From the Laboratory of Eukaryotic Gene Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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The PRT1, TIF34, GCD10, and SUI1 proteins of Saccharomyces cerevisiae were found previously to copurify with eukaryotic translation initiation factor 3 (eIF3) activity. Although TIF32, NIP1, and TIF35 are homologous to subunits of human eIF3, they were not known to be components of the yeast factor. We detected interactions between PRT1, TIF34, and TIF35 by the yeast two-hybrid assay and in vitro binding assays. Discrete segments (70-150 amino acids) of PRT1 and TIF35 were found to be responsible for their binding to TIF34. Temperature-sensitive mutations mapping in WD-repeat domains of TIF34 were isolated that decreased binding between TIF34 and TIF35 in vitro. The lethal effect of these mutations was suppressed by increasing TIF35 gene dosage, suggesting that the TIF34-TIF35 interaction is important for TIF34 function in translation. Pairwise in vitro interactions were also detected between PRT1 and TIF32, TIF32 and NIP1, and NIP1 and SUI1. Furthermore, PRT1, NIP1, TIF34, TIF35, and a polypeptide with the size of TIF32 were specifically coimmunoprecipitated from the ribosomal salt wash fraction. We propose that all five yeast proteins homologous to human eIF3 subunits are components of a stable heteromeric complex in vivo and may comprise the conserved core of yeast eIF3.
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INTRODUCTION |
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In the initiation phase of eukaryotic protein synthesis, mRNA and Met-tRNAiMet are placed in their proper positions on the 40 S ribosomal subunit to form the 48 S preinitiation complex (1). Binding of the 60 S ribosomal subunit to this complex forms the 80 S initiation complex that can enter the translation elongation cycle. These complex reactions in the initiation phase depend on numerous protein factors called initiation factors (eIFs),1 many of which bind directly or indirectly to 40 S ribosomes. Two well characterized heteromeric protein complexes, eIF2 and eIF4F, play important roles in the binding of Met-tRNAiMet and mRNA to 40 S subunits, respectively, in both yeast and mammals (2-5). The largest of the initiation factors, eIF3, is thought to have an important role in formation of the 48 S preinitiation complex, as early studies on the mammalian factor showed that it promoted dissociation of 80 S ribosomes and stimulated binding of both eIF2-GTP-Met-tRNAiMet ternary complex and mRNA to 40 S subunits (6, 7). Mammalian eIF3 binds to the 40 S ribosome independently of other eIFs at a location separate from the P site where ternary complex is presumed to bind (8, 9). Physical interactions between eIF4B and eIF3-p170 (10) and between eIF4F and the whole eIF3 complex (11, 12) have been demonstrated in mammalian systems, suggesting that eIF3 serves as a scaffold for these mRNA binding factors.
A mammalian eIF3 complex containing nine different subunits was purified by its ability to promote methionyl puromycin (Met-puromycin) synthesis by an 80 S initiation complex in an assay containing other purified eIFs and ribosomal subunits (13). The cDNAs encoding all nine subunits of this complex were recently cloned and sequenced (14-19).2 By comparing the sequences of these nine cDNAs to the entire Saccharomyces cerevisiae genome, it became apparent that only five of the known mammalian eIF3 subunits have identifiable homologues encoded in yeast; the products of the yeast genes TIF32, PRT1 (20), NIP1 (21), TIF34 (22), and TIF35 have similar molecular weights and show strong sequence similarities to the p170, p116, p110, p36, and p44 subunits, respectively, of mammalian eIF3. The 90- and 39-kDa products of PRT1 and TIF34, respectively, were shown to copurify with yeast eIF3 isolated by its ability to substitute for human eIF3 in the Met-puromycin synthesis assay (20, 22). The products of TIF32, NIP1, and TIF35 were not known to be components of yeast eIF3 purified in this fashion, although it contained a polypeptide similar in size to the predicted molecular weight of TIF35 (20).
The 62- and 16-kDa products of GCD10 (23) and SUI1 (24) also copurified with yeast eIF3 activity using the Met-puromycin synthesis assay. SUI1 was first identified genetically by mutations that permit increased utilization of UUG triplets as translation initiation codons (25) and is homologous to a poorly characterized mammalian factor called eIF1 (26). GCD10 was thought to be the yeast counterpart of human eIF3-p66 because of their similar molecular weights and in vitro RNA binding activities (16, 23); however, they do not have strong sequence similarity. Moreover, a putative human homologue of GCD10 can be predicted from human EST (expressed sequence tag) sequences3 that does not correspond to one of the known subunits of mammalian eIF3.
The above comparison of yeast and human eIF3 complexes suggests differences in subunit composition between the two species. In addition, a five-subunit complex containing PRT1 has been purified from yeast (27) with a composition quite different from that purified using the Met-puromycin assay (20). In view of these apparent discrepancies between different eIF3 preparations, we decided to pursue an alternative strategy for investigating the subunit composition of yeast eIF3. By using the cloned genes for all of the known or potential subunits of eIF3, we attempted to construct a protein linkage map (28) of the eIF3 complex with the yeast two-hybrid system. By combining this approach with in vitro binding assays, we identified protein-protein interactions which link together all five yeast homologues of human eIF3 subunits, TIF32, PRT1, NIP1, TIF34, and TIF35. Genetic suppressor analysis provided independent evidence that TIF34 and TIF35 interact with one another in vivo. In addition, we showed that PRT1, NIP1, TIF34, TIF35, and possibly TIF32 were the major polypeptides specifically coimmunoprecipitated with TIF34 from the ribosomal salt wash fraction. Based on these findings, we conclude that all five yeast proteins with sequence similarity to human eIF3 subunits physically interact with one another in vivo.
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MATERIALS AND METHODS |
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Plasmid Construction-- Plasmids carrying different yeast genes fused in-frame to the coding sequences for the DNA binding domain or activation domain of GAL4 in pGBT9 (TRP1 vector) or pGAD424 (LEU2 vector) (purchased from CLONTECH, Ref. 29), respectively, were constructed as follows. Oligonucleotides corresponding to the 5' and 3' ends of the ORFs in TIF32, NIP1 (21), PRT1 (30), TIF34 (22), TIF35, SUI1 (25), GCD10 (23) and GCD14,4 listed in Table I, were synthesized (Life Technologies, Inc.) and employed for PCR using yeast genomic DNA as template and the eLONGase kit, (Life Technologies, Inc.). Amplified DNA fragments were digested with the appropriate restriction enzymes corresponding to the terminal sequences (BamHI or SalI at 5' end, and PstI or BglII at 3' end) and subcloned into pGAD424 or pGBT9. The resulting plasmids are listed in Table II. For insertions into pGBT9, the BglII site at the 3' end of the amplified fragments was filled in with Klenow enzyme and ligated to the PstI site of pGBT9, after trimming the latter with mung bean nuclease.
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Yeast Strains--
Strains Y190 (MATa leu2-3,-112 ura3-52
trp1-901 his3- 200 ade2-101 gal4
gal80
URA3::GAL-lacZ
LYS::GAL-HIS3) and Y187 (MAT
gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3,-112
met
URA3::GAL-lacZ)
were employed as hosts for the pGBT9 and pGAD424 derivatives,
respectively, used in yeast two-hybrid analysis (40).
Construction of Libraries for Yeast Two-hybrid Screening for Interactions between Potential eIF3 Subunits-- Usage of libraries containing gene fragments for yeast two-hybrid screening has been described (28, 42). Two libraries of possible eIF3 subunit gene fragments fused to coding sequences for the GAL4 activation domain were constructed in pGAD424 as follows. DNA fragments containing the complete ORFs of the relevant genes were PCR-amplified using oligonucleotides listed in Table I, as described above. Equimolar amounts of the appropriate fragments were sheared by sonication, end-filled with T4 DNA polymerase, and inserted at the SmaI site of pGAD424. The ligation mixture was used for transformation of Escherichia coli DH10B cells on LB plates supplemented with 100 µg/ml ampicillin, and DNA was purified from pools of transformants by using a plasmid isolation kit (Qiagen) according to the vendor's instructions. For library 1, an aliquot of plasmid DNA derived from a pool of 104 transformants bearing a mixture of inserts from PRT1, TIF34, GCD10, and GCD14 with an average size of 0.3 kb was mixed with 1/20 of the same amount of plasmid DNA derived from 3 × 103 transformants containing SUI1 inserts with an average size of 0.15 kb. For library 2, plasmid DNA was prepared from a pool of 104 transformants containing a mixture of inserts from NIP1, TIF32, and TIF35 with an average size of 0.45 kb. In libraries 1 and 2, ~50% and 30% of the clones, respectively, were found to contain inserts. Taking into account the total numbers of independent clones comprising each library and the number of base pairs of coding sequence represented by the genes in the library, we estimated that the end points of overlapping clones for each gene in library 1 are spaced an average of 25 bp apart. The corresponding estimate for library 2 is an average spacing of 13 bp.
Interactions between Recombinant Proteins in Vitro-- Cell extracts were prepared from transformants of E. coli BL21(DE3) carrying the different GST fusion plasmids (Table II), and GST fusion proteins were purified on glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech), all as recommended by the vendor. After extensive washing, beads were resuspended in 0.75 ml of binding buffer (20 mM HEPES (pH 7.5), 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 1% skim milk, 1 mM dithiothreitol, 0.05% Nonidet P-40), to which 10 µl of rabbit reticulocyte lysate containing S-labeled proteins was added. The 35S proteins were synthesized by in vitro translation using [35S]methionine and pT7-7 derivatives (Table II) as template with the TnT transcription/translation kit (Promega). Binding assays were conducted as described in Fig. 2.
Isolation of Temperature-sensitive and Lethal Point Mutations in
TIF34--
YCpL-TIF34-HA, the single copy LEU2 plasmid
encoding HA-tagged TIF34 was randomly mutagenized through
transformation of E. coli XL1-Red cells (Promega) as
recommended by the manufacturer. Plasmid DNA was isolated from a pool
of 20,000 E. coli transformants and used to transform to
Leu+ strain KAY4, the tif34-1
yeast strain, bearing the URA3 TIF34 plasmid YCpU-TIF34. Two
thousand Ura+ Leu+ transformants were picked
and replica-plated to two 5-FOA plates that were incubated at 30 or
36 °C. Clones that did not grow on 5-FOA medium at both 30 and
36 °C after 5 days of incubation were selected as candidates for
containing lethal mutations in the TIF34 allele carried on
plasmid YCpL-TIF34-HA, whereas those that grew at 30 °C but not at
36 °C were selected as candidates for containing
temperature-sensitive (Ts
) tif34-HA alleles.
Total yeast DNA was isolated from the candidate strains, and used to
transform E. coli MC1066 (leuB trpC pyrF) (43) to
Leu+ on M9 medium (44) supplemented with tryptophan and
uracil, thereby isolating in E. coli the putative
YCpL-TIF34-HA derivatives containing tif34-HA mutant
alleles. Plasmid DNA was prepared from these E. coli
transformants and reintroduced into yeast strain KAY4 to verify the
phenotype after evicting YCpU-TIF34 with attendant growth on 5-FOA
medium. The sites of mutations in all 19 tif34-HA alleles
thus obtained were determined by sequencing the entire TIF34
ORF in each plasmid.
Preparation of Extracts for Immunoprecipitation of Epitope-tagged
TIF34--
Yeast cells from transformants of KAY1
(tif34-1 YCpL-TIF34 [TIF34]) and KAY8
(tif34
-1 YCpL-TIF34-HA [TIF34-HA]) carrying YEp-TIF35-FLAG (URA3 TIF35-FLAG) were grown in 50 ml of SC
medium lacking uracil to early log phase, harvested, and suspended in 0.1 ml of ice-cold lysis buffer (20 mM Tris-HCl, pH 7.4, 1 mM magnesium acetate, 100 mM KCl, 0.1% Triton
X-100, CompleteTM protease inhibitor (Boehringer Mannheim))
after washing with
volume of the same buffer. Whole cell
extracts (WCE) were prepared by homogenizing the washed cells by
vortexing with glass beads in a microcentrifuge tube for 15 s
three times. Cells were placed on ice for 30 s between each cycle
of vortexing. Cell lysates were clarified by centrifigation in a
microcentrifuge tube at maximum speed for 10 min at 4 °C. Ribosomal
salt wash (RSW) was prepared as described (27) except for a slight
modification (56) from 2-liter cultures
(A600 = ~10) of the same yeast strains employed above for WCE preparation, grown in SC medium lacking uracil.
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RESULTS |
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Two-hybrid Interactions between Possible eIF3 Subunits-- TIF32, TIF35, and NIP1 are homologous to mammalian eIF3 subunits but were not known to copurify with the yeast factor. We used the yeast two-hybrid assay (20, 23, 24) to investigate whether these three proteins can physically interact with the four previously reported subunits of yeast eIF3, PRT1, TIF34, GCD10, and SUI1. We also included in this analysis GCD14, which was recently linked both genetically and physically to GCD10.4,5 The complete coding sequences for these eight proteins were fused to the DNA binding and activation domains of the transcriptional activator GAL4, and the resulting constructs were tested in all combinations for two-hybrid interactions in yeast. As shown in Table III, we detected three groups of interactions, one between NIP1 and SUI1, a second between GCD10 and GCD14, and a third involving PRT1, TIF34, and TIF35. This last group of interactions indicated that TIF35, the putative yeast homologue of human eIF3-p44, is capable of interacting with two known subunits of yeast eIF3. We failed to detect any other interaction outside of these three groups. In particular, we detected no interaction involving TIF32, even though the TIF32 fusion proteins were expressed in amounts comparable to the other fusion proteins, based on immunoblot analysis of cell extracts with anti-GAL4 antibodies (data not shown). As described later, however, we show that TIF32 binds to PRT1 and NIP1, by in vitro binding assays (see Fig. 6).
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Intersubunit Binding Domains within PRT1, TIF34, and TIF35-- Failure to see interactions between full-length proteins by two-hybrid assay is not uncommon and can occur for a variety of reasons (29, 45). In an effort to overcome this limitation, and also to identify protein segments responsible for the two-hybrid interactions detected in Table III, we constructed two activation domain libraries containing random fragments of these eight genes and screened them for two-hybrid interactions with the DNA binding domain constructs containing full-length TIF34 and TIF35 coding sequences (see "Materials and Methods" and Refs. 28 and 42). By screening an activation domain library containing fragments of TIF34, PRT1, GCD10, GCD14, and SUI1 against the full-length TIF34 DNA binding domain fusion, we identified interactions between seven C-terminal segments of PRT1 (labeled A1-A16 in Fig. 1A) and full-length TIF34. An overlapping set of C-terminal segments of PRT1 from the same library (labeled B1-B17 in Fig. 1A) was found to interact with full-length TIF35. By screening the second activation domain library containing fragments of NIP1, TIF32, and TIF35 against the TIF34 DNA binding domain fusion, we also identified interactions between TIF34 and five N-terminal segments of TIF35 (labeled C1-C8 in Fig. 1B). These results confirmed the two-hybrid interactions involving full-length TIF34, PRT1, and TIF35 (Table III) and suggested that TIF34 and TIF35 both interact in vivo with the C-terminal ~80 amino acids of PRT1. In addition, they show that a binding domain for TIF34 resides within the N-terminal half of TIF35, upstream from the putative RNA-recognition motif in TIF35 (Fig. 1B). We also screened the fragment libraries with the full-length SUI1-, NIP1-, PRT1-, and GCD10-DNA binding domain fusions as bait; however, no additional interacting plasmids were obtained. This may indicate that the NIP1-SUI1 and GCD10-GCD14 interactions detected using full-length fusion proteins (Table III) are dependent on multiple non-contiguous segments in these proteins. Alternatively, fusions containing segments of these proteins may be too unstable in yeast cells to permit a detectable two-hybrid interaction (29, 45).
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In Vitro Binding between PRT1, TIF34, and TIF35-- The two-hybrid interactions between the C-terminal segment of PRT1, the N-terminal half of TIF35, and full-length TIF34 were confirmed by in vitro protein binding experiments. Recombinant GST fusions were constructed containing full-length TIF34 or TIF35 or the minimal interacting fragments of PRT1 or TIF35 identified by the two-hybrid analysis summarized in Fig. 1. The GST fusion proteins were expressed in bacteria and purified by immobilization on glutathione-Sepharose beads. As shown in Fig. 2A (upper panel), the slowest migrating species for each purified fusion had the predicted apparent molecular weight; however, a sizable proportion of the GST-TIF35 fusion proteins was truncated at various positions from the C terminus (the GST moiety is N-terminal), presumably due to proteolytic degradation (Fig. 2A, lane 6). The GST fusion proteins were mixed with 35S-labeled TIF34, PRT1, or TIF35 proteins synthesized by in vitro translation. After extensive washing, the radiolabeled proteins still bound to the beads were eluted, fractionated by SDS-PAGE, and visualized by autoradiography. In these reactions, the GST fusions are present in large molar excess over the radiolabeled protein. Therefore, at similar concentrations of the GST fusion proteins, the proportions of a given labeled protein recovered in complexes with the GST fusion proteins reflects its relative binding affinities for the different GST fusions (45).
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Isolation of Unconditional Lethal and Temperature-sensitive Mutations in TIF34-- TIF34, and its human homologue eIF3-p36, consist of 7 WD repeats (14, 22). Although WD repeats 3 and 6 in TIF34 are degenerate, their occurrence is supported by a sequence alignment of TIF34, human eIF3-p36, and three putative homologues of these proteins from Arabidopsis thaliana, Drosophila melanogaster, and Schizosaccharomyces pombe identified in GenBankTM (Fig. 3A). This alignment also reveals two regions of high sequence similarity (38-39% identity) spanning WD repeats 1-2 at the N-terminal ends (residues 1-84) and repeats 6-7 at the C-terminal ends (residues 232-332) of all five proteins. As other WD proteins are known to be involved in protein-protein interactions (47), we set out to isolate point mutations in TIF34 that would weaken its ability to interact with PRT1 or TIF35.
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Genetic Interactions between TIF34 and TIF35--
To test whether
the tif34-HA-1, -2, and -3 mutations weaken an
essential interaction between TIF34 and either TIF35 or PRT1 in
vivo, we asked whether overexpression of TIF35 or
PRT1 could suppress the growth defects of the
tif34-HA mutant strains. The TIF35 and
PRT1 genes were inserted into a high copy vector, and the
resulting plasmids were introduced into the tif34-HA mutant strains. As shown in Fig. 4, the
Ts phenotypes of tif34-HA-1 and
tif34-HA-2 were fully suppressed by multiple copies of
TIF35. Thus, transformants of these mutants containing the
high copy TIF35 plasmid grew as rapidly at 36 °C as did
the wild-type TIF34 strain (TIF34-HA) transformed
with empty vector, or the tif34 mutants transformed with a
plasmid bearing TIF34. The Slg
phenotype of
the tif34-HA-3 mutant was only weakly suppressed by the high
copy TIF35 plasmid, even though its growth defect at
36 °C was no more severe than that of the tif34-HA-1
mutant (Fig. 4). In contrast to the effects of the high copy
TIF35 plasmid, introducing a multicopy PRT1
plasmid did not suppress the phenotypes of any of the
tif34-HA mutants; however, it should be noted that high copy
PRT1 conferred an Slg
phenotype in the
wild-type TIF34-HA strain (Fig. 4). This effect could
complicate the ability of overexpressed PRT1 to compensate for the
growth defects conferred by the tif34-HA mutations. To explain these results, we suggest that the tif34-HA-1 and
tif34-HA-2 mutations weaken the physical interaction between
TIF34 and TIF35 at the non-permissive temperature due to an altered
conformation of the mutant TIF34 proteins and that overexpression of
TIF35 can restore the TIF34-TIF35 interaction by mass action. According to this model, the tif34-HA-3 allele is defective for a
function of TIF34 besides complex formation with TIF35 and thus cannot be fully suppressed by overexpression of TIF35.
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Effects of Point Mutations in TIF34 on Binding to TIF35 and PRT1 in
Vitro--
To test our model that the mutations in
tif34-HA-1 and tif34-HA-2 weaken a physical
interaction between TIF34 and TIF35, we synthesized
35S-labeled TIF34 proteins bearing these mutations and
compared them to wild-type TIF34 for binding to the GST fusion
containing the N-terminal half of TIF35. Because the
tif34-HA-1 and tif34-HA-2 alleles have a
Ts phenotype at 36 °C in vivo, these
in vitro binding reactions were carried out at 36 °C. As
shown in Fig. 5, A and
B, binding of wild-type 35S-TIF34 increased with
increasing amounts of the GST-TIF35 fusion protein added to the
reactions. Binding of the 35S-labeled Tif34-HA-1 and
Tif34-HA-2 proteins to the GST-TIF35 fusion protein was greatly reduced
compared with wild-type 35S-TIF34 binding at all
concentrations of the GST fusion protein tested. These results support
the idea that the tif34-HA-1 and tif34-HA-2
mutations reduce the affinity between TIF34 and TIF35 proteins and thus
can be suppressed by increasing the concentration of TIF35. In
contrast, binding of 35S-Tif34-HA-3 showed only a small
reduction in binding relative to wild-type 35S-TIF34,
consistent with our conclusion that tif34-HA-3 has
additional defects that cannot be corrected simply by overexpression of
TIF35. The fact that tif34 point mutations which are
suppressed in vivo by high copy TIF35 were found
to weaken binding between TIF34 and TIF35 in vitro strongly
suggests that the interactions we detected between these proteins are
relevant to the in vivo function of TIF34. The results in
Fig. 5, C and D, indicate that the Tif34-HA-1 and
Tif34-HA-2 proteins also were very defective for binding to the
GST-PRT1 fusion containing the C-terminal 83 residues of PRT1, whereas
Tif34-HA-3 again showed only a small defect in binding relative to
wild-type TIF34. The implication of these last findings in relation to
complex formation by TIF34, PRT1, and TIF35 in vivo will be
discussed later.
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PRT1 Binds Specifically to TIF32--
Although PRT1, TIF34, and
TIF35 did not interact with NIP1 or TIF32 in the two-hybrid assay, we
reinvestigated their possible interactions in vitro using
GST-TIF32 and GST-NIP1 fusions. SDS-PAGE analyses of these purified
fusion proteins revealed full-length products of the expected sizes
plus numerous degradation products truncated at the C terminus (Fig.
6, top panel). As shown in
Fig. 6, 35S-labeled full-length PRT1 and the M-(1-682)
and
S-(1-624) PRT1 proteins truncated at the C terminus bound
strongly to the GST-TIF32 fusion proteins but showed little or no
binding to the GST-NIP1 proteins (Fig. 6, panels 2-4 from
top, lanes 3 and 4; Table IV). In contrast,
neither 35S-TIF34 nor 35S-TIF35 interacted with
either set of GST fusion proteins (Fig. 6, panels 5 and
6 from top). The
M-(1-682) and
S-(1-624)
C-terminal truncations of PRT1 were shown above to abolish the binding
of PRT1 to GST-TIF34 and GST-TIF35 (Fig. 2B). Thus, it
appears that the binding domain for TIF32 in PRT1 is located N-terminal
to the binding domains for TIF35 and TIF34 (summarized in Fig.
8A).
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NIP1 Binds Specifically to TIF32 and SUI1-- Fig. 6 also shows that 35S-labeled NIP1 bound specifically to GST-TIF32 (panel labeled 35S-NIP1, lane 3), although the binding was weak compared with that seen between 35S-PRT1 and GST-TIF32 (see Table IV). This could indicate that NIP1 has a lower affinity than PRT1 for TIF32. Alternatively, it is possible that NIP1 can interact only with full-length GST-TIF32, whereas PRT1 is capable of binding to GST-TIF32 degradation products in addition to full-length GST-TIF32. These alternative possibilities remain to be addressed in the future. 35S-Labeled SUI1 bound to GST-NIP1 but not to GST-TIF32 or GST alone (Fig. 6, bottom panel, lanes 2-4; Table IV), confirming the interaction between SUI1 and NIP1 observed in the two-hybrid assay (Table III). Taken together, the results in Fig. 6 suggest that PRT1 binds to TIF32 and that NIP1 is capable of bridging an interaction between TIF32 and SUI1 (see Fig. 8A).
Complex Formation by TIF35, PRT1, NIP1, TIF34, and a 120-kDa
Protein in the Ribosomal Salt Wash Fraction--
To provide more
direct evidence for complex formation by the five yeast proteins
homologous to subunits of mammalian eIF3 in vivo, we asked
whether they could be coimmunoprecipitated from WCE. Toward this end,
we constructed an allele of TIF35 encoding a FLAG
epitope-tagged form of the protein on a high copy plasmid. This tagged
allele was judged to be functional because in high copy number it
suppressed the Ts phenotype of the tif34-HA-1
mutant to the same extent observed for high copy number wild-type
TIF35 (data not shown). We reasoned that overexpression of
FLAG-tagged-TIF35 would favor its incorporation into the eIF3 complex
in place of wild-type TIF35. Accordingly, the high copy plasmid bearing
TIF35-FLAG was introduced into strains containing wild-type
TIF34 or TIF34-HA, and WCEs from the resulting transformants were immunoprecipitated with anti-HA antibodies. When the
immune complexes were probed by immunoblot analysis for PRT1, NIP1,
TIF35-FLAG, and TIF34-HA, we found that all four proteins were
immunoprecipitated only from the strain containing HA-tagged TIF34
(Fig. 7A, panels 1-4, lanes 2 and 5). The yields of PRT1 and NIP1 in the immune complexes
from the TIF34-HA strain were similar to that observed for
TIF34-HA itself (~50% of the input amounts), suggesting that the
majority of these three proteins was present in the same heteromeric
complexes in vivo. The yield of TIF35-FLAG in the immune
complexes was less (15-20%), presumably because it was overexpressed
and the excess protein was not incorporated into the complex. In
contrast, we found that little or no GCD10 or the
-subunit of eIF2
(encoded by GCD11) was coimmunoprecipitated with the other
four proteins (Fig. 7A, bottom two panels).
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DISCUSSION |
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PRT1 and TIF34 are essential proteins in S. cerevisiae known to be required for the initiation of protein synthesis (22, 50, 51). They are homologous to the 116- and 36-kDa subunits, respectively, of the nine-subunit mammalian eIF3 complex purified by its activity in the Met-puromycin synthesis assay, and they were shown to be subunits of a yeast eIF3 complex purified with the same assay (20, 22). Thus, there is strong evidence that PRT1 and TIF34 carry out their essential functions in yeast as components of eIF3. The other three yeast proteins homologous to subunits of human eIF3, TIF32, NIP1, and TIF35 were not known to be subunits of yeast eIF3, although a 33-kDa polypeptide close in molecular weight to that of TIF35 copurified with eIF3 activity (20).
We considered the possibility that all five yeast proteins homologous
to human eIF3 subunits are components of a high molecular weight
complex in yeast cells, and we obtained several lines of evidence
supporting this hypothesis. We detected interactions between PRT1,
TIF34, and TIF35 in the yeast two-hybrid assay (Table III and Fig. 1),
and these interactions were confirmed by in vitro binding
assays using recombinant forms of the proteins (Fig. 2). We were able
to localize binding domains responsible for these interactions within
the C-terminal portion of PRT1 and the N-terminal half of TIF35 (Figs.
1 and 2). In addition to these physical interactions, we showed that
certain Ts mutations in TIF34 could be
suppressed in vivo by increasing the dosage of
TIF35 (Fig. 4). The suppressible tif34 mutations were found to weaken the interaction between recombinant TIF34 and
TIF35 proteins in vitro (Fig. 5). These findings suggested that an unstable interaction between TIF34 and TIF35 in vivo
was at least partially responsible for the inability of the mutant tif34 proteins to carry out their essential functions in
translation. Together, these data provide strong evidence that two
known subunits of eIF3, PRT1 and TIF34, interact in vivo
with TIF35, the yeast homologue of mammalian eIF3-p44.
We also found that recombinant forms of PRT1 and TIF32 interacted strongly in vitro and that NIP1 showed a weaker but significant interaction with TIF32 (Fig. 6). These data suggested that all five yeast homologues of mammalian eIF3 subunits might interact with one another in vivo. We obtained biochemical evidence for this hypothesis by showing that NIP1, PRT1, TIF34, and TIF35 were specifically coimmunoprecipitated from both WCEs and the RSW fraction using antibodies against an HA-tagged form of TIF34 (Fig. 7). In the coimmunoprecipitations shown in Fig. 7B, these four polypeptides (or degradation products of them), and a protein with the predicted molecular weight of TIF32, were the major proteins that specifically coimmunoprecipitated with HA-tagged TIF34. We conclude that at least four of the five homologues of human eIF3 subunits, and probably all five, interact with one another in vivo and are likely to be components of the same high molecular weight complex. These results support the physical interactions linking together all five yeast homologues of human eIF3 subunits summarized in Fig. 8A.
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The C-terminal portion of PRT1 (residues 641-724) was sufficient for both two-hybrid and in vitro interactions with TIF34. Full-length PRT1 interacted directly with TIF35 in vitro in a manner that depended on residues C-terminal to position 624 in PRT1 (Fig. 2B). As the C-terminal segment of PRT1 alone did not interact with TIF35 in vitro, we presume that more N-terminally located residues in PRT1 are additionally required for a direct interaction with TIF35 in the absence of TIF34 (Fig. 8A). PRT1 also showed direct binding to TIF32 in vitro, and this interaction was not dependent on the C-terminal segments of PRT1 required for its interaction with TIF35 and TIF34 (Fig. 6). Thus, the binding domain for TIF32 in PRT1 is probably located N-terminal to those for TIF35 and TIF34 (Fig. 8A). There is evidence that the mammalian homologues of PRT1 and TIF32, namely eIF3-p116 and eIF3-p170, also interact directly with one another (15) (Fig. 8B).
We obtained genetic and biochemical evidence that the WD repeat
elements of TIF34 are important for its interactions with TIF35 and
PRT1. The Ts phenotypes of point mutations in the 6th or
7th WD repeats in tif34-HA-2 and tif34-HA-1,
respectively, were suppressed by increased TIF35 gene dosage
(Fig. 4). Because the tif34-HA-3 mutant was not efficiently
suppressed by high copy TIF35, it is unlikely that
overexpression of TIF35 simply bypassed the function of TIF34 in
translation initiation. Based on the fact that both Tif34-HA-1 and
Tif34-HA-2, but not Tif34-HA-3, were very defective for interaction with TIF35 in vitro (Fig. 5), we proposed that
overexpression of TIF35 compensated for its reduced affinity for mutant
Tif34-HA-1 and Tif34-HA-2 and restored the TIF35-TIF34 interaction by
mass action. The tif34-HA-1 and tif34-HA-2
mutations also impaired binding between TIF34 and PRT1 in
vitro; in fact, there was a greater reduction in binding to PRT1
versus TIF35 (Fig. 5); however, increasing PRT1
dosage did not suppress the Ts
phenotypes of the
tif34 mutations (Fig. 4). To explain these observations, it
could be proposed that conformational changes caused by the
Ts
tif34 mutations led to dissociation of both
PRT1 and TIF35 from TIF34, but the reduction in PRT1 binding affinity
was too great to be overcome by PRT1 overexpression. In contrast, TIF35
overexpression would restore TIF34-TIF35 interaction and this, in turn,
would recruit PRT1 to the complex because PRT1 can interact with TIF35 as well as with TIF34 (Fig. 8A). It was shown previously
that a deletion in the C-terminal 147 amino acids of PRT1 destabilized the protein in vivo and had a lethal phenotype (52). These
previous findings are consistent with the idea that the physical
interactions we detected between TIF34 and the C-terminal end of PRT1
are important for the integrity of the eIF3 complex in
vivo.
While this manuscript was in preparation, Verlhac et al.
(48) also identified TIF35 as a protein that interacts with TIF34 in vivo by using yeast two-hybrid assay and
coimmunoprecipitation analysis and by showing that increased
TIF35 dosage suppressed the Ts phenotype of a
TIF34 allele containing two substitutions in WD repeats 3 and 6 but not one containing three substitutions in WD repeats 2, 4, and 5. They also cited unpublished data indicating that direct peptide
sequencing of the 33-kDa polypeptide that copurified with yeast eIF3
activity in the Met-puromycin assay (20) had established its identity
as TIF35. These findings, in combination with the coimmunoprecipitation
results in Fig. 8, provide strong evidence that TIF35 is an integral
subunit of yeast eIF3.
The eIF3 complex purified using the Met-puromycin synthesis assay contains PRT1 (20), TIF34 (22), GCD10 (23), SUI1 (24), and apparently TIF35 (48); however, it lacked NIP1 and TIF32 (53). Our finding that a large percentage of the NIP1 in whole cell extracts was coimmunoprecipitated with HA-TIF34 (Fig. 7A) and that this association was maintained in high salt buffers (Fig. 7B) suggests that NIP1 is stably associated with the yeast eIF3 complex in vivo. The in vitro interaction between GST-TIF32 and 35S-PRT1 was among the strongest we detected (Table IV). In addition, a protein with molecular weight similar to that of TIF32 was coimmunoprecipitated with HA-TIF34 from the RSW at roughly the same stoichiometry as the other four yeast homologues of mammalian eIF3 subunits (Fig. 7B). These findings suggest that TIF32 is also associated with yeast eIF3. Supporting this conclusion, we recently purified a high molecular mass complex of ~600 kDa from a strain expressing a polyhistidine-tagged form of PRT1 and showed by mass spectrometric analysis that it contains TIF32, NIP1, TIF34, and TIF35 as the major proteins specifically associated with the tagged PRT1 protein (56). A PRT1-containing complex has been purified previously by conventional chromatography containing major polypeptides with relative molecular weights similar to those of TIF32, NIP1, TIF34, and TIF35 (27). This complex was not tested in the Met-puromycin assay; however, it was shown to rescue binding of Met-tRNAiMet to 40 S ribosomes in extracts prepared from a temperature-sensitive prt1 mutant (27), consistent with a known function of eIF3. We recently succeeded in demonstrating the same activity for the complex we purified containing His-tagged PRT1 and the other four yeast homologues of mammalian eIF3 subunits.6
It was shown previously that a temperature-sensitive sui1 mutant grown at the nonpermissive temperature lacks SUI1 protein and eIF3 activity (24), suggesting that SUI1 is required for eIF3 function in the Met-puromycin assay. We detected interactions between NIP1 and SUI1 with both yeast two-hybrid and in vitro protein binding assays. Unlike NIP1, however, we found that SUI1 did not coimmunoprecipitate with HA-TIF34 from the RSW fraction (Fig. 7B). Thus, SUI1 may dissociate more readily from the yeast eIF3 complex than do other eIF3 subunits, consistent with the fact that the mammalian homologue of SUI1 (eIF1) and eIF3 were purified as separate factors from the RSW fractions of rabbit reticulocytes and HeLa cells (6, 13). It is noteworthy that the active fraction of mammalian eIF3 apparently does not contain eIF1 (13, 16).
GCD10 was found previously to coimmunoprecipitate with PRT1 using polyclonal antibodies against PRT1 or GCD10 (23); however, GCD10 did not coimmunoprecipitate with HA-TIF34 in our experiments (Fig. 7). In addition, we found that GCD10 did not copurify with a polyhistidine-tagged form of PRT1 (56). These recent results suggest that GCD10 is not tightly bound to other subunits of yeast eIF3. On the other hand, GCD10 interacted with GCD14 in the two-hybrid assay, confirming observations that GCD10 is stably associated with GCD14 in a nuclear complex that functions in the expression of initiator tRNAiMet.5 More work is needed to determine whether GCD10 also functions in the cytoplasm in association with eIF3.
The results presented in this report suggest that all five yeast homologues of human eIF3 subunits are physically associated with the eIF3 complex of yeast. It appears that NIP1 and TIF32 are not required for eIF3 activity in the Met-puromycin assay. In addition, it was recently shown that the mammalian counterpart of TIF32 (eIF3-p170) is dispensable for stimulating tRNAiMet binding to 40 S ribosomes (19). Thus, NIP1 and TIF32 may be required only for the function of eIF3 in promoting ribosomal subunit dissociation or mRNA binding. A high degree of evolutionary conservation has been observed for most of the translation initiation factors characterized in both yeast and mammals (1). Nevertheless, human eIF3 contains four subunits, p40, p47, p48, and p66, with no apparent counterparts in yeast. In particular, eIF3-p48 is encoded by int-6, a site of frequent integration by mouse mammary tumor viruses and, therefore, is suspected to be a regulatory subunit of mammalian eIF3 (18). It will be interesting to learn whether the functions of these unique subunits in mammalian eIF3 are carried out in yeast by other nonconserved proteins not tightly associated with the eIF3 complex.
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ACKNOWLEDGEMENTS |
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We are indebted to John Hershey and Parisa Hanachi for pNOTAp39 and pNOp33 and for communicating results prior to publication and to David Goldfarb for anti-NIP1 antibodies. We also thank Tom Dever for critical reading of the manuscript; Weimin Yang for sharing materials used in two-hybrid screening; and Hanse Chung and Fan Zhang for technical assistance.
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FOOTNOTES |
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* 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.
Supported by the Japan Society for the Promotion of Science
Research Fellowships for Japanese Biomedical and Behavioral Researchers at the National Institutes of Health.
§ To whom correspondence should be addressed. Tel.: 301-496-4480; Fax: 301-496-6828; E-mail: ahinnebusch{at}nih.gov.
1 The abbreviations used are: eIF, eukaryotic translation initiation factor; PCR, polymerase chain reaction; ORF, open reading frame; kb, kilobase(s); GST, glutathione S-transferase; UTR, untranslated region; SC, synthetic complete (medium); 5-FOA, 5-fluoroorotic acid; 3-AT, 3-aminotriazole; bp, base pair(s); WCE, whole cell extract(s); RSW, ribosomal salt wash; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.
2 K. Block, H.-P. Vornlocher, K. Asano, and J. Hershey, manuscript in preparation. The amino acid sequence of human eIF3-p44 was made available by J. Hershey.
3 J. Anderson, K. Asano, and A. G. Hinnebusch, unpublished observations.
4 R. Cuesta, O. Calvo, J. Anderson, M. T. Garcia Barrio, A. G. Hinnebusch, and M. Tamame, manuscript in preparation.
5 J. Anderson, M. Pak, L. Phan, R. Cuesta, K. Asano, M. Tamame, and A. G. Hinnebusch, submitted for publication.
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