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. 1998 Aug;18(8):4935-46.
doi: 10.1128/mcb.18.8.4935.

Identification of a Translation Initiation Factor 3 (eIF3) Core Complex, Conserved in Yeast and Mammals, That Interacts With eIF5

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Free PMC article

Identification of a Translation Initiation Factor 3 (eIF3) Core Complex, Conserved in Yeast and Mammals, That Interacts With eIF5

L Phan et al. Mol Cell Biol. .
Free PMC article

Abstract

Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni2+ affinity and gel filtration chromatography and obtained a complex of approximately 600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNAiMet binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.

Figures

FIG. 1
FIG. 1
Affinity purification of a high-molecular-weight complex containing yeast homologs of mammalian eIF3 subunits and eIF5. Equal amounts of RSW fractions prepared from strains LPY200 and LPY201 containing wild-type Prt1p or His-Prt1p, respectively, were bound to Ni2+-NTA agarose, eluted, and separated on a Pharmacia Superose-6 FPLC column precalibrated with known size standards (Std) (4 to 670 kDa; Bio-Rad). The masses of the standards are shown above the fractions in which they eluted. (A) Aliquots (20 μl) of column fractions (numbered across the top) were separated by SDS-PAGE using 4 to 20% gradient gels, and the proteins were visualized by silver staining. Identical fractions derived from LPY200 and LPY201 were loaded in adjacent lanes (His-Tag − and +, respectively). The molecular masses of SDS-PAGE size standards (Novex) are shown on the right. The six major polypeptides whose amounts peaked in fraction 18 from LPY201 and were absent in the corresponding fraction from LPY200 are indicated on the left. (B) A gel identical to that in panel A was subjected to immunoblot analysis with antibodies against the proteins listed on the right, used at the following dilutions: Nip1p, 1:1,000; Prt1p, 1:1,000; eIF5, 1:2,500; and Tif34p, 1:500.
FIG. 2
FIG. 2
Identification of proteins specifically associated with His-Prt1p in whole-cell extracts. Equal amounts of total protein in whole-cell extracts prepared from strains LPY200 and LPY201 containing wild-type Prt1p or His-Prt1p, respectively, were bound to Ni2+-silica, eluted, resolved by SDS-PAGE using 4 to 20% gradient gels, and subjected to immunoblot analysis using antibodies against the proteins listed on the left of each panel, used at the following dilutions: Prt1p, 1:1,000; Tif34p, 1:500; Nip1p, 1:1,000; eIF5, 1:2,500; Sui1p, 1:1,000; Gcd10p, 1:500; eIF4G, 1:1,000; and Gcd6p, 1:1,000. Lanes 1 and 2 contain 20% of the input amounts of whole-cell extract from LPY200 (His-Tag −) and LPY201 (His-Tag +), respectively; lanes 3 and 4 contain the entire Ni2+-silica eluates (Ni2+-EL) from LPY200 and LPY201, respectively.
FIG. 3
FIG. 3
Coimmunoprecipitation of Prt1p, Nip1p, and eIF5 with HA-tagged Tif34p. Whole-cell extracts from strains KAY8 (TIF34-HA) and KAY1 (TIF34) were immunoprecipitated with monoclonal antibody 12CA5 against the HA epitope. Aliquots containing 25% of the input whole-cell extracts (I), 25% of the supernatant fractions (S), and the entire immunoprecipitated pellets (P) were separated by SDS-PAGE using 12% gels and subjected to immunoblot analysis using antibodies against the proteins listed to the right of each panel.
FIG. 4
FIG. 4
Complementation of a heat-treated prt1-1 mutant or Ubi-Nip1p-depleted extract for translation of exogenous luciferase mRNA. Whole-cell extracts were tested for the ability to translate capped luciferase mRNA. (A) Extracts prepared from isogenic strains H1616 (prt1-1) or LPY200 (PRT1) were incubated at 37°C for 5 min prior to performance of the assay; 35-μl aliquots of extract were mixed with an equal volume of 2× translation buffer containing 1 mM GTP, 4 μg of mRNA, 0.1 mM complete amino acid mix (Promega), and 10 U of RNasin (Promega) RNase inhibitor and then incubated at 26°C. Reactions designated (+) were performed in the presence of ∼4 pmol of purified His-Prt1p complex from Superose-6 column fraction 18 (shown in Fig. 1A) from strain LPY201; reactions designated (−) received an equivalent proportion of column fraction 18 lacking the Prt1p complex from strain LPY200. At the indicated times, 8-μl aliquots were withdrawn and diluted with 22 μl of distilled H2O and frozen in a dry ice-ethanol bath. The amount of luciferase produced (in relative light units [RLU]) in each sample was measured subsequently as described in Materials and Methods. (B) NIP1KR4R1 (UBI-R-NIP1) and its isogenic parent Ad (NIP1) were grown in galactose-containing medium for 17 h and shifted to glucose-containing medium and allowed to double three to four times before harvesting. Extracts were prepared and tested for the ability to translate capped luciferase mRNA as described for panel A.
FIG. 5
FIG. 5
Complementation of a heat-treated prt1-1 mutant and Ubi-Nip1p-depleted extracts for [3H]Met-tRNAiMet binding to 40S ribosomal subunits. The binding of [3H]Met-tRNAiMet to 40S ribosomal subunits was assayed by mixing 15 μl of extract with an equal volume of 2× translation buffer containing ∼10 pmol of [3H]Met-tRNAiMet (0.77 μCi) and 2.4 mM nonhydrolyzable GTP analog GMPPNP. As for Fig. 4, duplicate reactions were carried out for each extract containing an aliquot of fraction 18 (Fig. 1A) from strain LPY201 containing ∼2 pmol of purified His-Prt1p complex (+) or an equivalent proportion of fraction 18 from LPY200 lacking Prt1p complex (−). The reactions were incubated at 26°C for 20 min, fixed by addition of formaldehyde to 0.3%, and resolved by velocity sedimentation on 10 to 30% sucrose gradients by centrifugation at 41,000 rpm for 5 h in an SW41 rotor. Fractions (0.7 ml) were collected starting at the top of the gradient and analyzed by filter assay and counted for [3H]Met-tRNAiMet by liquid scintillation (dashed line). The positions of the 40S and 60S ribosomal subunits and 80S ribosomes were determined by monitoring the OD254 while collecting fractions from the gradient. (A) Extracts prepared from isogenic strains H1616 (prt1-1) (bottom) and LPY200 (PRT1) (top) were incubated at 37°C for 5 min prior to performance of the assay. (B) Extracts prepared from isogenic strains Ad (NIP1) (top panel) and NIP1KR4R1 (UBI-RNIP1) (bottom) grown under conditions described for Fig. 4B to deplete Ubi-Nip1p from the NIP1KR4R1 extract.
FIG. 6
FIG. 6
Efficient Met-tRNAiMet binding to 40S ribosomal subunits in the absence of Gcd10p. Whole-cell extracts were prepared from isogenic strains YJA146 (gcd10Δ) and YJA158 (GCD10), and binding of [3H]Met-tRNAiMet to 40S ribosomal subunits was assayed by mixing 100 μl of extract with an equal volume of 2× translation buffer containing ∼10 pmol of [3H]Met-tRNAiMet (80 pCi/pmol) and 2.4 mM nonhydrolyzable GTP analog GMPPNP and then incubating the mixture for 5 or 15 min at 26°C. Reactions were stopped and resolved by velocity sedimentation essentially as described for Fig. 5 except that 0.4-ml fractions were collected from the gradients.
FIG. 7
FIG. 7
Evidence that eIF5 interacts with Nip1p. (A) Analysis of interactions between full-length eIF5 and the five yeast proteins homologous to subunits of eIF3 in the yeast two-hybrid assay. Fusions between GBD and full-length Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p encoded by the plasmids listed in the first column were tested for interactions with a fusion between full-length eIF5 and GAD encoded by pGAD-TIF5 or with GAD alone (Vector). The pGBT9 derivatives and pGBT9 alone were introduced into yeast strain Y190, and the resulting Trp+ Leu transformants were mated to Trp Leu+ transformants of strain Y187 containing pGAD-TIF5 or pGAD424 alone. The resulting Trp+ Leu+ diploids were tested for growth on SC medium lacking leucine, tryptophan, and histidine and containing different concentrations of 3-AT. The extent of two-hybrid interaction is indicated by the degree of 3-AT resistance (22, 27). −, no growth at 5 mM 3-AT; +++, growth at 30 mM 3-AT. (B) The GST-eIF5 fusion protein or GST alone was immobilized on glutathione-Sepharose beads and incubated with 35S-labeled Nip1p synthesized by in vitro translation. After extensive washing, proteins bound to the beads were eluted and separated by SDS-PAGE. The gel was stained with Coomassie blue to visualize the eluted GST proteins (top), followed by autoradiography to detect the [35S]Nip1p (bottom). The species of greatest apparent molecular weight (molecular weights are indicated in thousands on the left) visible in the GST-TIF5 lane (top) migrated with the mobility expected for the full-length GST-TIF5 fusion. The less abundant species migrating more rapidly are presumed to be degradation products of the full-length fusion. Lane In in the bottom panel contains 100% of the input amount of 35S-labeled Nip1p used in the binding reactions. Phosphorimaging analysis of the bottom panel showed that 61% of the input amount of 35S-labeled full-length Nip1p bound to GST-TIF5, whereas only 0.3% bound to GST alone.

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