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. 2005 Nov 8;102(45):16164-9.
doi: 10.1073/pnas.0507960102. Epub 2005 Oct 27.

The eukaryotic initiation factor (eIF) 5 HEAT domain mediates multifactor assembly and scanning with distinct interfaces to eIF1, eIF2, eIF3, and eIF4G

Yasufumi Yamamoto  1 , Chingakham Ranjit Singh, Assen Marintchev, Nathan S Hall, Ernest M Hannig, Gerhard Wagner, Katsura Asano
Affiliations

The eukaryotic initiation factor (eIF) 5 HEAT domain mediates multifactor assembly and scanning with distinct interfaces to eIF1, eIF2, eIF3, and eIF4G

Yasufumi Yamamoto et al. Proc Natl Acad Sci U S A. .

Abstract

Eukaryotic translation initiation factor (eIF) 5 is crucial for the assembly of the eukaryotic preinitiation complex. This activity is mediated by the ability of its C-terminal HEAT domain to interact with eIF1, eIF2, and eIF3 in the multifactor complex and with eIF4G in the 48S complex. However, the binding sites for these factors on eIF5-C-terminal domain (CTD) have not been known. Here we present a homology model for eIF5-CTD based on the HEAT domain of eIF2Bepsilon. We show that the binding site for eIF2beta is located in a surface area containing aromatic and acidic residues (aromatic/acidic boxes), that the binding sites for eIF1 and eIF3c are located in a conserved surface region of basic residues, and that eIF4G binds eIF5-CTD at an interface overlapping with the acidic area. Mutations in these distinct eIF5 surface areas impair GCN4 translational control by disrupting preinitiation complex interactions. These results indicate that the eIF5 HEAT domain is a critical nucleation core for preinitiation complex assembly and function.

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Figures

Fig. 1.
Fig. 1.
Identification of MFC partner-binding surfaces on eIF5-CTD. (A) Positions of mutations are located on the surface of the eIF5-CTD structure model (Center); and after rotating 60° to the left (Left) and to the right (Right). Areas I and II, defined in the text, are circled. Residues changed in the AN1, BN1, and BN2 mutants are colored in red and dark and light blue, respectively, and labeled in Left and Right, respectively. Residues deleted by W391Δ and E396Δ are colored in orange and yellow, respectively, with the first deleted residues labeled in Center. The last nine residues of eIF5 (residues 397–405) are not present in this model. (B) Yeast eIF5 amino acids from position 339 to 400 are arranged to show the predicted secondary structure. Residues predicted to participate in α-helices 5–8 (defined in Fig. 5) are boxed with the helix numbers in boldface. Highlighted with orange are AA-box amino acids (AA box 1 in helices 6 and 7; AA box 2 in helix 8), whereas highlighted with green in blue letters are basic residues highly conserved in all eIF5. Conserved acidic residues are shown in red. Arrows indicate the site of mutations. Thick arrows indicate regions deleted by W391Δ and E396Δ. Circled in red are the residues altered by Ts– point mutations (11). (C) GST pull-down assays. GST alone (lanes 2) or GST-eIF5 (lanes 3 and 10) and its mutants (lanes 4–8 and 11) were allowed to bind the 35S partners indicated in the middle. The complex was analyzed by SDS/PAGE and autoradiography. Five or 3 μg of GST or its fusion protein was used in lanes 1–8 or 9–11, respectively. Percent of 35S protein pulled down is shown below each gel. Lanes 1 and 7, 20% in-put amounts; N. T., not tested. (D) Summary of effects of eIF5 mutations, indicated to the right, on GST-eIF5 binding to eIF2β-N (a), eIF3c-N (b), and eIF1 (c). Binding assays as in C were performed several times, and the fractions of 35S partners bound to GST-eIF5 mutants were compared with the fraction of the same partners bound to wild-type GST-eIF5. Average values (boxes) and SDs (bars) are presented. (E) Hypothetical model of MFC assembly. eIF5-CTD is depicted as the same orientation as in A Center, with areas I and II in red and blue, respectively. Parts of eIF1, eIF2β-K-box, eIF3c, and eIF4G are drawn as differently colored objects.
Fig. 2.
Fig. 2.
Effect of eIF5-CTD mutations on MFC formation in vivo. (A) Coimmunoprecipitation of HA-eIF3. Whole-cell extracts prepared from KAY37 (TIF34 TIF5; Control), KAY113 (TIF34-HA TIF5: TIF5), and its derivatives listed in Table 3 with indicated mutations were used for immunoprecipitation with anti-HA affinity resin. The entire pellet fractions (P) were analyzed together with 10% input (I) and 10% supernatant (S) fractions by immunoblotting with antibodies indicated to the right (see Supporting Text). TIF34-HA encodes the HA-tagged eIF3i subunit. (B) Coimmunoprecipitation of FLAG-eIF2. KAY17 (SUI3 TIF5; Control) (4), KAY128 (FL-SUI3 TIF5; TIF5), and its derivatives listed in Table 3 with indicated mutations were used for immunoprecipitation with anti-FLAG affinity resin, and the immune complex was analyzed as in A. Numbers below anti-eIF5 blots of the P fractions indicate percent of eIF5 found in these fractions, as measured with nih image software (National Institutes of Health). (C) The fraction of eIF5 found in the FL-eIF2 pellet fractions from indicated strains was compared with that of eIF5 found in the pellet fraction from the wild-type strain. Average (filled box) and SD (empty box) from at least three independent experiments are presented. (D) Models of MFC assembly in the strains tested. Circles, individual eIFs. Filled circle, mutant eIF5. Thick solid or dotted lines, MFC partner interfaces strongly or weakly impaired by the mutation introduced, respectively, as judged by in vitro binding studies. Direct contact, strong interactions. No contact, defective interactions, as judged by coimmunoprecipitation studies. Thin lines, interaction eliminated in the coimmunoprecipitation via HA-eIF3 but not affected in that via FL-eIF2.
Fig. 3.
Fig. 3.
Effect of eIF5-CTD mutations on general control phenotypes. (A) Gcd– phenotype test. The same amount of the overnight culture of transformants of gcn2Δ strains (KAY24 derivatives in Table 3) carrying the indicated TRP1 and URA3 plasmids, grown in SC-ura-trp, and their 1/10 and 1/100 dilutions were spotted onto SD medium containing required supplements with (b and d) or without (a and c) 30 mM 3AT and incubated for 4 or 3 days, respectively. TRP1 plasmids used are: YEplac112 (Vector, odd-numbered rows) and YEpW-SUI1 (eIF1, even-numbered rows). URA3 plasmids used are YEplac195 (Vector, a and b) and p1780-IMT (formula image, c and d) (see Table 1). (B) Gcn– phenotype test. TIF5 GCN2 strains with indicated mutations (Table 3 and isogenic tif5-F364S strain KAY315 in row 2; ref. 11) were grown in yeast extract/peptone/dextrose, diluted and spotted as in A, onto SC-his medium with (c and e) or without (a, b, and d) 50 mM 3AT and 40 mM leucine. SC-his plates were incubated for 2 days at indicated temperatures, and SC-his containing 3AT were incubated for 4 and 6 days at 33°C and 36°C, respectively.
Fig. 4.
Fig. 4.
Effect of eIF5-CTD mutations on GCN4 translational control. Transformants of KAY314 (WT), KAY364 (BN1), KAY351 (AN1), and KAY405 (AN1 E396Δ) carrying the indicated GCN4-lacZ plasmid (Table 1) were grown in the following media and assayed for β-galactosidase activity, as described and expressed in ref. . Bars indicate β-galactosidase activities from several experiments by using at least two independent transformants, with SD in lines. Schematics at the top of A–D depict the GCN4 leader structure drawn to scale, with positive regulatory uORF1 (filled boxes), other negative regulatory uORFs (open boxes), and the GCN4 coding region (gray boxes). In the table to the left, column 1 (temp.) indicates temperature at which yeast cultures were incubated, column 2 (TIF5) indicates TIF5 mutations, and column 3 (ind.) indicates the ratio of LacZ activity in the presence of 3AT to that in the absence of 3AT. (A and B) Transformants were grown in SC-his-ura medium supplemented with (gray bars) or without 10 mM 3AT (filled bars). (C and D) Transformants were grown in SC-ura medium.
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