reading frame (ORF), tentatively called TIF31 (for translation initiation factor
3, 1st subunit), the gene was PCR-amplified from total
yeast genomic DNA to generate a 4.6-kb DNA fragment that includes 519- and 257-bp flanking the 5' and 3' ends of the ORF. The PCR product was
cloned into the SmaI site of pBluescript KSII to yield
pKS-TIF31. pKS-TIF31 was digested with BstBI and HpaI to remove 98% of the TIF31 coding region
and a 1.7-kb BamHI DNA fragment containing the
HIS3 gene was inserted (see Fig. 1). The resulting plasmid,
named pKS- TIF31::HIS3, was digested with SspI
and AseI to release a 2.5-kb DNA fragment containing the tif35::HIS3 allele. The fragment was transformed
into the diploid yeast strain W303 to create a one-step gene
deletion/disruption (23), confirmed by Southern blot analysis, to yield
PH135D-8. PH135D-8 was sporulated, tetrads were dissected, and a
His+ haploid spore colony containing
tif31::HIS3 was selected and named PH135H-B.
For the cloning of the p110 gene (TIF32), the two p21
peptide sequences (Table II) matched two regions of a putative 110-kDa protein derived from the yeast genome data base (ORF YBR079c). To
obtain TIF32 DNA, yeast genomic DNA (200 µg) was digested
with NheI and PstI, subcloned into pBluescript
KS+ (Stratagene) and transformed into E. coli
XL1-blue. Colonies containing the desired plasmid were identified by
hybridization with a labeled 3.9-kb PCR product amplified from genomic
DNA. One of the plasmids carrying TIF32 was named pHV110-1
and the sequence of the 4565-bp insert was determined and submitted to GenBank under accession number AF004912. A
SstI/Asp718I fragment from pHV110-1 carrying the
putative TIF32 gene was subcloned into the same sites of the
centromeric/URA3 plasmid pRS316 (24) to generate
pHV110-2.
To disrupt a chromosomal TIF32 gene by a one-step gene
replacement, the 3.9-kb PCR product described above, which includes 557 and 462 bp of DNA flanking the 5' and 3' ends of the TIF32 coding region, respectively, was inserted into the SmaI site
of pBluescript KS+ to generate pKSp110. The
TIF32 coding region was deleted by digestion with
StyI and BspMI (see Fig. 1) and the 1.7-kb
BamHI fragment carrying the HIS3 gene was ligated
to form pHV110-3. The tif32::HIS3 allele was
excised by digestion of pHV110-3 with AseI and
PacI and the resulting 2.3-kb DNA fragment was transformed
into the diploid yeast strain W303. One of the His+
transformants was selected and named HV110-D20/D. The correct disruption of one of the TIF32 genes was confirmed by
Southern blot analysis (not shown).
Construction of Plasmids Expressing TIF31--
p414Gal1-NH135, a
CEN4 TRP1 plasmid which allows expression of the
N-terminal-(His)6-tagged p135 under a GAL1
promoter, was produced as follows. The p135 coding sequence (3.8 kb)
was PCR amplified from pKSCos-TIF31 and subcloned into pNoTA (5 Prime Construction of Plasmids and Strains Expressing TIF32--
To
express TIF32 under a regulatable promoter, plasmids
directing expression of wild type p110 and N- or C-terminal
(His)6-tagged p110 were constructed as follows. The 5' and
3' halves of TIF32 were PCR amplified from pHV110-1 with
suitable primers to generate DNAs encoding either wild type or
(His)6-tagged fragments. These were combined to encode
full-length p110 proteins, then subcloned into p414Gal1 to yield
pHPp110EX1 (wild type), pHPp110EX2 (N-terminal tag), and pHPp110EX3
(C-terminal tag) under control of the GAL1 promoter. The
wild type construct also was cloned into p414GalS (25) which contains a
weakened GAL1 promoter to give pHPp110EX1-S. All four
constructs were verified by sequencing. They were transformed into the
yeast strain HV110-D20/D, cells were sporulated, and tetrads were
dissected. The resulting His+Leu+ haploid
strains HV110-24, HV110-29, HV110-33, and HV110-42 express, respectively, an untagged p110 from the GAL1 promoter, an
untagged p110 from the GALS promoter, an N-terminal
His-tagged p110, and a C-terminal His-tagged p110 as the sole source of
eIF3-p110.
Antibodies--
(His)6-p135 Coimmunoprecipitations--
Yeast cells expressing various
(His)6-or Myc-tagged subunits of eIF3 were grown in YPG to
an OD600 of 1.0. A 50-ml aliquot of each culture was
harvested and cells were resuspended in 300 µl of buffer
C50+ (50 mM Tris/HCl, pH 7.5, 50 mM KCl, 0.1% (v/v) Triton X-100, 10% (v/v) glycerol, and
1 × CompleteTM (Roche Molecular Biochemicals)
protease inhibitors). Cells were lysed with glass beads, lysates were
clarified by centrifugation, and the protein concentration was
determined (27) and adjusted with buffer C350+
(buffer C50+, but containing 350 mM
KCl) to 2 mg/ml. Lysates (500 µl) precleared with Protein A/G-agarose
beads (Santa Cruz Biotechnology) were incubated for 90 min at 4 °C
with 20 µl of Protein A/G-agarose beads loaded with
anti-(His)6 antibody (CLONTECH) or
anti-c-Myc antibody 9E10 (Santa Cruz Biotechnology). Beads were washed
3 times with 500 µl of buffer C350+ and
resuspended in SDS-PAGE sample buffer. The bead-extracted proteins were
separated by SDS-PAGE, blotted onto a polyvinylidine difluoride
membrane, and probed with the antibodies described in the figure legends.
For radiolabeling of cell proteins for subsequent immunoprecipitation,
cells were grown in 10 ml of SC/galactose medium lacking methionine to
an OD600 of 0.8, labeled with 0.5 mCi of
[35S]methionine and [35S]cysteine
(Tran35S-label, ICN) for 2 h at 30 °C and lysed in
200 µl of buffer C350+ as described above.
The lysate volume was adjusted with C350+ to
500 µl and a lysate from the protease-deficient strain YAS538 was
added to a final protein concentration of 2 mg/ml.
Coimmunoprecipitation was as described above.
Ni2+ Affinity Purification of
eIF3--
(His)6-tagged eIF3 subunits from PH1-135 and
HV110-33 were purified on HIS-BindTM resin essentially as
described (15), except that the binding buffer contained 10 mM imidazole and the eluting buffer, 250 mM imidazole. Column fractions were analyzed by SDS-PAGE followed by
immunoblotting with anti-eIF3 antibodies and/or Coomassie staining. In
the case of (His)6-p110, fractions containing the eIF3
complex were pooled, concentrated, and proteins were separated at
4 °C by size exclusion chromatography on a HiLoad 16/60 Superdex
200-pg column (Pharmacia) in 20 mM HEPES/KOH, pH 7.5, 300 mM KCl, 5% (v/v) glycerol, 0.2 mM EDTA, and 7 mM Far Western Blot Analysis--
To generate the
35S-labeled proteins used as probes, the full-length open
reading frames of the following genes were PCR amplified and cloned
into pET28c to allow coupled in vitro
transcription/translation in a rabbit reticulocyte lysate (Promega):
PRT1 (p90), TIF32 (p110), TIF31
(p135), and TIF3 (eIF4B). eIF3 proteins obtained from the gel filtration step described above or purified recombinant full-length p33 or p33 Identification and Cloning of TIF31 and TIF32 Encoding p135 and
p110--
To complete the identification of genes encoding possible
subunits of eIF3, we report the cloning and characterization of TIF31 and TIF32. Each of these genes was cloned
by obtaining partial peptide sequences derived from the 135- and 21-kDa
proteins present in our preparations of eIF3. The sequences reported in
Table II were used to search yeast data bases, an open reading frame
was identified that encodes each set of peptides, and the genes were cloned as described under "Materials and Methods." pKSCos-TIF31 carries an insert with a 3834-bp coding region (assuming the first AUG
serves as the initiator codon), with 1752 and 1638 bp of DNA flanking
the 5' and 3' regions, respectively. An in-frame stop codon exists 21 nucleotides upstream of the first AUG, ruling out initiation sites
upstream. However, AUGs found 62 and 88 codons downstream of the first
AUG possibly may serve as initiator codons. The absence of a strict
consensus sequence surrounding the initiator AUG in yeast (28) does not
allow a firm prediction of which AUG is actually used. The hypothetical
p135 product of TIF31 translated from the first AUG contains
1277 amino acid residues with a calculated mass of 145,165 Da and a pI
of 6.0. A search of data bases indicates that p135 has no mammalian
homolog (but see "Discussion" below).
For p110, plasmid pHV110-1 was constructed as described under
"Materials and Methods" which contains a 2892 bp ORF with 920 and
748 bp of 5'- and 3'-flanking DNA, respectively. The first AUG is
preceded by an in-frame stop signal 16 codons upstream, whereas the
second in-frame AUG is 169 codons downstream. TIF32 mRNA
translated from the first AUG generates a protein with 964 amino acid
residues, a mass of 110,329 Da and a calculated isoelectric point of
6.1. The two peptide sequences from the 21-kDa protein are derived from
a stable C-terminal fragment of p110. Apparently, the p110 subunit is
readily degraded in yeast strains derived from W303, since little or no
p110 is detected in eIF3 prepared by classical protein fractionation
techniques (11). Northern blot hybridizations (not shown) with probes
derived from either the N- or C-terminal coding regions of
TIF32 show a single 3.2-kb band indicative of only one size
class of mRNAs. Further evidence that TIF31 and
TIF32 encode the p135 and p110 proteins in eIF3 is provided
below and is summarized under "Discussion."
The p110 amino acid sequence has a high content of charged residues
(33.5%) evenly distributed over the entire protein. Data base searches
identify homologous proteins from human, mouse, Caenorhabditis
elegans, and Nicotina tabacum with amino acid sequence identity/similarity values around 26/38%. The mammalian homologs encode the p170 subunit of eIF3. An alignment of these sequences has
been published elsewhere (5).
The p110 Subunit Is Essential for Cell Growth, but p135 Is
Not--
To explore the physiological role of p135 and p110 in eIF3,
we attempted to construct haploid strains lacking these proteins. Individually, most of the coding region of one of the TIF31
or TIF32 genes in the diploid strain W303 was deleted and
disrupted by insertion of HIS3 as described under
"Materials and Methods" and depicted in Fig.
1. The presence of both the wild type and disrupted allele was confirmed in each case by Southern blotting (not
shown). Following sporulation and tetrad dissection, four viable spore
colonies were obtained from the TIF31-disrupted diploid PH135D-8 (results not shown). The four spore colonies are of equal size, indicating that p135 is not required for rapid growth of yeast.
The presence of only the disrupted tif31::HIS3
allele and the absence of p135 protein in the two His+
spore colonies of one of the tetrads were confirmed by Southern blotting (not shown) and by Western immunoblotting of cell lysates (Fig. 2).
In contrast, when the disrupted TIF32 diploid strain,
HV110-D20/D, was sporulated and tetrads were dissected, a
2+:2
To demonstrate that the disruption of TIF32 is the sole
cause of defective cell growth, we cloned TIF32 with its
flanking regions into the centromeric plasmid pRS316 to generate
pHV110-2. The plasmid was transformed into HV110-D20/D and
transformants were sporulated and tetrads dissected. For all dissected
asci two large colonies were observed with the additional presence of
zero to two slower growing colonies (not shown). Marker analysis revealed that all fast growing colonies are His Depletion of p110 Inhibits Cell Growth and Protein
Synthesis--
The cellular level of eIF3-p110 was reduced by placing
TIF32 under control of glucose-repressible promoters.
pHPp110EX1 and pHPp110EX1-S were constructed to express
TIF32 from the strong GAL1 promoter and a weaker
GALS promoter as described under "Materials and
Methods." Each plasmid was transformed into the diploid strain HV110-D20/D, followed by tetrad dissection, to yield haploid strains HV110-24 and HV110-29, respectively, that express TIF32 only
from the plasmid. Both haploid strains grow rapidly (100 min doubling times) in YPG liquid medium and no overexpression of p110 is detected by Western immunoblotting (results not shown), even though these expression vectors normally produce higher levels of protein. This
suggests that p110 is rapidly degraded when not incorporated into the
eIF3 complex. The absence of intact p110 in our purified eIF3
preparations (11) also indicates that p110 is an unstable protein
readily susceptible to degradation. Because no difference was detected
in either growth rate or p110 expression level for strains HV110-24 and
HV110-29 (results not shown), all subsequent experiments were carried
out with HV110-29.
To determine the effects of p110 depletion, we compared the growth
rates of strains HV110-29 and HV110-13 (expressing TIF32 under control of its own promoter) in both galactose and glucose media
(Fig. 3A). Although both
strains grow equally well in galactose medium, only HV110-29 shifted to
glucose grows progressively more slowly, exhibiting a doubling time of
220 min after 3 h and cessation of growth by about 20-25 h (after
4 generations). The depletion results are consistent with the
requirement of p110 for cell division.
Depletion of an eIF3 subunit is expected to result in reduced protein
synthesis. The rate of protein synthesis in strain HV110-29 after the
shift to glucose was measured by [35S]methionine pulse
labeling as described previously (15). A substantial reduction of the
rate of protein synthesis is seen by 4 h, and after 10 h of
depletion the rate has dropped to about 20% of the non-depleted level
(Fig. 3B, solid circles). Little or no inhibition
of protein synthesis is seen in strain HV110-13 in glucose
(closed squares) or HV110-29 in galactose (open
circles), where p110 depletion does not occur; the slight decrease
in protein synthesis rates at later times likely is due to their
approach to stationary growth. The continued slow growth and low-level protein synthesis with HV110-29 after 12 h in glucose suggest either that p110 is not absolutely essential, or that low levels are
sufficient to provide some eIF3 function. Maintenance of low p110
levels may be due to incomplete glucose suppression of TIF32 expression.
Effects of p110 Depletion on Polysome Profiles and eIF3 Subunit
Composition--
To determine the phase of protein synthesis that is
inhibited upon p110 depletion, we analyzed the distribution of
ribosomes on mRNAs. Reducing the rate of translation initiation
without changing the rate of elongation leads to a reduction of
polysome size (number of ribosomes per mRNA) and an increase in
free 80 S ribosomes. When HV110-29 cells are shifted to glucose for
2 h, a time when protein synthesis already is inhibited over
2-fold (Fig. 3B), a reduction of polysome size and an
increase in 80 S ribosomes are observed compared with cells grown in
galactose for the same time (Fig. 4).
After 6 h the polysome size is severely reduced and after 12 h polysomes are hardly detectable. When the same cells are grown in
galactose, little or no reduction of polysome size is apparent after
6 h. The more pronounced effect on polysome size after 12 h
again may be due to exit from the exponential growth phase (see Fig.
3A). The results demonstrate that the reduced protein
synthesis rate observed after depletion of p110 (Fig. 3B) is
due to decreased efficiency of translation initiation, indicating that
eIF3-p110 plays an important role in the initiation pathway.
To demonstrate that p110 actually is depleted from the HV110-29 cells
after the shift to glucose, we analyzed the levels of p110 and some
other eIF3 subunits in crude cell lysates prepared at various times
after the carbon source shift. eIF3 subunit levels in equal amounts of
cell lysates were determined by Western blotting with specific
antibodies as described under "Materials and Methods." As shown in
Fig. 5A, an immunoreactive
protein of 110 kDa is indeed reduced over the time course of glucose
repression of TIF32 expression. This is seen with blots
subjected to treatment with a crude anti-eIF3 antiserum (upper
panel), and also with antibodies affinity purified from the
anti-eIF3 antiserum with recombinant p110 protein. Specific detection
of a 110-kDa protein in undepleted cells with the affinity-purified antibodies demonstrates that TIF32 indeed encodes a 110-kDa
protein.
The crude anti-eIF3 antiserum also detects the p33 subunit, whose level
does not change following p110 depletion. The p39 and p90 subunits are
not readily detected in lysates by the antiserum to eIF3, but analyses
with affinity-purified anti-p39 antibodies and an antiserum specific
for p90 show that p39 levels remain constant, whereas p90 levels
decrease. Quantitation of the Western blot signals (Fig. 5B)
reveals that the p110 level drops following the shift to glucose and by
12 h is only 10% of the normal concentration. The p90 level
decreases similarly. The quantitations also confirm that p39 and p33
levels, as well as that of eIF5A which is not associated with eIF3, do
not significantly change over the time course of the experiment.
Whether p110 depletion regulates the synthesis of p90 or makes p90 more
accessible to degradation remains to be determined. The observed
co-depletion of p90 and p110 suggests an interaction between the two
subunits. Since p90 is a proven component of eIF3, the finding supports
the view that p110 is associated with the eIF3 complex.
p110 Is a Integral Part of the eIF3 Complex--
Further evidence
that p110 is in fact a subunit of yeast eIF3 was obtained by
immunoprecipitation of the eIF3 complex. Strain HV110-33 was
constructed which overexpresses an N-terminal (His)6-tagged p110 protein as the sole source of p110. Since the growth rate of the
strain is normal, we conclude that the (His)6 tag is not detrimental to the function of the protein. Strains expressing (His)6-tagged p33 and p135, and Myc-tagged p39, were
subjected to protein labeling with [35S]methionine and
immunoprecipitation with (His)6-specific or anti-Myc antibodies as described under "Materials and Methods." To control for nonspecific immunoprecipitation, strain W303-1A, which contains no
(His)6- or Myc-tagged protein, was treated similarly. A
number of radiolabeled protein bands are seen in the autoradiogram of the tagged immunoprecipitates that also are seen with W303-1A (Fig.
6A), and these are labeled
with the letter u, indicating nonspecific precipitation. The
anti-(His)6 antibodies specifically precipitate proteins in
the HV110-33 lysate that have masses of 110 and 105 kDa, the former
corresponding to (His)6-tagged p110 and the latter probably
being its partial degradation product (labeled p110* in Fig.
6A). Additionally, proteins of 90, 39, and 33 kDa are
coimmunoprecipitated in the HV110-33 strain but not in strain W303-1A,
except that the 39-kDa protein is precipitated in both strains (see
"Discussion" below). A similar pattern of labeled
immunoprecipitated proteins is seen with strain PHS33 containing
(His)6-tagged p33 and strain PH1-39 containing Myc-tagged p39 (Fig. 6A). Interestingly, PH1-39 produces an additional
weak band of ~95 kDa (labeled with a dot) which might
correspond to the p93 (Nip1p) subunit of eIF3. The results indicate
that p110 is found in complexes with the 90, 39, and 33 kDa proteins.
Since all of these proteins correspond in size to subunits of eIF3, the
results imply that p110 is a part of the eIF3 complex. It is noteworthy
that in Fig. 6A, the bands corresponding to the (His)6- and Myc-tagged p33 and p39 subunits are very
intense, indicating substantial accumulation of these proteins in the
cells, whereas that for (His)6-tagged p110 is not intense,
consistent with an innate lability of p110.
To confirm the identity of the coimmunoprecipitated proteins detected
by autoradiography, nonradiolabeled lysates from strains W303-1A,
HV110-33, and PHS33 were subjected to immunoprecipitation with the
anti-(His)6 antibodies as described above. Following SDS-PAGE fractionation of the proteins in the immunoprecipitates, we
used anti-eIF3 antiserum (Fig. 6B, upper panel) as well as highly specific antibodies (lower panels) to p110, p90, p39,
and p33 to detect these eIF3 subunits in the immunoprecipitates. The p110, p90, and p33 subunits are not detected when the
immunoprecipitating antibody is omitted or in the precipitate from
W303-1A. However, they are present in the anti-(His)6
precipitates from strains HV110-33 and PHS33 when analyzed by anti-eIF3
and the specific antibodies. Detection of the prominent 105-kDa band
(just below the faint p110 band labeled by dots) from strain
HV110-33 with the anti-p110 antibodies indicates that this protein is
very likely a (His)6-p110 degradation product. In the case
of p90, its absence in the W303-1A precipitate is obscured by a defect
in the blot with the highly specific antibody; however, it clearly is
not present in the anti-eIF3 blot or in the labeled precipitate
analyzed in panel A. Unexpected is the apparent
precipitation of p39 from W303-1A by the anti-(His)6
antibodies seen in panel A and confirmed with the anti-p39
antibodies (panel B). Noteworthy is the fact that the p39
subunit from W303-1A also co-purifies by IMAC (see Fig. 8 below).
Although this might suggest that p39 contains an oligo-histidine tract,
no such tract longer than two residues is present in the protein (14).
We are not able to explain the presence of p39 in the W303-1A
precipitates. In summary, the coimmunoprecipitation experiments
demonstrate that p110 is part of a multisubunit complex containing at
least two of the known subunits of eIF3, namely p33 and p90.
A second approach to obtaining eIF3 complexes rapidly is to employ
IMAC. The eIF3 complex was purified on a HIS-BindTM resin
by utilizing the N-terminal (His)6-tagged p110 from strain HV110-33, as described under "Materials and Methods." Fractions from each elution step were subjected to SDS-PAGE and Western immunoblotting with the anti-eIF3 antiserum (Fig.
7A). The majority of eIF3
elutes with 250 mM imidazole. Because the 250 mM imidazole fraction contains numerous proteins as
detected by Coomassie staining (result not shown), it was subjected to
size exclusion chromatography on a Superdex 200 column as described
under "Materials and Methods." Aliquots of various fractions were
subjected to SDS-PAGE, and eIF3 was detected with the anti-eIF3
antiserum (Fig. 7B, upper panel) and by Coomassie staining
(middle panel). The strongest signals with the antibody are
seen in fractions 11-13. Coomassie staining also shows the presence of
33, 39, 90, and 110 kDa proteins in fractions 11-13. A complex of
identical subunit composition was obtained from strain HV110-42, which
expresses p110 with a C-terminal (His)6 tag (results not
shown). The proteins in fractions 11-13 elute with an apparent
molecular mass of 530 kDa (Fig. 7B, lower panel), consistent
with the size of the eIF3 complex.
p135 Also May be Present in a Complex with Other eIF3
Subunits--
Further evidence that TIF31 encodes a protein
associated with eIF3 was obtained by constructing strain PH1-135 which
expresses (His)6-tagged p135 as the sole source of this
protein from a GAL1 promoter. Ribosomal salt wash fractions
were prepared from strain PH1-135 and from strain PH1-414 which
expresses wild type p135 (untagged) from its own promoter. The
preparations were subjected to IMAC and analyzed by SDS-PAGE and
Western immunoblotting as described under "Materials and Methods."
When the blots are analyzed with antiserum to eIF3, a number of bands
corresponding to eIF3 subunits (135, 110, 90, 39, and 33 kDa) are
detected with strain PH1-135 which are not present with PH1-414
(results not shown). To demonstrate that these bands correspond to eIF3
subunits, the blot was probed with antibodies affinity purified against
recombinant p135, p110, p39, and p33, and with an antiserum specific to
p90 (Fig. 8). Each of these proteins is
found in the eluate fractions from the (His)6-tagged p135
strain, PH1-135, but barely (e.g. p39) or not at all in the
eluate fractions from the non-tagged strain PH1-414 (labeled
Wt in the figure). The faint p110 immunoreactive band with
PH1-135 suggests either partial degradation during purification or
dissociation from the eIF3 complex under the conditions used. However,
an association of p135 with the eIF3 complex is not always seen, as
immunoprecipitation of (His)6-p135 (Fig. 6A,
lane 5) does not bring down other eIF3 subunits. The
relationship of p135 to eIF3 is discussed below.
Interactions of p110 or p135 with Other eIF3 Subunits and
Initiation Factors--
To obtain further evidence for the presence of
p135 and p110 in the eIF3 complex, and to generate information leading
to a structural model of eIF3, we attempted to define subunit-subunit interactions by two methods: far Western blotting and two-hybrid analyses. Because depletion of p110 leads to depletion of p90 as well,
it seemed likely that these two proteins interact directly in the eIF3
complex. We therefore asked whether p110 might bind to p90 directly,
rather than through other subunits in the eIF3 complex. As probes for
the far Western blot analyses, p110 and p90 were synthesized in
vitro in reticulocyte lysates in the presence of
[35S]methionine as described under "Materials and
Methods." Full-length p110 and p90, as well as numerous smaller
proteins likely due to degradation during the translation incubation,
are seen when analyzed by SDS-PAGE and autoradiography (Fig.
9A). The radiolabeled p110 and
p90 proteins were used to probe eIF3 subunits fractionated by SDS-PAGE.
The eIF3 complexes were purified from strain HV110-33 by IMAC and
Superdex chromatography as described above (Fig. 7B). Radiolabeled p110 binds only to p90 (Fig. 9B, lane
2), whereas radiolabeled p90 binds most strongly to p110
(lane 1), confirming the interaction. p90 also binds to
another protein of ~95 kDa, possibly a degradation product of p110,
and less strongly to p39. Interactions of p90 with p39 and p110 also
have been detected by two-hybrid analyses and by glutathione
S-transferase pull-down experiments (29, 30).
Possible interactions of p135 with other eIF3 subunits were sought by
far Western blotting. Radiolabeled p135 binds to recombinant p33 (Fig.
9C, lane 3), but not to recombinant p39 or p62
under these conditions (results not shown). To further substantiate this interaction, the two-hybrid system was employed as described in
the legend to Table III. A positive
interaction is seen between p135 and p33 (Table III), but not with p93
or p39 although the fusion proteins are expressed as detected by
Western immunoblotting (not shown). Thus a p135-p33 interaction is
confirmed.
Because mammalian eIF4B has been shown by far Western blotting to bind
to the p170 subunit of human eIF3 (31), we asked whether or not yeast
eIF4B might bind to the yeast homolog of human p170, namely p110.
Radiolabeled eIF4B (Fig. 9A) was used to probe the eIF3
complex that contains (His)6-p110, but no binding was
detected (Fig. 9B, lane 3). Instead, labeled eIF4B binds to recombinant p33 (Fig. 9C, lane 1), but not to recombinant
p39, p93, or p110 (results not shown). The binding interaction also is
seen in a two-hybrid analysis (Table III). The eIF4B-p33 interaction appears to depend on the C-terminal 71 amino acid residues of p33 which
contain the RNA recognition motif (29), as the mutant form lacking this
region fails to bind to eIF4B (Fig. 9C, lane 2). The result
suggests that eIF4B binds to the C-terminal region of p33. We cannot
rule out the possibility, however, that the C-terminal deletion of p33
causes misfolding of other parts of the protein and thereby its
loss of eIF4B binding activity.
We report here the cloning and analysis of TIF31 and
TIF32 encoding p135 and p110, the largest and second largest
proteins associated with eIF3. The conclusion that TIF31
encodes the 135-kDa protein found in eIF3 preparations is based on 4 major lines of evidence. 1) The encoded protein contains regions that
match 51 out of 55 residues of 5 sequenced peptides derived from the
135-kDa protein in purified eIF3. 2) The calculated molecular mass of the protein encoded by TIF31 (145,165 Da) is consistent with
the apparent mass of 135 kDa determined by SDS-PAGE. 3) Antibodies in
an antiserum raised against purified eIF3 recognize a polypeptide of
135 kDa in crude extracts prepared from TIF31-expressing
cells but not in extracts of cells lacking the TIF31 gene.
Affinity-purified antibodies raised against recombinant p135 give the
same result (data not shown). 4) When (His)6-tagged p135
is expressed in cells where the chromosomal copy of TIF31 is
disrupted, the protein that is immunoreactive with anti-p135 antibodies
possesses an apparent mass of 135 kDa. We conclude that the cloned
TIF31 gene encodes the 135-kDa protein found in our eIF3 preparations.
The role of p135 in yeast cells is not known. TIF31
deletion/disruption displays no obvious phenotype when cells are grown at temperatures lower or higher than 30 °C (data not shown) and thus
is not an essential gene. Furthermore, analysis of polysome profiles of
strain PH135H-B carrying the disrupted gene reveals no defect in
protein synthesis (results not shown). It is noteworthy that p135 is
not related to any subunit of mammalian eIF3, but is 27% identical and
50% similar to a 150-kDa protein encoded in the
Dictyostelium genome. Recently, Zhu et al. (32)
isolated the Dictyostelium gene and named it
cluA. Disruption of cluA impairs cytokinesis and
results in the clustering of mitochondria near the cell center. While
this manuscript was in preparation, Fields et al. (33)
cloned the S. cerevisiae homologue of Dictyostelium cluA and named it CLU1. CLU1 and TIF31 are
identical. Consistent with the results reported here, deletion of
CLU1 from S. cerevisiae does not affect cell
viability even when cells are exposed to stress conditions such as heat
and osmotic shock. However, disruption does result in the clustering of
mitochondria as was observed in Dictyostelium. It is
difficult to envision a mechanism whereby eIF3 directly affects
cytokinesis and the clustering of mitochondria. The results suggest
instead that p135 has a role in cells other than, or in addition to,
initiation of protein synthesis. Nevertheless, there is strong evidence
that p135 can associate with eIF3: it is present in highly fractionated
eIF3 preparations; it co-purifies with other eIF3 subunits by IMAC when
either p135 (Fig. 8) or p33 (15) is (His)6-tagged; and it
binds specifically to p33 as determined by far Western blotting and
two-hybrid analyses. In effect, the precise physiological function of
p135 and why it is sometimes found associated with the eIF3 complex are unclear.
Evidence that TIF32 encodes the p110 subunit of eIF3 also is
strong. 1) The sequence of two internal peptides from p21 match precisely the C-terminal region of the putative product of
TIF32. Although no 110-kDa protein is detected in our
initial preparations of eIF3 (11), the 21-kDa protein in such
preparations is derived from the p110 subunit through partial
proteolysis, likely during the purification process. 2)
TIF32 encodes a putative protein of 110 kDa which is present
in eIF3 preparations prepared rapidly by IMAC. 3) Antibodies affinity
purified from anti-eIF3 with recombinant p110 expressed from
TIF32 recognize a 110-kDa protein in eIF3 rapidly purified
by IMAC. Taken together with other experiments discussed below, the
data clearly establish that the 110-kDa protein in eIF3 is encoded by
TIF32.
In contrast to p135, the p110 subunit plays an important role in
protein synthesis. The protein is essential for growth of yeast, and
depletion results in slowed growth and an inhibition of protein
synthesis. Polysome profile analyses of p110-depleted cells indicate
that p110 is required for efficient initiation, consistent with its
being a subunit of eIF3. Similar effects are observed when other eIF3
subunits are depleted (14, 15).
Considerable additional evidence has been generated to demonstrate that
p110 is a component of the eIF3 complex. Coimmunoprecipitates from
lysates containing epitope-tagged p110 contain p33 and p90 previously
characterized as eIF3 subunits. In addition, precipitates from lysates
with other tagged eIF3 subunits contain p110. The p110 subunit
interacts with p90 as shown by far Western blotting experiments.
Depletion of p110 causes degradation of p90 as well, consistent with a
direct interaction within the eIF3 complex. Furthermore, the p110
subunit is homologous to the p170 subunit of mammalian eIF3 (5), thus
supporting the notion that it is present in yeast eIF3. In work
published while this manuscript was in preparation, the p110 subunit
was identified by mass spectroscopic analysis of preparations of eIF3
obtained from cells expressing (His)6-tagged p90 (16) and
was shown to interact with p90 by glutathione S-transferase
pull-down experiments (30).
The experiments above establish that p110 is an integral part of the
eIF3 complex and that it plays an important role in the initiation
phase of protein synthesis. A gene identical to TIF32, called RPG1, was cloned independently through a screen for
yeast proteins that cross-react with antibodies to mammalian
microtubule-associated protein 2 (34). The protein product of
RPG1 was not characterized then, but analysis of a
temperature-sensitive rpg1-1 allele demonstrated that cells
shifted to nonpermissive temperature arrest in the G1 phase
(34). A similar phenotype was observed for mutant alleles of
PRT1 (encoding p90) (35) and TIF34 (encoding p39)
(29). These data suggest that a properly functioning eIF3, presumably providing efficient translation initiation and protein synthesis, is
required for progression through START and the cell cycle (36, 37). In
a recent publication (38), RPG1 was further characterized by
employing the temperature-sensitive mutant to show that initiation of
protein synthesis is diminished. Their results and the results reported
here are entirely compatible and support and extend each other.
The composition of yeast eIF3 has been controversial, as different
preparations appear to contain different subunits with different
apparent masses (11, 39). (His)6-tagging of eIF3 subunits
followed by IMAC has shed light on the composition of eIF3. When
(His)6-tagged p90 is employed, a complex is isolated that
contains p33, p39, p90, p93, and p110 as well as eIF5 (16). This may
represent a core complex of eIF3, as it possesses activity in
vitro. In the IMAC and immunoprecipitation experiments reported here, tagged p33, p39, or p110 lead to co-purification of the same 5 subunits, except for p93 which is known to be unstable in strains
derived from W303 (17) and presumably is degraded in the lysates
examined. It is noteworthy that only the 5 core subunits have homologs
in mammalian eIF3 (see below).
The results presented above indicate that p135 also may associate with
the eIF3 complex, albeit likely with lower affinity than the core
subunits. Since p16 and p62 are co-immunoprecipitated with antibodies
specific for other eIF3 subunits (12, 13), these proteins may be
present in eIF3 as well, although p62 is not required for eIF3 activity
in vitro (16). We lack suitable antibodies to p16, p62, and
p93, and thus are not able to determine unambiguously whether or not
they are present in our (His)6-tagged preparations. Taking
into account all of the evidence available, we believe that yeast eIF3
may contain up to 8 subunits, although the p16, p62, and p135 subunits
appear not to be as firmly associated with the complex as the core
subunits. The more loosely associated subunits may not play essential
roles in all aspects of eIF3 function.
The 5 core subunits in yeast eIF3 are homologous with 5 proteins
identified as subunits of mammalian eIF3: yeast p110 is homologous with
mammalian p170 (29% identity); p93, with p110 (31%); p90, with p116
(31%); p39, with p36 (46%); and p33 with p44 (33%). Thus yeast and
mammalian eIF3 subunit structures are conserved, although more weakly
than most of the other initiation factors (1). This conservation is
reflected in the ability of yeast eIF3 to substitute for mammalian eIF3
in an in vitro initiation assay (11). On the other hand,
there is considerable structural diversity in that the less tightly
bound yeast subunits are not related to mammalian eIF3 subunits
(although yeast p16 is homologous with mammalian eIF1). Since the
overall eIF3 structures appear to be related, interactions of eIF3 with
other translational components might be conserved as well. One such
interaction concerns mammalian eIF4B, which binds to the p170 subunit
of eIF3 (31). The interaction involves the DRYG-rich motif in the
central region of eIF4B as demonstrated by far Western blotting. It has
been postulated that the C-terminal repeat region of p170 also may be
involved in this interaction (5). Our attempts to detect a yeast eIF4B
interaction with yeast p110, the homolog of mammalian p170, were not
successful. Close examination of the yeast and mammalian eIF4B
sequences (40, 41) indicates that the two proteins are poorly conserved
(17% identity), especially in the DRYG region implicated in the
interaction with eIF3. Furthermore, yeast p110 differs considerably
from the mammalian p170 subunit. The N-terminal third of yeast p110
shares 35% sequence identity with eIF3-p170, but the C-terminal
two-thirds is less well conserved (20% identity) and the C-terminal
repeat region of human p170 is lacking entirely in yeast p110. Our
two-hybrid and far Western blotting results indicate that yeast eIF4B
interacts instead with the p33 subunit of eIF3. Thus the interaction of yeast eIF4B with eIF3 may differ from that occurring in mammalian cells. Along similar lines, mammalian eIF4G binds to eIF3, but no such
interaction has been demonstrated in yeast although numerous attempts
have been made to do so. Further work is required to determine the
three-dimensional structure of eIF3 in both yeast and mammals and to
define the subunits responsible for its interaction with other
translational components. Until then, caution must be exercised when
extrapolating results from one system to the other.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Yeast strains used in this study
Partial peptide sequences from p135 and p110
3 Prime Inc., Boulder, CO) to generate pNo-NH135F. A
BamHI fragment from pNo-NH135F was subcloned into the
BamHI site of p414Gal1 (25) to generate p414Gal1-NH135.
Transformation of PH135H-B with this plasmid yielded PH1-135. An
N-terminal-(His)6-tagged 151-amino acid fragment of p135
was constructed by PCR amplification and ligation into pET28c to yield
pET-NH135
C.
C was purified with
HIS-BindTM resin (Novagen) from E. coli BL21
(DE3) transformed with pET-NH135
C, and used to raise antibodies
against p135 in rabbits (BAbCo). Antibodies specific for p110 were
obtained from rabbit anti-yeast eIF3 antiserum (11) by affinity
purification (26) against a urea-purified His-tagged C-terminal
607-amino acid fragment of p110 fused to the TRX protein expressed in
E. coli. Affinity-purified antibodies to p135 were prepared
similarly. Rabbit antisera against p33, p39, and Prt1p (a gift from
Alan Hinnebusch (National Institutes of Health)) have been described
previously (15).
-mercaptoethanol.
C (15) were separated by SDS-PAGE and blotted onto polyvinylidine difluoride membranes. Membranes were washed 4 times for
10 min in buffer D (20 mM HEPES, pH 7.4, 10% (v/v)
glycerol, 1 mM dithiothreitol, 150 mM KCl, 5 mM MgCl2) and blocked by incubation for 1 h in buffer D containing 5% (w/v) bovine serum albumin. Membranes were
incubated for 5 h at the indicated temperatures with the in
vitro-translation extracts containing a radiolabeled protein
supplemented with 3% (w/v) bovine serum albumin in buffer D, unbound
protein was removed by washing the membranes 2 times for 10 min in
buffer D containing 0.1% (v/v) Nonidet P-40 and the membranes were
subjected to autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Disruption of TIF32 and
TIF31. The figure shows schematic descriptions of
the TIF32 (3.9 kb, upper portion) and
TIF31 (4.6 kb, lower portion) regions, with the
ORFs shown as stippled rectangles translated from left to
right. Restriction enzyme sites mentioned in the text also are
indicated. The inner dotted lines mark the DNA deleted and
replaced by HIS3 (shown as an open rectangle),
whereas the outer dotted lines depict the region excised
(shown below the gene) and used for transformation of the diploid W303
as described under "Materials and Methods." The gray vertical
bar at the 3'-end of TIF32 and TIF31
identifies the coding regions not deleted. PCR primers used to generate
the 3.9-kb DNA fragment are indicated by arrows in the
TIF32 scheme.
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Fig. 2.
Identification of eIF3-p135 expressed from
TIF31. Yeast lysates were prepared from strain
PH135D-8 (TIF31 disrupted diploid, lane 1) and
from the four spore colonies obtained by dissection of one of its
tetrads (lanes 2-5). Protein concentrations were determined
by the BCA assay (Pierce). Equal amounts of lysate proteins (30 µg)
were fractionated by SDS-PAGE (42), and the gel was subjected to
Western immunoblotting with a rabbit anti-eIF3 antiserum. The figure
shows a scan of the blot developed with nitro blue tetrazolium and
5-bromo-4-chloro-3-indoyl phosphate (Sigma). eIF3 subunits are
identified on the right; positions of molecular mass markers
are shown on the left in kDa. PH135H-A and D are the
His haploids carrying TIF31; strains PH135H-B
and -C are His+ haploids carrying
tif31::HIS3.
segregation pattern for cell growth was
observed for all dissected spores (results not shown). All viable
spores were His
, demonstrating the absence of the
disrupted tif32::HIS3 allele. Microscopic analysis
of the nonviable spores showed that the spores are capable of
undergoing two to four cell divisions before ceasing to grow. The
observed segregation pattern together with the microscopic analysis
demonstrate that a functional TIF32 gene is required for
cell growth, but not for germination.
and
therefore carry a wild type chromosomal allele. All slow growing
colonies are His+ and Ura+, demonstrating
disruption of the chromosomal copy of TIF32 and the presence
of the plasmid carrying a wild type TIF32 gene. One His+/Ura+ haploid strain was named HV110-13.
The reduced growth rate of cells carrying pHV110-2 as sole source of
p110 might be due to the lack of TIF32 regulatory elements
in the plasmid, but such elements would have to be located more than
920 bp upstream from the translational start codon. TIF32
carried on the plasmid is a recessive allele, because several of the
normal growing His
cells are also Ura+ and
therefore carry both TIF32 alleles. The slow growth
phenotype is not seen in liquid cultures and was not analyzed further.
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Fig. 3.
Effects of p110 depletion on cell growth and
protein synthesis rates. Panel A, exponentially growing
cultures of strains HV110-13 (squares) and HV110-29
(circles) were grown in YPG medium to an OD600
of 2.0. Cells were diluted into galactose-containing medium (YPG,
open symbols) or glucose-containing medium (YPD, solid
symbols) to an OD600 of ~0.07 and cell growth was
monitored well into the saturation phase. Panel B,
determination of in vivo protein synthesis rates by pulse
labeling with [35S]methionine. At the indicated time
points, cells from strain HV110-13 grown in YPD (solid
squares) and strain HV110-29 grown in YPG (open
circles) or YPD (solid circles) were removed and
labeled as described previously (15). The results shown are
representative of independent experiments performed three times.
Protein synthesis rates are expressed as counts/min × 10 4 of incorporated methionine × µg
1 protein min
1.
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Fig. 4.
Effect of p110 depletion on polysome
profiles. Strain HV110-29 was shifted into medium containing
either galactose (right panels) or glucose (left
panels), cells were chilled and harvested with cycloheximide (100 µg/ml) at the indicated times, lysed and analyzed by sucrose gradient
centrifugation essentially as described (15). Absorption (254 nm) was
monitored during upward displacement with a gradient fractionator
(ISCO). The figure shows A254 scans of the
gradients, with sedimentation from left to right.
The large peak at about fraction 5 corresponds to 80 S ribosomes.
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Fig. 5.
Quantitation of eIF3 subunits in
p110-depleted cells. Panel A, lysates (30 µg protein)
from HV110-29 cells shifted to galactose (left 2 lanes) or
glucose (right 8 lanes) for the times in hours indicated
above the lanes were analyzed by SDS-PAGE and immunoblotting
with anti-eIF3 antibodies. Lysate protein concentrations were
determined by the BCA assay (Pierce). Loading of equal amounts of
protein was confirmed by Coomassie Blue staining of parallel gels (not
shown) and by probing with anti-eIF5A. Blots were probed with anti-eIF3
antiserum (top panel) or with affinity-purified antibodies
against the indicated subunits (lower panels) as described
in the legend to Fig. 2. Molecular weight standards are indicated to
the left of the upper panel and eIF3 subunits are
identified on the right. Panel B. The signal
intensities for the individual subunits shown in panel A
were quantitated with a GS-505 Imager System (Bio-Rad). Signal
intensities for p110 ( ), p90 (
), p39 (
), p33 (
), and eIF5A
(
) for the various times after the shift to glucose are reported as
relative values compared with the signal obtained after shifting to
galactose for 8 h (100%).
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Fig. 6.
Coimmunoprecipitation of p110 with other eIF3
subunits. Panel A, coimmunoprecipitation of eIF3 from
35S-labeled proteins in crude lysates from strains W303-1A
(control), PHS33 ((His)6-tagged p33), PH1-39 (c-Myc-tagged
p39), HV110-33 ((His)6-tagged p110), and PH1-135
((His)6-tagged p135). Coimmunoprecipitation was carried out
with anti-(His)6 antibody (CLONTECH)
for lanes 1, 2, 4, and 5 and anti-c-Myc antibody
(Santa Cruz Biotechnology) for lane 3 as described under
"Materials and Methods." The immunoprecipitates were fractionated
by SDS-PAGE; an autoradiogram of the gel is shown. Migration positions
of molecular mass markers are shown on the left and eIF3
subunits are identified on the right. A putative p110
degradation product is labeled p110*, the 95-kDa band is labeled with a
dot, and nonspecifically bound proteins are denoted by the
symbol u. Panel B, coimmunoprecipitations with or
without anti-(His)6 antibodies (labeled above
the gel lanes) of eIF3 subunits from lysates derived from strains
W303-1A (control), HV110-33 (expressing (His)6-p110), and
PHS33 (expressing (His)6-p33). Proteins were
coimmunoprecipitated as described under "Materials and Methods,"
separated by SDS-PAGE, and detected with anti-eIF3 serum (upper
panel) or affinity-purified antibodies against individual eIF3
subunits as indicated. Migration positions of molecular mass markers
and eIF3 subunits are indicated on the left and
right, respectively. The positions of full-length p110 are
indicated by dots.
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Fig. 7.
Affinity purification of eIF3 by IMAC.
Panel A, analysis of different fractions during eIF3
purification on a HIS-BindTM resin. Proteins were analyzed
by SDS-PAGE and immunoblotting with anti-eIF3 antiserum. The gel
contains the indicated subcellular fractions (lanes 1-3) as
follows: S-30 and S-100 (30 µg of protein); high salt wash (15 µg).
After the flow-through (fourth lane) was collected, proteins were
eluted from the IMAC column as described under "Materials and
Methods" with 5 mM (lanes 5-8), 30 mM (lanes 9-10), and 250 mM
(lanes 11-13) imadazole, and 10-µl aliquots were analyzed
(fraction numbers are shown above each gel lane). Panel B,
size exclusion chromatography of eIF3 from the IMAC column. Proteins
eluted with 250 mM imidazole (fractions 1 and 2) were
subjected to fractionation on a Superdex 200 column as described under
"Materials and Methods." Aliquots (10 µl) of column fractions
were analyzed by SDS-PAGE and proteins were detected with anti-eIF3
antiserum (upper panel), or 100-µl aliquots of each
fraction were precipitated with trichloroacetic acid, separated by
SDS-PAGE, and proteins were stained with Coomassie Blue (middle
panel). Column fractions are labeled above the
upper panel. The lower panel shows the column
absorption profile at 280 nm, with fraction numbers indicated
below. Elution positions of molecular mass markers are
indicated by arrows: thyroglobulin (669 kDa), apoferritin
(443 kDa), -amylase (200 kDa), bovine serum albumin (66 kDa).
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Fig. 8.
Analysis of eIF3 subunits associated with
(His)6-tagged p135. Equal amounts of protein in the
high salt wash fractions (Load) prepared from strain PH1-414 (labeled
Wt, expressing untagged p135) and PH1-135 (expressing
(His)6-p135) were fractionated by IMAC as described (15).
Unbound (wash, with 10 mM imidazole) and bound (eluate,
with 250 mM imidazole) fractions were analyzed by 7.5%
SDS-PAGE and immunoblotting with the affinity-purified antibodies
indicated on the right. The figure shows relevant portions
of the immunoblots developed by nitro blue tetrazolium and
5-bromo-4-chloro-3-indoyl phosphate color reagents.
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Fig. 9.
Far Western blot analyses with eIF3 subunits
and eIF4B. Panel A, analysis of the radiolabeled
probes. In vitro transcription/translation reactions were
employed to label p90, p110, p135, and eIF4B as described under
"Materials and Methods." A 2-µl aliquot of each translation
reaction was fractionated by SDS-PAGE, proteins were blotted on a
polyvinylidine difluoride membrane, and radiolabeled products were
detected by autoradiography. Migration positions of molecular mass
markers are indicated on the left. Panel B, eIF3
purified by IMAC and Superdex 200 size exclusion chromatography as
described in the legend to Fig. 7 was subjected to SDS-PAGE and blotted
on a polyvinylidine difluoride membrane. The blot was probed with the 3 different in vitro labeled proteins as described under
"Materials and Methods." Migration positions of eIF3 subunits are
indicated on the right. The panel shows a scan of the
autoradiogram. Panel C, radiolabeled eIF4B was used to probe
equal amounts of purified recombinant p33 (lane 1) and
p33 C (lane 2) and radiolabeled p135 was used to probe
recombinant p33 (lane 3) as described above. A scan of the
autoradiogram is shown. Coomassie Blue-stained gels with purified
recombinant p33 and p33
C have been shown elsewhere (29).
Two hybrid interactions within yeast eIF3 subunits
-galactosidase activity according to the manufacturer's protocol.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Rani Agrawal and Susan MacMillan for excellent technical assistance, Alan Hinnebusch for the anti-p90 antibodies, Alan Sachs for yeast strain YAS538, Marie-Hélène Verlhac for plasmids and antibodies, and Margaret Clarke and Alan Hinnebusch for communicating results prior to publication.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM22135 from the United States Public Health Service (to J. W. B. H.) and a postdoctoral fellowship from the German Research Society (to H. P. V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Institut für Molekularbiologie und
Tumorforschung, Philipps-Universität Marburg, 35037 Marburg, Germany.
§ Present address: Genelabs Technologies, Inc., Redwood City, CA 94063.
¶ To whom correspondence syould be addressed. Tel.: 530-752-3235; Fax: 530-752-3516; E-mail: jwhershey{at}ucdavis.edu.
2 G. L. Mayeur and J. W. B. Hershey, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: eIF, eukaryotic initiation factor; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; IMAC, immobilized metal affinity chromatography; kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction.
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REFERENCES |
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