Eukaryotic translation initiation factor 3 (eIF3) and eIF2 can promote mRNA binding to 40S subunits independently of eIF4G in yeast

Abstract

Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was reduced by eIF5 depletion. Hence, stimulation of the GTPase activity of the ternary complex, a prerequisite for 60S subunit joining in vitro, is likely the rate-limiting function of eIF5 in vivo. Depleting eIF2 or eIF3 impaired mRNA binding to free 40S subunits, but depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, it appears that eIF3 and eIF2 are more critically required than eIF4G for stable binding of at least some mRNAs to native preinitiation complexes and that eIF4G has a rate-limiting function at a step downstream of 48S complex assembly in vivo.

FIG. 1.

Degron mutants display impaired growth and rapid degradation of the tagged eIFs under nonpermissive conditions. (A) WT strain YAJ3 and degron mutants YAJ18-3 (sui3-td), YAJ34 (tif32-td prt1-td), YAJ23 (tif5-td), and YAJ41 (tif4632Δ tif4631-td) were streaked on solid synthetic complete (SC) medium with 2% raffinose as carbon source and 0.1 mM copper sulfate at 25°C (SC_Raf + Cu²⁺, 25°C; permissive conditions) and on SC containing 2% raffinose and 2% galactose lacking copper and containing the copper-chelating agent bathocuproinedisulfonic acid (BCS) at 36°C (SC_Raf/Gal + BCS, 36°C; nonpermissive conditions) and incubated for 6 days. (B) Cultures of the same strains described for panel A were grown in liquid SC_Raf + Cu²⁺ at 25°C to an optical density at 600 nm (OD₆₀₀) of ∼1.0, split into halves, and grown under the permissive and nonpermissive conditions defined above, as indicated. (C) Strains described for panel A were cultured under the same conditions as for panel B, and WCEs were prepared from cells fixed with trichloroacetic acid and subjected to Western analysis using antibodies against the indicated proteins. The immune complexes were visualized by fluorescence and chemiluminescence using the ECL Plus kit (Amersham).

FIG. 2.

Depletion of degron-tagged initiation factors leads to polysome runoff. The WT strain (A) or indicated degron mutants (B to E) described for Fig. 1 were grown in SC_Raf plus Cu²⁺at 25°C to an optical density (OD) at 600 nm of ∼1.0. Galactose was added to 2% for 30 min (to induce P_GAL-UBR1-myc), and cells were shifted to prewarmed SC_Raf/Gal plus BCS and cultured for ∼16 h at 36°C. Cycloheximide was added to 50 μg/ml just prior to harvesting. To better visualize the half-mer shoulder on the 80S peak in the tif5-td and tif4631-td mutants, cycloheximide was omitted and cells were cross-linked with 1% HCHO. WCEs were separated on a 4.5-to-45% sucrose gradient by centrifugation at 39,000 rpm for 2.5 h in an SW41Ti rotor (Beckman). Gradients were collected while scanning at 254 nm to visualize the indicated ribosomal species. The tif5-td and tif4631-td mutants exhibited less severe polysome runoff when examined with cycloheximide (data not shown).

FIG. 3.

Simultaneous depletion of TIF32/eIF3a and PRT1/eIF3b in the tif32-td prt1-td mutant reduces levels of 43S and 48S complexes. Strains YAJ3 (TIF32 PRT1) and YAJ34 (tif32-td prt1-td) (A and B) and the corresponding rpl11bΔ derivatives YAJ43 and YAJ47 (D), respectively, were cultured under nonpermissive conditions as described in the legend for Fig. 2, except that cells were cross-linked with 1% HCHO (or 2% HCHO when analyzing eIF1) before lysis. (C) Strains BY4741 (RPL11B) and 4715 (rpl11bΔ) were grown in yeast extract-peptone-dextrose at 30°C to an optical density at 600 nm of 1.0 to 2.0. WCEs were separated on a 7.5-to-30% sucrose gradient by centrifugation at 41,000 rpm for 5 h in an SW41Ti rotor. (A) A 2% portion of each gradient fraction (numbered beneath the blots) and an aliquot of the starting WCE (“In” for input) were subjected to Western analysis using antibodies against the proteins listed between the blots. (B and C) Total RNA was extracted from 70% of each fraction and from an aliquot of the WCE and subjected to Northern analysis using probes for RPL41A mRNA or tRNA_i^Met, as indicated. (D) The 40S fractions from an experiment of the type described for panels A and B were pooled and resedimented through a second sucrose gradient, and the resulting fractions were analyzed by Northern blotting as described above. (E) Amounts of each initiation factor and tRNA_i^Met in the 40S fractions were quantified by phosphorimaging or fluorescence imaging analyses from three to eight replicate experiments (depending on the factor) for the tif32-td prt1-td (A and B) or tif32-td prt1-td rpl11bΔ (data not shown) mutant. The results were normalized to the corresponding WT values measured in parallel, combined, and averaged. The data for all five eIF3 subunits were combined and averaged (eIF3), as were the data for eIF2α, eIF2γ, and tRNA_i^Met (TC). The means and standard errors are plotted in the histogram. (F) The amounts of RPL41A mRNA in the gradient fractions shown in panel D were quantified and plotted. The average mRNA level in 40S fractions of the degron mutant (as a percentage of WT) was calculated from replicate experiments and is indicated below the 40S peak.

FIG. 4.

Distributions of initiation factors and tRNA_i^Met in sucrose gradients following velocity sedimentation of WCEs from degron mutants and the parental WT strain cultured under nonpermissive conditions. The amounts of each factor in the gradient fractions were calculated in arbitrary units from phosphorimaging or fluorescence imaging analysis of the appropriate Northern or Western blots, respectively, for eIF2α (A), tRNA_i^Met (B), eIF3b (C), eIF1 (D), eIF5 (E), and eIF1A (F) for the indicated degron mutants analyzed in Fig. 3, 6, 7, and 9. The results for tRNA_i^Met were expressed as a percentage of the total signal across the gradient.

FIG. 5.

Western analysis of initiation factors in resedimented 40S fractions from the degron mutants. (A to C) Strains YAJ43 (WT), YAJ47 (tif32-td prt1-td), and YAJ46 (sui3-td) were cultured under nonpermissive conditions and analyzed as described in the legend for Fig. 3D, except that Western analysis was performed on the resedimented 40S fractions using antibodies against the proteins listed on the left of the blots. (D and E) Data from replicate experiments of the type shown in panels A to C were quantified, and the results for the tif32-td prt1-td (D) and sui3-td (E) mutants were normalized to the values measured in parallel for the WT strain and plotted as described for Fig. 3E. (F to H) Strains YAJ43 (WT) and YAJ45 (tif5-td) were analyzed identically, and the results shown in panels G and H together with data from replicate experiments were quantified and are summarized in panel F. All of the strains used for these resedimentation experiments lack the RPL11B gene in order to increase the concentration of 48S PICs (see text for further explanation).

FIG. 6.

Depletion of eIF2β in the sui3-td mutant reduces the levels of 43S and 48S complexes in vivo. (A and B) Strains YAJ3 (SUI3) and YAJ18-3 (sui3-td) were cultured under nonpermissive conditions and analyzed as described for Fig. 3A and B. (C) The 40S fractions were isolated from WCEs of the rpl11bΔ SUI3 and rpl11bΔ sui3-td strains YAJ43 and YAJ46, respectively, resedimented, and analyzed as described for Fig. 3D. (D) The results from three to seven replicate experiments for sui3-td (A and B) or sui3-td rpl11bΔ (data not shown) were quantified and normalized to the WT values determined in parallel and are plotted as described for Fig. 3E. (E) Quantification of the amounts of RPL41A mRNA in the gradient fractions shown in panel C.

FIG. 7.

Depletion of eIF5 in the tif5-td mutant reduces the level of 43S complexes but leads to accumulation of 48S complexes in vivo. (A and B) Strains YAJ3 (TIF5) and YAJ23 (tif5-td) were cultured under nonpermissive conditions and analyzed as described for Fig. 3A and B. (C) The 40S fractions were isolated from WCEs of the rpl11bΔ TIF5 and rpl11bΔ tif5-td strains YAJ43 and YAJ45, respectively, resedimented, and analyzed as described for Fig. 3D. (D) Data from three to six replicate experiments for tif5-td (A and B) or tif5-td rpl11bΔ (data not shown) were quantified and normalized to the WT values determined in parallel and are plotted as described for Fig. 3E. (E) Quantification of the amounts of RPL41A mRNA in the gradient fractions shown in panel C.

FIG. 8.

Depletion of TIF32/eIF3a or eIF2β simultaneously with eIF5 impairs mRNA binding to 40S subunits in vivo. (A and C) WT strain YAJ3 and degron mutant YAJ23 (tif5-td) together with either YAJ31 (tif5-td tif32-td) (A) or YAJ29 (tif5-td sui3-td) (C) were cultured under nonpermissive conditions and analyzed as described for Fig. 3B. (B and D) The amounts of RPL41A mRNA in gradient fractions shown in panels A and C were quantified and plotted. (E and F) WCEs from the experiments depicted in panels A and C were separated by velocity sedimentation and analyzed as described for Fig. 2C and E. Only a portion of each optical density at 254 nm (OD₂₅₄) tracing is shown, to facilitate evaluation of the half-mer shoulder and free 40S fractions. In both panels E and F, two independent experiments are shown for each pair of mutant strains compared.

FIG. 9.

Depletion of eIF4G1 in the tif4631-td tif4632Δ mutant lacking eIF4G2 has little effect on levels of 43S or 48S complexes in vivo. (A to C) Strains YAJ3 (TIF4631 TIF4632) and YAJ41 (tif4631-td tif4632Δ) (A and B) and the corresponding rpl11bΔ derivatives YAJ43 and YAJ48 (C), respectively, were cultured under nonpermissive conditions and analyzed as described for Fig. 3A and B. (D) The experiment shown in panel C was repeated, and 40S fractions were resedimented and analyzed as described for Fig. 3D. (E) Data from three to seven replicate experiments for tif4631-td tif4632Δ (A and B) or tif4631-td tif4632Δ rpl11bΔ (data not shown) were quantified and normalized to the WT values determined in parallel and are plotted as described for Fig. 3E. (F) Quantification of the amounts of RPL41A mRNA in the gradient fractions shown in panel D.