From the Department of Biological Chemistry, University of California School of Medicine, Davis, California 95616
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Eukaryotic translation initiation factor-3 (eIF3)
is a large multisubunit complex that binds to the 40 S ribosomal
subunit and promotes the binding of methionyl-tRNAi
and mRNA. The molecular mechanism by which eIF3 exerts these
functions is incompletely understood. We report here the cloning and
characterization of TIF35, the Saccharomyces
cerevisiae gene encoding the p33 subunit of eIF3. p33 is an
essential protein of 30,501 Da that is required in vivo for
initiation of protein synthesis. Glucose repression of
TIF35 expressed from a GAL1 promoter results in
depletion of both the p33 and p39 subunits. Expression of
histidine-tagged p33 in yeast in combination with Ni2+
affinity chromatography allows the isolation of a complex containing the p135, p110, p90, p39, and p33 subunits of eIF3. The p33 subunit binds both mRNA and rRNA fragments due to an RNA recognition motif near its C terminus. Deletion of the C-terminal 71 amino acid residues
causes loss of RNA binding, but expression of the truncated form as the
sole source of p33 nevertheless supports the slow growth of yeast.
These results indicate that the p33 subunit of eIF3 plays an important
role in the initiation phase of protein synthesis and that its
RNA-binding domain is required for optimal activity.
There are five major steps involved in initiation of eukaryotic
protein synthesis: dissociation of ribosomes into 40 S and 60 S
subunits, binding of Met-tRNAi to the 40 S ribosomal
subunit; binding of mRNA to the 40 S preinitiation complex;
scanning and initiation codon recognition, and the joining of the 60 S
subunit to the 40 S initiation complex (1, 2). The initiation phase is
promoted by at least 10 soluble proteins known as eukaryotic initiation
factors (eIFs).1 eIF3 is the
largest and most complex initiation factor, comprising 10 or more
subunits in mammalian cells (3) and up to eight subunits in yeast (4,
5). Several functions have been assigned to eIF3 in the translation
initiation pathway in mammalian cells (1). It promotes dissociation of
80 S ribosomes into 40 S and 60 S subunits and stabilizes
Met-tRNAi binding to the 40 S ribosomal subunit. eIF3 also
is required for mRNA binding to 40 S and 80 S ribosomes in part
through its interaction with eIF4G (6) and/or eIF4B (7). Thus, eIF3
plays a central role in the initiation process.
To assist in elucidating the function of eIF3, attention has been given
to the corresponding factor in yeast, where genetic approaches are
feasible. The yeast eIF3 complex was purified on the basis of its
stimulation of methionylpuromycin synthesis dependent on formation of
80 S initiation complexes in an assay composed of mammalian components
(5). The active eIF3 preparation contains eight subunits with apparent
molecular masses of 16, 21, 29, 33, 39, 62, 90, and 135 kDa (5).
However, further studies revealed that the 29-kDa protein is a
truncated form of p39 (8) and that the p21 protein originates by
partial degradation of a 110-kDa protein.2 A somewhat similar
preparation was isolated and purified by using its stimulation of
protein synthesis in lysates prepared from a
prt1-1 temperature-sensitive strain of yeast. The
reported molecular masses of the subunits in this preparation of
putative eIF3 are 130, 80, 75, 40, and 32 kDa (9).
The following yeast genes already have been characterized as encoding
subunits of eIF3: SUI1 (p16) (10), TIF34 (p39)
(8), GCD10 (p62) (11), PRT1 (p90) (5), and
NIP1 (p93) (12). SUI1, encoding the smallest
subunit of eIF3 (p16), was first identified genetically by recessive
mutations that allow utilization of a UUG triplet as a translation
initiation codon (13). TIF34 was cloned by obtaining a
partial amino acid sequence of p39 and matching it to translated
sequences in the data base (8) or by identifying the yeast homolog of
the human TRIP1 protein (eIF3-p36) (14). The gene encoding p62 was
identified through mutations that constitutively derepress the
expression of GCN4 in rich medium (11). The p90 (Prt1)
subunit was shown to affect Met-tRNAi binding to the 40 S
ribosomal subunit (15, 16). The gene for p93 (NIP1) was first identified through a mutation that affects nuclear import of
proteins (17). Analysis of strains carrying temperature-sensitive mutations in each of these five genes demonstrated that the proteins are required for initiation of protein synthesis in vivo
(11-15).
Genes encoding the p135 (TIF31) and p110 (TIF32)
subunits have been identified,2 and a preliminary account
of their cloning has been published (4). To complete the cloning of the
genes coding for yeast eIF3 subunits, we focused on the p33 subunit of
eIF3 encoded by TIF35 (for translation
initiation factor 3, the
5th subunit). TIF35 was first cloned through a
two-hybrid analysis with p39 (TIF34) as bait (14). In this
report, identification of the gene product as a component of eIF3 was
based in large part on the work described here. During the preparation
of this manuscript, reports were made identifying p33 in eIF3 complexes
prepared by affinity chromatography or immunoprecipitation (18, 19).
However, further characterization of p33 and its role in protein
synthesis were not addressed in the reports. We describe here the
cloning of TIF35, characterization of the function of p33 in
protein synthesis, and analysis of its RNA recognition motif (RRM).
This completes the detailed characterization of the genes encoding
eight eIF3 subunits in yeast.
Strains and Media--
Escherichia coli strain
XL1-Blue was used for plasmid propagation. The various
Saccharomyces cerevisiae strains used in this work are based
on strain W303-1A (MATa,
leu2-3,112 his3-11,15 ade2-1
trp1-1 can1-100) or its isogenic diploid,
W303 (20). Yeast cells were grown at 30 °C in YP or synthetic (S)
medium supplemented with the relevant amino acids and 2% glucose (YPD or SD) or 2% galactose (YPG or SG) as described previously (21); growth was monitored by measuring absorbance at 600 nm
(A600). Sporulation was carried out at room
temperature on plates containing 0.3% potassium acetate, 0.02%
raffinose, and 10 µg/ml each amino acid. Tetrad dissections and DNA
transformations were carried out by standard procedures (22).
Cloning and Disruption of TIF35--
The yeast gene encoding
eIF3-p33 was tentatively identified as a homolog of the gene for
mammalian eIF3-p44. The mouse eIF3-p44 sequence (34) was used to
conduct a TblastN search of the entire yeast genome
sequence,3 and a putative
protein was identified whose amino acid sequence exhibits 33.3%
identity to mouse eIF3-p44. The coding sequence, together with flanking
regions, was amplified from total yeast genomic DNA by PCR. The
upstream (5'-CTCTTCACGATCTGCAAAAGTCCCAACATT-3') and downstream,
(5'-GCTTATGGTGGTGGTGCTTCTTATAGCGCC-3') primers generate a single 2.1-kb
DNA fragment. The fragment was gel-purified and subcloned into pNoTA (5 Prime
To disrupt the TIF35 gene, pNo-TIF35 was digested with
BsaAI and BsmI to remove 91% of the
TIF35 coding region, and a 1.7-kb BamHI DNA
fragment containing HIS3 was inserted to generate
pNoTAtif35::HIS3. The upstream and downstream cloning
primers described above were used to generate a 3.1-kb
tif35::HIS3 PCR fragment, which was transformed
into the diploid yeast strain W303 to create a one-step gene
deletion/disruption (23). One of the stable His+
transformants (PH33D-7) was selected, and the disruption of one of the
TIF35 genes was confirmed by Southern blot analysis (data not shown).
Plasmid Constructions--
pRS316-TIF35 was constructed by
digesting pNo-TIF35 with BamHI and subcloning the resulting
fragment into BamHI-cleaved pRS316 (American Type Culture
Collection). p415Gal1-NH33 is a CEN4 LEU2 plasmid that
allows expression of N-terminal His6-tagged p33 under the
control of the GAL1 promoter. For its construction, the p33 coding sequence was PCR-amplified from pNo-TIF35 using
5'-CCCGGATCCGCCATGGGTAGAGGTTCTCACCATCACCATCACCATATGAGTGAACATATTCTGTGCATCTA-3' (tagged with BamHI and NcoI sites (underlined);
the bases corresponding to the initiation codon of wild-type
TIF35 are in boldface) and 5'-CCCGTCGACCTCGAGCATATTCTGTGCATCTA-3' (tagged
with SalI and XhoI sites (underlined); the bases
corresponding to the stop codon are in boldface). The resulting 0.9-kb
DNA fragment was subcloned into pNoTA and sequenced, yielding pNo-NH33
(NH represents N terminus tagged with His6). To construct
plasmids p415GalL-NH33 and p415GalS-NH33, pNo-NH33 was digested with
BamHI and SalI, and the 0.9-kb fragment was
subcloned into the BamHI/SalI sites of p415GalL
and p415GalS, respectively (24). p415p33
To express a recombinant form of His-tagged p33 in E. coli,
pET-NH33 was constructed by inserting the
NcoI/XhoI fragment from pNo-NH33 into the
corresponding sites in pET28c (Novagen). To generate pET-NH33 Construction of Haploid Yeast Strains PHS33, PHL33, and
PH133--
Strain PH33D-7 was transformed with p415GalS-NH33,
p415GalL-NH33, or p415p33 Antibodies--
Rabbit antiserum against yeast eIF3 has been
described previously (5). Rabbit anti-Prt1 antibody was a gift from
A. G. Hinnebusch (National Institutes of Health), and
affinity-purified rabbit anti-p39 antibody (14) was kindly provided by
M.-H. Verlhac. To obtain anti-p33 antiserum, rabbit antibodies were
raised against purified His6-p33 (BAbCo). For
affinity-purified anti-recombinant p33 antibodies, His6-p33
was overexpressed from pET-NH33 in E. coli BL21(DE3),
partially purified by Ni2+ affinity chromatography
(Novagen), and fractionated by SDS-PAGE, followed by transfer to a
polyvinylidene difluoride membrane (Millipore Corp.). The anti-p33
antiserum was incubated with a piece of the membrane containing
His6-p33, and antibodies bound to His6-p33 were
eluted with 0.2 ml of low-pH buffer (0.2 M glycine HCl and 1 mM EGTA, pH 2.5). The eluate was quickly neutralized with
0.2 ml of 100 mM Tris-HCl, pH 8.8; diluted with 1 volume of
Blotto (0.5% (w/v) nonfat dry milk in 10 mM Tris-HCl, pH
7.4, 150 mM NaCl, and 0.075% (v/v) Tween 20); and stored
frozen at Analysis of Polysome Profiles--
Strains PHS33 and W303-1A
were grown in YPG medium at 30° C to early log phase and shifted
into YPD medium. Five, nine, and twelve hours after the shift to
glucose, cycloheximide was added to a final concentration of 100 µg/ml, followed by quick cooling of the cultures on ice. The cells
were harvested by centrifugation and washed with buffer A (10 mM Tris-HCl, pH 7.4, 100 mM KCl, 10 mM MgCl2, and 1 mM dithiothreitol)
plus 100 µg/ml cycloheximide. Cells were broken by vortexing with
glass beads in lysis buffer (20 mM HEPES, pH 7.5, 5 mM MgCl2, 150 mM KCl, 5% (v/v)
glycerol, and 1× CompleteTM protease inhibitors
(Boehringer Mannheim)), and cell lysates were clarified by
centrifugation at 20,000 × g for 10 min at 4° C.
Aliquots (10 A260 units) from each extract were
fractionated on 15-45% sucrose gradients in buffer A by
centrifugation at 38,000 rpm in a Beckman SW 40 rotor for 2.25 h
at 4° C. The gradients were analyzed by upward displacement, and
A254 profiles were obtained with a density
gradient fractionator and UV monitor (Isco Model 185).
Measurement of Protein Synthesis Rates--
Strains W303-1A and
PHS33 were grown overnight in SG complete minus methionine medium.
Cells were harvested, washed in water, and resuspended in either SG
complete minus methionine or SD complete minus methionine medium at a
density of 0.05 A600. During incubation at
30° C, cells corresponding to 1 A600 unit
were withdrawn at the indicated time points, harvested, washed in
buffer A, and resuspended in 300 µl of the same medium containing 100 µCi of [35S]methionine (1 × 106
Ci/mol). The cells were incubated at 30° C for 5 min, followed by
addition of 1 ml of a "stop buffer" containing 1.2 mg/ml
nonradioactive methionine and 0.1 mg/ml cycloheximide in lysis buffer.
Cells were disrupted with glass beads, and proteins from the cleared lysate were precipitated with 10% trichloroacetic acid. The pellet was
washed with acetone and dissolved in 200 µl of 1% SDS. The incorporation of [35S]methionine into total protein was
determined by counting radioactivity in an aliquot of the SDS extract.
The protein concentration of the SDS extract was determined by the
micro-BCA protein assay reagent kit (Pierce) as described by the
manufacturer. The rate of protein synthesis is expressed as cpm × min Immobilized Metal Affinity Chromatography--
Cells harvested
at an A600 of 0.9-1 were disrupted with glass
beads in lysis buffer by eight 30-s pulses in a Bead Beater (BioSpec
Products, Inc.). The lysate was centrifuged for 15 min at 12,000 × g at 4° C, and the supernatant was centrifuged at 65,000 rpm in a Beckman TL100.4 rotor for 80 min at 4° C. The ribosomal pellet was suspended in 500 mM KCl in lysis
buffer and centrifuged as described above. The resulting ribosomal salt
wash enriched in eIF3 was incubated in batch for 1 h at 4° C
with 0.8 ml of HIS-bindTM resin (Novagen) equilibrated with
binding buffer (20 mM Tris-HCl, pH 7.9, 10% (v/v)
glycerol, 30 mM imidazole, and 500 mM NaCl). After pouring the resin into a column, unbound proteins were removed by
washing with 40 bed volumes of binding buffer, and eIF3 was eluted with
the same buffer containing 500 mM imidazole. Eluted fractions were analyzed by SDS-PAGE and Western immunoblotting.
Northwestern Blot Analysis--
Plasmid pRIB-S1 (kindly provided
by J. Warner, Albert Einstein University) carrying a copy of the yeast
rDNA gene was used as template to amplify two fragments of yeast 18 S
rDNA overlapping at the unique SacI site. rDNA nucleotides
1-1248 were amplified with primers
5'-CCCCTCGAGTATCTGGTTGATCCTGCCAG-3' (introducing an
XhoI site (underlined) upstream of the first rDNA
nucleotide) and 5'-CCCGAATTCGAGCTCTCAATCTGTCAATC-3'
(introducing an EcoRI site downstream of the SacI
site in the rDNA). Nucleotides 1243-1800 were amplified with primers
5'-CCCTCGAGGAGCTCTTTCTTGATTTTGTG-3' (introducing an
XhoI site upstream of the SacI site) and
5'-CCCATCGATTAATGATCCTTCCGCAGGTT-3' (introducing a
ClaI site after the last rRNA nucleotide). PCR products were
cloned into pNoTA to generate pNo18S-5' and pNo18S-3', and the sequence
was verified for both constructs. To construct templates for in
vitro transcription under control of the T7 RNA polymerase
promoter, the XhoI/EcoRI fragment from pNo18S-5'
was cloned into pSP73 digested with XhoI/EcoRI to
generate pSP18S-5'. pSP18S-3' was created by ligating the
XhoI/ClaI fragment from pNo18S-3' into
XhoI/ClaI-digested pSP73. Ligating a
SacI/ClaI fragment from pNo18S-3' into the
SacI/ClaI sites of pSP18S-5' resulted in
pSP18S-fl. pSP18S-fl and pSP18S-3' were linearized with ClaI
and transcribed with T7 RNA polymerase to yield rRNA-(1-1800) and
rRNA-(1243-1800), respectively. pSP18S-5' digested with
EcoRI and BstBI generated rRNA-(1-1248) and
rRNA-(1-264), respectively. To synthesize rRNA-(263-1248), pSP18S-5'
was digested with XhoI and BstBI, blunt-ended
with Klenow DNA polymerase, religated, and digested with
EcoRI prior to transcription. All T7 transcripts carry the
sequence 5'-GGGAGACCGGCCUCGAG at the 5'-end of the rRNAs, corresponding
to the pSP73 sequence following the transcription start site. In
vitro transcription was carried out in the presence of 50 µCi of
[
For Northwestern RNA binding experiments, purified His6-p33
(3 µg), His6-p33 Cloning and Characterization of TIF35--
When this work was
initiated, the gene encoding the 33-kDa subunit of eIF3 had not been
identified. An attempt to obtain partial amino acid sequences from
tryptic digests of p33 from purified yeast eIF3 was not successful.
However, at this time, partial amino acid sequence information was
being developed in the laboratory (34) for one of the last subunits of
mammalian eIF3 to be characterized, namely eIF3-p44. The mouse p44
amino acid sequence was used to search the yeast data base as described
under "Materials and Methods," and an ORF encoding a putative
homolog of mammalian eIF3-p44 was identified. Since the yeast ORF
appeared to encode a 30.5-kDa protein, we considered it to be a good
candidate for the gene for yeast eIF3-p33, which we named
TIF35. Evidence reported below and elsewhere (18) shows that
the protein product of TIF35 is a 33-kDa protein that is
present in a complex with other known eIF3 subunits, confirming that
the gene encodes the p33 subunit.
A 2.1-kb fragment of DNA containing TIF35 was amplified by
PCR from yeast genomic DNA and cloned into the pNoTA vector to yield
pNo-TIF35 as described under "Materials and Methods." The 2.1-kb
DNA contains an ORF of 825 base pairs that codes for a protein of 274 amino acid residues with a calculated mass of 30,501 Da. The sequence
context of the first AUG in the ORF, AUAAUG (the initiation
codon is underlined), resembles the yeast consensus context,
A(A/U)AAUG (26). This AUG is preceded by an in-frame UAG
termination codon, whereas the next in-frame AUG is found far
downstream at codon 117. Thus, the first AUG very likely serves as the
initiation codon. Hybridization of 32P-labeled DNA probes
(derived from the coding region) to a single band of genomic DNA
individually digested with four different restriction enzymes suggested
the presence of a single gene locus (data not shown).
Sequence comparisons revealed amino acid sequence
identities/similarities of 35.8/46.9, 33.1/42.5, 33.3/43.1, and
33.6/47.3% when yeast eIF3-p33 was compared with the corresponding
homologous proteins from Schizosaccharomyces pombe
(GenBankTM/EBI accession number AB011823), human (U96074),
mouse (AA109090, AA270800, and W18370), and Caenorhabditis
elegans (Z50044; protein F22B5.2), respectively. When restricting
the sequence comparison to the C-terminal 93-amino acid region of p33
that contains the RRM, identity/similarity values range between
43.7/56.3% for S. pombe and 36.4/47.7% for C. elegans. Therefore, eIF3-p33 appears to be present and moderately
conserved in essentially all eukaryotic cells. In contrast, no homolog
is found in Archaea sequences.
TIF35 Is an Essential Gene--
To examine whether or not
TIF35 is an essential gene, we constructed a diploid strain,
PH33D-7, in which one of the TIF35 genes is nearly entirely
deleted and is replaced by HIS3, as described under
"Materials and Methods." Tetrad analysis of PH33D-7 revealed that
only two of the four spores in each of 30 asci formed colonies on rich
medium (YPD), even after a long incubation at 30° C (data not
shown). All viable spores were unable to grow on SD-His medium, suggesting that the phenotype of
tif35::HIS3 is lethal. The segregation pattern of the tetrad spores (2+:2
To confirm that the lethal phenotype is due to disruption of
TIF35, plasmid pRS316-TIF35, which expresses
TIF35 from its own promoter on a centromeric plasmid
carrying a URA3 marker gene, was constructed and transformed
into PH33D-7. Ura+ transformants were selected; two were
sporulated; and the resulting asci were dissected. Two, three, or four
viable spore colonies per ascus were obtained (data not shown). Only
one spore from the three viable tetrads or two from the four viable
tetrads grew on SD-His plates, and all His+ spore colonies
were also Ura+. Thus, spores containing the
tif35::HIS3 allele must harbor the URA3 plasmid, which carries TIF35. The results
show that TIF35 is the only gene affected and that the
disruption is complemented by the cloned gene.
p33 Is Present in a Complex with Other eIF3 Subunits--
To
obtain further evidence that TIF35 encodes a subunit of
eIF3, we fused six histidine residues to the N terminus of p33 to
create His6-p33. Strain PHL33, expressing
His6-p33 as the sole source of this subunit, grows at the
wild-type rate, indicating that the histidine tag is not deleterious to
p33 function. Ribosomal salt wash fractions were prepared from strains
expressing the wild-type (strain W303-1A) and histidine-tagged (strain
PHL33) forms of p33, and the preparations were fractionated on
Ni2+ affinity columns as described under "Materials and
Methods." Bound proteins were eluted with 500 mM
imidazole and analyzed by SDS-PAGE and Western immunoblotting (Fig.
1). When the blot was analyzed with
antiserum to eIF3, no p33 or other eIF3 subunit was detected in the
eluted fraction prepared from the strain expressing the wild-type form
of p33 (W303-1A). However, with histidine-tagged p33 (strain PHL33),
His6-p33 and numerous other eIF3 subunits were detected.
Bands with mobilities corresponding to p135, p110, p90, p39, and p33
were readily identified. To confirm that these bands correspond to true
eIF3 subunits, the blot was probed with antibodies affinity-purified
against recombinant p135, p110, p39, and p33 and with an antiserum
highly specific for Prt1p (p90). Each of these proteins was found bound
to the Ni2+ affinity column only when p33 was
histidine-tagged. We do not have antisera to the Nip1p (p93), Gcd10p
(p62), and Sui1p (p16) subunits of eIF3, so we could not determine
whether or not these proteins were present as well. However, it is
apparent that p33 is present in complexes that contain most if not all
of the putative eight subunits of eIF3.
Effect of TIF35 Depletion on Cell Growth and eIF3 Subunit
Levels--
To investigate the function of eIF3-p33 in
vivo, we depleted yeast cells of endogenous p33 and measured the
effect of such depletion on cell growth and eIF3 subunit levels. As
described under "Materials and Methods," we placed the
TIF35 ORF under the glucose-repressible GALS
promoter, an attenuated form of the GAL1 promoter (24). The
strain carrying this construct as the sole source of TIF35,
called PHS33, was grown in galactose-containing medium to early
exponential growth phase and then transferred to glucose medium to turn
off transcription of TIF35 from the GALS
promoter. PHS33 in liquid cultures containing galactose as the carbon
source grew with a doubling time of 90 min, comparable to that of the
parental strain, W303-1A (Fig.
2B). When shifted to
glucose-containing medium, strain W303-1A (in which p33 expression occurs from its own promoter) grew with a doubling time of ~90 min
(Fig. 2A). In contrast, the growth rate of PHS33 began to decrease after about four generations (6 h), and the apparent growth
rate was drastically reduced after 12 h (Fig. 2A). The very slow rate of growth seen thereafter may be due to extremely low
levels of expression of TIF35 resulting from incomplete
repression of transcription by glucose. The near-cessation of growth is
consistent with an essential role for eIF3-p33.
The levels of p33 and other eIF3 subunits in lysates prepared from
W303-1A and PHS33 cells were determined at 5, 9, and 12 h after
shift from galactose to glucose medium. Equal amounts of whole cell
lysates were subjected to SDS-PAGE and Western immunoblotting with
anti-eIF3 antibodies (Fig. 3, upper
panel). The level of p33 in cell lysates prepared from the W303-1A
strain remained constant up to 12 h (lane 8). In
contrast, the level of p33 in strain PHS33 was greatly diminished after
9 h (lane 3), and the protein was nearly undetectable
at 12 h (lane 4) after the shift to glucose.
Interestingly, depletion of p33 was accompanied by a strongly reduced
amount of p39, but not of the other eIF3 subunits examined here. This
is seen clearly in the Western immunoblots made with antibodies
affinity-purified against recombinant p135, p110, p39, and p33 and with
a highly specific anti-Prt1p (p90) antiserum (Fig. 3, lower
panels). The levels of p135, p110, and p90 did not change, whereas
p39 and p33 were strongly reduced at 9 h (lane 3) and
12 h (lane 4). The experiments were performed at least
five times, each time resulting in a depletion of only p33 and p39. The
results suggest an important role of p33 in stabilizing the p39 subunit
in the cell. The observation is consistent with our earlier results
(14) and the results of Asano et al. (19) that demonstrate
that p39 interacts with p33 in the yeast two-hybrid assay and that
overexpression of p33 suppresses the slow growth phenotype of
tif34-ts mutants. These observations support the view that
p33 is physically associated with p39 in the eIF3 complex.
p33 Depletion Inhibits Protein Synthesis in Vivo--
The
observation that TIF35 is an essential gene that encodes the
p33 subunit of eIF3 suggests that p33 is required for initiation of
translation. To test this idea, we first analyzed the effect of p33
depletion on protein synthesis in vivo. The rate of protein synthesis in strain PHS33 was measured by pulse-labeling cells for 5 min at various times following the shift from galactose- to
glucose-containing medium (Fig. 4).
During the first 3 h of growth in glucose, the rate of protein
synthesis remained constant, probably due to the presence of
pre-existing p33 in the cells. Subsequently, the rate of protein
synthesis decreased to ~20% by 9 h, the time when the level of
p33 was low as judged by immunoblot analysis (Fig. 3). In contrast,
when W303-1A was grown under the same conditions or PHS33 cells were
grown in galactose-containing medium, the rate of protein synthesis
remained constant and decreased only slightly as cells reached late
exponential phase. The results establish that p33 is required for
optimal translation in yeast cells.
To gain further insight into the role played by eIF3-p33 in protein
synthesis, polysome profiles were examined from PHS33 and W303-1A cells
shifted to glucose medium. Lysates prepared from p33-depleted and
non-depleted cells were subjected to sucrose gradient centrifugation,
and the gradients were scanned for absorbance as described under
"Materials and Methods." PHS33 cells harvested 5 h after the
shift to glucose showed large polysomes (Fig.
5A), consistent with little or
no inhibition of protein synthesis rates at this time (Fig. 4). The
gradient profiles from p33-depleted PHS33 cells analyzed at 9 and
12 h (Fig. 5, B and C) showed a marked
reduction in the amount of large polysomes, with a proportionate increase in the amount of 80 S ribosomes when compared with parental W303-1A cells (Fig. 5D). The percentage of ribosomes
remaining in polysomes in the p33-depleted cells was ~10-15%
compared with 65% in non-depleted W303-1A cells, consistent with the
~6-fold inhibition of protein synthesis observed at those times (Fig. 4). The data demonstrate that ribosomes run off mRNAs following depletion of p33. Such behavior is indicative of a severe decrease in
the rate of translation initiation.
eIF3-p33 Is An RNA-binding Subunit--
One function of the eIF3
complex is to promote the binding of mRNA to the 40 S ribosomal
subunit (27, 28). Of the eight subunits of yeast eIF3, three contain
RNA-binding motifs in their sequences: p90, p62, and p33. p62 (Gcd10p)
binds RNA strongly when assayed by Northwestern blotting with a globin
mRNA fragment (5). Both p90 (Prt1p) and p33 contain the RNP-1 and
RNP-2 motifs found in a family of proteins possessing the RRM (29);
however, RNA binding to these subunits was not detected by Northwestern blotting (10, 21). To assess the RNA-binding ability of p33, purified
His6-p33 was subjected to Northwestern analysis with radiolabeled 18 S rRNA (Fig.
6B, lane 2) or
The C-terminal RNA-binding Domain of p33 Is Not Essential for Cell
Growth--
To assess whether or not the RRM of p33 is essential, the
C-terminal 71 amino acid residues were deleted, thereby removing the
RNP-1 element and most of the RRM (14). p415p33 Yeast eIF3 was purified in this laboratory based on its activity
in an eIF3-dependent mammalian assay for the synthesis of methionylpuromycin (5). To elucidate the role of eIF3 in the initiation
process, we have sought to determine the primary structures of all the
eIF3 subunits by cloning the genes that encode them. Descriptions of
the genes for p93, p90, p62, p39, and p16 have been published, whereas
those for p135 and p110 have been completed.2 The studies
described here on the p33 gene, TIF35, extend earlier work
that identified p33 as part of eIF3 (14, 18, 19). Thus, the genes for
all eight of the subunit proteins have been identified and
characterized in detail.
The cloning of TIF35 was based initially on its homology
(33% sequence identity and 43% similarity) to mouse or human
eIF3-p44. Further evidence that the putative cloned gene actually
encodes the p33 subunit of eIF3 follows. 1) TIF35 encodes a
protein with a calculated mass of 30.5 kDa, close to the apparent mass
of the 33-kDa subunit of eIF3. It should be noted, however, that the apparent mass of this protein depends on the gel system used, and
values range from 32 to 36 kDa. 2) Antibodies raised against the
product of TIF35 expressed in E. coli react with
the p33 subunit in purified eIF3 preparations. 3) By using the yeast
two-hybrid system and co-immunoprecipitations, p33 was shown to
interact with a previously characterized subunit of eIF3, namely p39
(14, 19). 4) When p33 tagged with His6 is expressed in
yeast as the sole source of p33, a complex is isolated by
Ni2+ affinity chromatography that contains numerous other
subunits of eIF3, namely p135, p110, p90, and p39. 5) The
TIF35 gene product was identified by mass spectroscopy in a
complex with p110, p93, His6-p90, and p39, which was
isolated by Ni2+ affinity chromatography (18). 6) Depletion
of p33 results in lowered levels of p39. 7) Finally, overexpression of
p33 from TIF35 suppresses the temperature-sensitive
phenotype of mutant forms of p39 (14, 19). These findings provide
convincing evidence that p33 expressed from TIF35 is a
subunit of yeast eIF3.
Deletion/disruption of TIF35 is lethal and, together with
depletion experiments, shows that p33 is required for cell growth. Depletion also causes an inhibition of the initiation phase of protein
synthesis as deduced from polysome profiles. This is consistent with
p33 being a subunit of eIF3, and comparable effects on protein synthesis are seen when other eIF3 subunits are depleted or
inactivated. However, a precise role for p33 in initiation cannot be
determined from these experiments. Because p39 is depleted along with
p33, the loss of eIF3 function could be due to the lack of p39, rather than p33 itself. The availability of cloned TIF35 should
expedite the isolation of mutant forms of p33 that may shed light on
its function.
One of the possible functions of p33 is to bind RNA. Possible targets
are the 18 S rRNA in the 40 S ribosomal subunit, mRNA, and
Met-tRNAi residing in the ternary complex with eIF2 and
GTP. Northwestern blotting was used to examine binding to radiolabeled 18 S rRNA and For further insight into the RNA-binding properties of yeast eIF3-p33,
a comparison was made of the RRM sequences of four other p33 homologs
derived from S. pombe, C. elegans, Homo sapiens, and Mus musculus (Fig. 8). The
RRM regions of p33 from these species share sequence
identities/similarities with the RRM of S. cerevisiae p33 of
41/63, 32/53, 39/55, and 40/56%, respectively. In particular, each of
the four putative
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3 Prime, Inc., Boulder, CO ) to create pNo-TIF35.
Sequencing confirmed that the 2.1-kb insert contains an 825-base pair
open reading frame (ORF) with 607 and 674 base pairs of DNA flanking
the 5'- and 3'-ends, respectively.
C (kindly provided by M.-H.
Verlhac, University of California, San Francisco) (14) is identical to
p415Gal1-NH33, except that the encoded p33 lacks the C-terminal 71 amino acids.
C,
p415p33
C was digested with BamHI and SalI, and
the 0.7-kb fragment was subcloned into pET28c digested with BamHI/SalI.
C, and transformants were selected on
SD-His-Leu plates. The resulting transformants were sporulated, and
their asci were dissected. Two, three, and four viable spores were
obtained on YPG plates and were streaked on SG-His-Leu plates to
identify TIF35-disrupted cells carrying p415GalS-NH33,
p415GalL-NH33, and p415p33
C. The corresponding haploid strains were
named PHS33, PHL33, and PH133, respectively.
80° C. A second batch of affinity-purified anti-p33
antibodies was prepared similarly, but from the anti-eIF3 antiserum,
and was used as indicated in the figure legends. Anti-p135 and
anti-p110 antibodies were affinity-purified against recombinant
His6-tagged p135 and p110 as described
elsewhere.2
1 × µg of protein
1.
-32P]UTP (800 Ci/mmol) with the T7/SP6 transcription
MAXIscript kit (Ambion Inc.) according to the manufacturer's
recommendations. Unincorporated nucleotides were removed using
MicroSpinTM S-200 HR columns (Amersham Pharmacia Biotech).
The 32P-labeled
-globin mRNA was prepared as
described previously (25). Isotopically labeled transcripts were
analyzed on denaturing 4% (for long transcripts) or 5% (for shorter
transcripts) polyacrylamide gels.
C (3 µg), and yeast lysate (10 µg)
were subjected to SDS-PAGE and electrotransferred to polyvinylidene
difluoride membranes. The membranes were treated for 20 min with
binding buffer containing 20 mM HEPES-KOH, pH 7.5, 2 mM Mg(OAc)2, 75 mM KOAc, 1 mM EDTA, 1 mM dithiothreitol, 0.2% (w/v)
CHAPS, 1 mg/ml E. coli tRNA, and 200 units of ribonuclease
inhibitor (Amersham Pharmacia Biotech). Membranes were then incubated
for 20 min with 200,000 cpm/ml of the 32P-labeled 18 S rRNA
or
-globin transcripts in 8 ml of binding buffer. The blots were
washed three times for 5 min each with binding buffer and subjected to autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) and the
fact that no His+ segregants were found indicate that
TIF35 is necessary for germination and/or cell viability.
View larger version (72K):
[in a new window]
Fig. 1.
Analysis of eIF3 subunits associated with
histidine-tagged p33. Equal amounts of protein in the ribosomal
salt wash (RSW) fractions prepared from strains W303-1A
(expressing untagged p33) and PHL33 (expressing His6-p33)
were fractionated by Ni2+ affinity chromatography as
described under "Materials and Methods." Unbound (Wash)
and bound (Eluate) fractions were analyzed by 7.5% SDS-PAGE
(32), followed by immunoblotting onto polyvinylidene difluoride
membranes with crude rabbit anti-eIF3 antiserum (upper
panel) as well as affinity-purified antibodies (lower
panels) as indicated on the right. The immunoblots were developed
by nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Sigma)
color detection. wt, wild type.
View larger version (17K):
[in a new window]
Fig. 2.
Effect of p33 depletion on cell growth.
Strains W303-1A (solid lines) and PHS33 (dashed
lines) were initially grown in YPG medium and then diluted to an
A600 of 0.03 in either YPD (A) or YPG
(B) medium. Cultures were maintained in logarithmic growth
phase throughout the experiment by diluting them to an
A600 of 0.05 once they reached an absorbance of
0.5. The readings above an absorbance of 0.5 are calculations, taking
into account the dilutions made. The results are representative of 5 independent experiments.
View larger version (82K):
[in a new window]
Fig. 3.
Immunoblot analysis of eIF3 subunits.
Lysates were prepared from strains W303-1A and PHS33 harvested after
shifting from galactose (G) to glucose (D) at the
times indicated and as shown by arrows in Fig. 2 as
described under "Materials and Methods." Lane 1, 0 h; lanes 2 and 7, 5 h; lane 3,
9 h; lanes 4 and 8, 12 h. Similarly,
strain PHS33 was diluted into galactose medium. Lane 5,
5 h; lane 6, 12 h. The concentration of protein in
each cell lysate was determined (33), and equal amounts (30 µg) were
examined by 7.5% SDS-PAGE and immunoblotting as described in the
legend to Fig. 1. Equal loading of lysate protein was confirmed by
Coomassie Blue staining of identical gels run in parallel (data not
shown). The antibodies used are indicated on the right, and the
migration positions of the eIF3 subunits are labeled on the left. Only
the relevant portions of the membrane are shown. rc,
recombinant.
View larger version (18K):
[in a new window]
Fig. 4.
Inhibition of protein synthesis in
p33-depleted cells. W303-1A cells grown in YPD medium
(D; ), PHS33 cells grown in YPG medium (G;
), and PHS33 cells shifted from YPG to YPD medium (
) were
subjected to a 5-min pulse labeling at the indicated times as described
under "Materials and Methods." The rate of
[35S]methionine incorporation into protein was calculated
as cpm × min
1 × µg of
protein
1.
View larger version (18K):
[in a new window]
Fig. 5.
Analysis of polysome profiles.
Exponentially growing cultures of strains W303-1A and PHS33 in YPG
medium were shifted to YPD medium at the indicated times, and lysates
were processed for polysome analysis by sucrose gradient centrifugation
as described under "Materials and Methods." A, PHS33 (5 h in YPD medium); B, PHS33 (9 h in YPD medium);
C, PHS33 (12 h in YPD medium); D, W303-1A (12 h
in YPD medium). The results are representative of five independent
experiments. Shown are absorbance profiles with sedimentation from left
to right. The positions of the 80 S ribosomes are indicated by
arrows.
-globin mRNA (Fig. 6C, lane 5). The
protein bound either type of RNA, suggesting a lack of specificity (but
see "Discussion" below). The possibility that p33 binds a specific
region of the 18 S rRNA was examined by Northwestern blotting with
32P-labeled fragments of 18 S rRNA (Fig. 6, A
and C). All fragments bound, again suggesting little or
no specificity. The role of the RRM in RNA binding was examined by
testing purified recombinant His6-p33
C. No binding to 18 S rRNA was detected (Fig. 6B, lane 3). The
results demonstrate that p33 has nonspecific RNA-binding activity and
that its C-terminal RRM is essential for the RNA-binding function.
View larger version (30K):
[in a new window]
Fig. 6.
Northwestern blot analysis of p33 binding to
RNA. A, scheme of the in vitro transcripts
of 18 S rRNA used. Radiolabeled transcripts were prepared with T7 RNA
polymerase as described under "Materials and Methods." The numbers
on the right describe the range of nucleotides in the transcript.
B, upper panel: Northwestern blotting of
His6-p33 and His6-p33 C. His6-p33
and His6-p33
C were produced in E. coli
BL21(DE3) and purified as described under "Materials and Methods."
Purified recombinant (rc) proteins (2-3 µg) and 20 µg
of yeast lysate were subjected to 7.5% SDS-PAGE, followed by blotting
onto a polyvinylidene difluoride membrane and probing with
32P-labeled 18 S rRNA-(1-1800) as described under
"Materials and Methods." Lower panel: Coomassie Blue
staining of recombinant His6-p33 and
His6-p33
C in the blot above. The migration positions of
full-length and truncated recombinant tagged p33 proteins are shown;
only a portion of the blot is shown. C, Northwestern
blotting of His6-p33 (2-3 µg) with truncated forms of 18 S rRNA and
-globin mRNA. Only the portions of the blots
containing tagged p33 are shown.
C, which expresses
the C-terminal truncated p33 (p33
C) from a GAL1 promoter, was transformed into strain PH33D-7, and transformants were sporulated and analyzed by tetrad dissection. From each ascus, two fast growing haploid cells plus none, one, or two very slow growing cells were obtained in galactose-containing medium (Fig.
7A). The slow growing haploid
cells were His+, indicative of carrying the disrupted
tif35::HIS3 gene on a chromosome, and
Leu+, due to the presence of p415p33
C (Fig.
7B). One of the haploid slow growing colonies was selected
and called PH133. Strain PH133 did not grow on glucose-containing
medium, consistent with the plasmid being the only source of
TIF35 expression. It exhibited a doubling time of 6 h
in galactose-containing medium, thereby suggesting that the RRM is not
absolutely essential when p33
C is expressed from a GAL1
promoter. The slow growth phenotype could be caused either by a reduced
specific activity or by a reduced level of p33
C. To distinguish
between these possibilities, the cellular levels of p33
C and p33
were measured by Western immunoblotting of lysates derived from strain
PH133 and the parental strain, W303-1A (Fig. 7C). The band
corresponding to full-length p33 was absent in PH133, and instead a
strong band with the expected apparent molecular mass of 25 kDa was
seen that was absent in W303-1A. The intensity of the p33
C band in
PH133 is comparable to that of p33 in W303-1A, indicating that p33
C
is stable and accumulates to wild-type levels. We conclude that the
slow growth phenotype is caused by an appreciable loss of p33 activity
due to the removal of its RRM.
View larger version (40K):
[in a new window]
Fig. 7.
Effect of deleting the C-terminal RNA-binding
domain of p33 on cell growth. A, dissection
of seven tetrads from PH33D-7 transformed with p415p33 C was carried
out as described under "Materials and Methods." Spores are arranged
vertically on a YPG plate as indicated by letters on the left and were
allowed to germinate and grow at 30 °C for 5 days. A photograph of
the plate is shown. B, the dissected tetrad shown in
A (lane 3), which gives four viable
spore colonies, was selected, and the four colonies were streaked onto
SG-His-Leu and SD-His-Leu plates as indicated. The plates were
incubated at 30 °C for 5 days and then photographed. C,
equal amounts of protein (10 µg) from strain W303-1A (lane
1) and PH133 (lane 2) were subjected to SDS-PAGE and
immunoblotting with anti-p33 antiserum as described in the legend to
Fig. 1. Equal loading of the lysates was confirmed by Amido Black
staining of a parallel gel and by the equal intensities of
cross-reacting protein. A scan of a portion of the blot is shown; the
bands corresponding to p33 and p33
C are identified.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-globin mRNA. Because p33 binds to all fragments of 18 S rRNA and to mRNA, it appears that the RNA-binding activity is nonspecific for both sequence and structure. The human homolog, eIF3-p44, also binds RNA nonspecifically (34). An important caveat is
that there could be significant differences in RNA-binding affinities
between the different RNA probes that are not detected by the method
employed. Deletion of most of the C-terminal RRM results in loss of RNA
binding as determined by Northwestern blotting. It is surprising,
however, that the C-truncated protein, p33
C, nevertheless supports
growth (albeit slow) of yeast as the sole source of the p33 subunit.
Therefore, the RRM does not perform an essential function, but clearly
is required for optimal activity of the protein.
-sheet structures appears well conserved, suggesting common functions. The RRMs of the five p33 homologs also are
similar to the RRM in the spliceosomal protein U1A (Fig. 8). Structural
data for a U1A-RNA interaction are available from crystallographic (30)
and NMR (31) studies. The U1A RRM contains a four-stranded
-sheet
that is stabilized by two
-helices. The single-stranded loop of the
RNA binds to the surface formed by the
-sheets, interacting mainly
with
-sheets 1 and 3. Most amino acids required to form the
hydrophobic core of the U1A RRM (Leu-17, Leu-26, Phe-34, Phe-59, and
Ala-68) are also conserved in p33 and its homologs (residues denoted by
solid squares in the figure). Half of the residues
implicated in U1A binding to RNA also are conserved in p33 (solid
circles), whereas half are not (open circles), especially those in
-sheet 4. A striking difference corresponds to
Tyr-13 in
-sheet 1 of U1A, which is a basic amino acid residue in
all of the p33 homologs. In U1A, Tyr-13 stacks with a C base in the
RNA, whereas in p33, this interaction presumably is replace by ionic
attraction between the basic amino acid and a phosphate in the RNA. The
stacking interaction seen with U1A Gln-54 could be accomplished with
the Leu or Phe residues in the p33 homologs. Although
-sheets 2 and
4 differ in sequence from the corresponding regions in U1A,
-sheet 2 in particular contains numerous basic residues that may contribute to
RNA binding. The sequence similarities and differences between U1A and
the p33 homologs suggest that slightly different modes of RNA binding
may be involved that may contribute to different binding
specificities.
View larger version (56K):
[in a new window]
Fig. 8.
Sequence comparisons of the RRM-containing C
terminus of p33 from five species. The C-terminal sequences of
S. cerevisiae (S.ce.) p33 and its homologs from
S. pombe (S.po.), C. elegans
(C.el.), human (H. sapiens (H.sa.)),
and mouse (M. musculus (M.mu.)) are compared with
the RRM sequence in the human U1A protein. Locations of the RNP motifs
and secondary structure elements of U1A are indicated above the
sequences. Numbering is according to the U1A protein. Residues
important for forming the hydrophobic core of the RRM are denoted by
solid squares. Solid and open circles
indicate residues involved in U1A-RNA interactions that are conserved
or not conserved, respectively, between U1A and p33 or its
homologs.
Work from several laboratories has identified eight possible subunits in yeast eIF3: p135, p110, p93, p90, p62, p39, p33, and p16. Evidence for the presence of p135 and p110 in eIF3 will be published elsewhere.2 The p93 subunit, encoded by NIP1, has been seen in a complex with p110, p90, p39, and p33 obtained by Ni2+ affinity chromatography with His6-tagged p90 (18). As reported elsewhere, p90, p62, p39, and p16 are present in the various immunoprecipitates of eIF3 formed by antibodies to these proteins (5, 8, 10, 11). The sum of the evidence therefore indicates that a complex of eight subunits exists in yeast.
We show here that His6-tagged p33 allows the isolation of a complex in which p135, p110, p90, and p39 are positively identified with affinity-purified antibodies. Lacking antibodies to p93, we have not detected p93 in these preparations. Furthermore, the p93 subunit is readily degraded in extracts derived from strain W303 (12). Our results differ somewhat from those obtained by Phan et al. (18) with His6-tagged p90, where five eIF3 subunits were detected, namely p110, p93, p90, p39, and p33, but not p135. This complex of five subunits is active in stimulating Met-tRNAi binding to 40 S subunits in vitro and therefore may constitute the active "core" of eIF3. It is interesting that of the eight eIF3 subunits, only the five core subunits are homologous to mammalian eIF3 subunits. We believe that p135 associates with the eIF3 core complex, although its affinity is not so strong as that of the core subunits. The issue of the presence of p135 in eIF3 is addressed in detail elsewhere.2
Depletion of p33 causes loss of p39, but not of the p90, p110, and p135
subunits. On the other hand, depletion of p110 causes p90 to be
degraded, whereas p39 and p33 are stable.2 These
experiments suggest that eIF3 may have two major domains, one with p39
and p33 and the other with the larger subunits. The two domains might
be connected by an interaction between p39 and p90, as shown by
two-hybrid analyses (14, 19). Work is in progress to further elucidate
the structure of eIF3 by employing a number of methods that detect
protein-protein interactions. The characterization of TIF35
contributes to these studies. We anticipate that further detailed
structural studies of eIF3 will help to elucidate the function of this
key initiation factor.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. Warner, A. G. Hinnebusch, and M.-H. Verlhac for providing clones and antibodies. We also thank A. G. Hinnebusch for communicating results prior to publication and S. Ribeiro and L. Phan for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM22135 and by a research fellowship from the Deutsche Forschungsgemeinschaft (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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF004913.
To whom correspondence should be addressed. Tel.: 530-752-3235;
Fax: 530-752-3516; E-mail: jwhershey{at}ucdavis.edu.
§ Present address: Institut für Molekularbiologie und Tumorforschung, Philipps Universitaet Marburg, 35033 Marburg, Germany.
2 H.-P. Vornlocher, P. Hanachi, S. Ribeiro, and J. W. B. Hershey, manuscript in preparation.
3 http://genome-www.stanford.edu/Saccharomyces/.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: eIFs, eukaryotic initiation factors; RRM, RNA recognition motif; PCR, polymerase chain reaction; kb, kilobase pair; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; RNP, ribonucleoprotein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|