An eIF5/eIF2 complex antagonizes guanine nucleotide exchange by eIF2B during translation initiation

Abstract

In eukaryotic translation initiation, the eIF2.GTP/Met-tRNA(i)(Met) ternary complex (TC) binds the eIF3/eIF1/eIF5 complex to form the multifactor complex (MFC), whereas eIF2.GDP binds the pentameric factor eIF2B for guanine nucleotide exchange. eIF5 and the eIF2Bvarepsilon catalytic subunit possess a conserved eIF2-binding site. Nearly half of cellular eIF2 forms a complex with eIF5 lacking Met-tRNA(i)(Met), and here we investigate its physiological significance. eIF5 overexpression increases the abundance of both eIF2/eIF5 and TC/eIF5 complexes, thereby impeding eIF2B reaction and MFC formation, respectively. eIF2Bvarepsilon mutations, but not other eIF2B mutations, enhance the ability of overexpressed eIF5 to compete for eIF2, indicating that interaction of eIF2Bvarepsilon with eIF2 normally disrupts eIF2/eIF5 interaction. Overexpression of the catalytic eIF2Bvarepsilon segment similarly exacerbates eIF5 mutant phenotypes, supporting the ability of eIF2Bvarepsilon to compete with MFC. Moreover, we show that eIF5 overexpression does not generate aberrant MFC lacking tRNA(i)(Met), suggesting that tRNA(i)(Met) is a vital component promoting MFC assembly. We propose that the eIF2/eIF5 complex represents a cytoplasmic reservoir for eIF2 that antagonizes eIF2B-promoted guanine nucleotide exchange, enabling coordinated regulation of translation initiation.

Figure 1

Determination of TC and eIF assembly intermediate levels in vivo. (A) and (C) Quantitative immunoprecipitation of FL-eIF2 and eIF5-FL complexes, respectively. A 1 mg portion of whole-cell extract (WCE) prepared from (A) KAY17 (Control), KAY128 (sc TIF5), and KAY482 (hc TIF5), and (C) KAY24 (Control), KAY35 (sc TIF5), and KAY39 (hc TIF5) was used for anti-FLAG immunoprecipitation, and 80% (top panel) and 20% (bottom panels) of the precipitated fractions (lanes under IP) were analyzed by Northern and Western blotting, respectively, with 2% input amount (lanes under Input) and purified tRNA_i^Met and eIF2 as the standard (not shown), as described in Materials and methods. The molar amounts of precipitated components, calculated in comparison with the band densities of purified components, are listed below each panel. N/D, not determined as the bond density was out of linearity range. (B) TC and MFC levels. Percentage of tRNA_i^Met- or eIF3-bound eIF2 compared to total eIF2, observed in KAY128 (sc TIF5) and KAY482 (hc TIF5), is represented by bars with s.d. in lines.

Figure 2

Analyses of eIF complexes by sucrose gradient-velocity sedimentation. KAY35 (TIF5-FL) (A, C) and KAY39 (hc TIF5-FL) (B, D) were grown in YPD at 30°C, treated with cycloheximide for 5 min, and then harvested for preparation of WCE, as described previously (Asano et al, 2001). Twenty five A₂₆₀ units of the WCE were fractionated on 5–45% sucrose gradients by centrifugation at 39 000 r.p.m. for 2 h. Top panels: A₂₅₄ profile, indicating the positions of ribosomal species. A quarter of the first five fractions was precipitated with ethanol and analyzed by SDS–PAGE and immunoblotting with antibodies indicated to the left (middle to bottom panels in (A) and (B)). The reminder of the fractions was incubated with anti-FLAG affinity resin (Sigma) for 30 min, followed by washing four times. A third of the immunoprecipitated fractions was analyzed similarly by immunoblotting (C and D, top to second to the last panels). The reminder was analyzed by Northern blotting for tRNA_i^Met (C and D, bottom panels). For accurate comparison, proteins in the gradient samples, either ethanol or immunoprecipitated, were analyzed on the same gel and with each antibody simultaneously. In, 1% of the in-put amount, except that 0.5% was used for Northern blotting (bottom gels).

Figure 3

Analyses of different eIF complexes by co-immunoprecipitation. A 200 μg portion of WCE prepared from the following strains, grown in YPD, was subjected to HA-eIF3 or eIF5-FL immunoprecipitation with anti-HA (A, B) or anti-FLAG (D) antibodies, respectively, as described previously (Asano et al, 1999; Singh et al, 2004b). The entire pellet fractions (P) were analyzed by immunoblotting with antibodies indicated to the left of each panel. Percentage of input (I), pellet (P), and supernatant (S) fractions used to analyze the reaction is indicated besides the panel. (A–C) Extra copies of eIF5 are found in MFC formed in hc eIF5 cells. To preserve intact eIF association, we added formaldehyde to the culture before cell disruption, as described previously (Nielsen et al, 2004). Strains used are KAY35 (a), KAY50 (b), and KAY471 (c). In (B), the P fractions from a repeat of the experiment in (A) were analyzed side by side to show the increase in eIF5 binding to HA-eIF3. The graph to the right shows the relative amount of eIF5 found in the P fraction from KAY471 (hc WT) compared to that in KAY50 bearing sc TIF5 allele with s.d. in line. In (C), 1 mg of WCE was subjected to anti-HA precipitation and 40% of the P fraction was analyzed by Northern blotting with the probe specific to tRNA_i^Met, as in Figure 1A. Bottom gel: The EtBr staining pattern of total tRNAs in these fractions. The position of tRNA was determined by running side by side with purified total yeast tRNA (Sigma). (D) Formation of aberrant eIF2/eIF5/eIF2B complex in hc eIF5 cells. Strains used are KAY24 (lanes 1–3), KAY35 (lanes 4–6), KAY39 (lanes 7–9), and KAY40 (lanes 10–12). Data from top to third panels of lanes 1–9 are adapted from Asano et al (1999). NT, not tested.

Figure 4

Effect of eIF5-CTD mutations on the Gcd⁻ phenotype caused by hc eIF5. (A) Yeast growth assay on 3AT media. The gcn2 deletion strains KAY128 (sc WT) and KAY482 (hc WT) and its derivatives (Supplementary Tables S2) overexpressing different mutant forms of eIF5 were grown in the YPD medium and diluted to A₆₀₀=0.15. Then, 5 μl of this and indicated dilutions were spotted onto SD medium containing uracil with (panels 2 and 3) or without (panel 1) 3AT (concentration shown) and incubated for the time indicated. Hc WT cells are 3AT resistant owing to general control derepression independent of Gcn2p (Gcd⁻ phenotype) (panels 2 and 3, row 2). Column 1, TIF5 alleles expressed from sc or hc plasmid. Column 2 indicates the factor(s) whose in vitro interaction with eIF5-CTD was perturbed for each of eIF5 mutants, based on the data from Yamamoto et al (2005). (B) Structure and interactions of eIF5. Gray ovals, the NTD involved in GTPase activation (GAP) and the C-terminal HEAT domain (HEAT) as a core for MFC assembly. Thick lines with ‘N' and ‘C' at the ends, unstructured regions indicating amino and carboxyl termini, respectively. The positively and negatively charged areas II and I, respectively, of eIF5-CTD are indicated by + and − signs. Arrows denote interaction with designated partners at the corresponding surface. (C) Expression levels of eIF5-CTD mutants. Indicated amounts of WCE prepared from strains used in (A), grown in YPD, were subjected to immunoblotting with antibodies specific to eIF5 and eIF2α, as indicated. (D) Summary of TC levels in indicated strains (defined as in (A)). All of the strains tested carry FL-SUI3 allele as the sole source of eIF2β, for anti-FLAG immunoprecipitation of FL-eIF2. Presented are the average (thick bar) and s.d. (line) for relative TC levels compared to sc WT strain (KAY128) from three independent experiments for each strain.

Figure 5

The effect of hc eIF5 on eIF2Bɛ mutations. (A) Transformants of KAY33 (gcn2Δ GCD6) and KAY34 (gcn2Δ gcd6-7A) carrying YEplac195 (rows 1 and 3) and YEpU-TIF5 (Supplementary Tables S2) (rows 2 and 4) were grown overnight in the SC-ura medium and diluted and spotted, as in Figure 5A, onto SD medium with (panels 2 and 3) or without (panel 1) 20 mM 3AT and incubated for times indicated on the top. (B) Expression levels of eIF5 in the strains used in (A) were measured and presented, as in Figure 4C, except that yeast was grown in SC-ura. (C) HIS4-lacZ expression. Yeast strains used in (A) were grown in the SC-ura medium and assayed for β-galactosidase as described previously (Hannig and Hinnebusch, 1988). The enzyme activities from strains at corresponding rows in (A) were presented with s.d. in line. (D) Transformants of GP3751 (WT, wild type) and its designated eIF2Bɛ mutant derivatives (listed to the left) carrying an empty vector (sc eIF5) or hc eIF5 plasmid were grown to A₆₀₀=0.3 and 2 μl of five-fold serial dilutions spotted onto SD medium and incubated at 30°C for 2 days (panel 1). The same cells were spotted onto SD medium with 50 mM 3AT and incubated at 30°C for 4 days (panel 2). (E) Transformants of GP4214 (WT) and GP4203, F98, and H625 altering indicated eIF2B subunits (Supplementary Tables S2) were assayed exactly as in (D), panel 1, except that the spots at the top six rows were incubated at 37°C.

Figure 6

The mini-eIF2Bɛ derepresses the general control without inhibiting eIF2B activity. (A) Transformants of KAY16 carrying p(LEU2 GCD6) (row 1) and p(LEU2 GCN2) (row 2) and those of GP3889 carrying pAV1427 (row 3), pAV1689 (row 4), pEG(KT) (row 5), pAV1694 (row 6), and pAV1695 (row 7) encoding indicated products under the GAL promoter (see Supplementary Tables S1 and S2) were spotted onto synthetic galactose/raffinose (SGal) medium with (2) or without (1) 25 mM 3AT for indicated times. Spots are five-fold dilutions starting at A₆₀₀=0.02. (B) A 10 μg portion of WCE prepared from GP3889 transformants carrying plasmids a–c as defined in the table in (A), which had been grown in SGal medium to A₆₀₀∼1, was subjected to immunoblotting with αGST mouse monoclonal antibodies. (C) TC level (% in square) was determined for KAY128 (FL-SUI3) transformants carrying pEG(KT) (a) and pAV1694 (b). −, negative control using KAY113 (SUI3) transformant carrying a URA3 vector. (D) Transformants of KAY113 (wild type) and KAY282 (tif5-7A) (Singh et al, 2005) carrying pEG(KT) (a), pAV1694 (b), and pAV1695 (c) were spotted onto SGal medium with (2) or without (1) 50 mM 3AT for indicated times. Spots are 10-fold dilutions starting at A₆₀₀=0.15.

Figure 7

Impact of excess eIF5 or eIF2Bɛ on MFC and eIF2/eIF2B complex assembly. Numbers in circles represent eIFs. (A, B) A standard initiation cycle is presented focusing on eIF2–factor interactions. Factors are numbered in black and connected in a currently accepted sequence with black arrows, but with the addition of an eIF2·GDP/eIF5 complex that is shown here as forming upon eIF release from initiating ribosomes. (A) Additional competing complexes, identified in this study, form in the presence of excess eIF5 (gray circles labeled 5) and are shown in gray outlined square boxes with connecting gray arrows to indicate possible connection routes with the main pathway. However, TC+5 complex may transiently form as a natural MFC intermediate (Singh et al, 2004b). Likewise, it is possible that the eIF2B/eIF2/eIF5 complex is a transient intermediate before eIF5 displacement from the eIF2/eIF5 complex (see Discussion). (B) By analogy with hc eIF5, proposed complexes formed by excess eIF2Bɛ (gray diamonds labeled ɛ) are shown in gray outlined boxes connected with gray arrows to the main pathway (black factors and arrows).