Phosphorylation of initiation factor eIF2 in response to stress conditions is mediated by acidic ribosomal P1/P2 proteins in Saccharomyces cerevisiae

Abstract

Eukaryotic cells contain an unusually large cytoplasmic pool of P1/P2 phosphoproteins, which form the highly flexible 60S subunit stalk that is required to interact with and activate soluble translation factors. In cells, cytoplasmic P1/P2 proteins are exchanged for ribosome-bound proteins in a process that can modulate ribosome function and translation. Here, we analysed different S. cerevisiae stalk mutants grown under stress conditions that result in eIF2α phosphorylation. These mutants either lack a cytoplasmic pool of stalk proteins or contain free but not ribosome-bound proteins. Only cells that contain free P1/P2 proteins induce eIF2 phosphorylation in vivo in response to glucose starvation or osmotic stress. Moreover, we show that free S. cerevisiae P1/P2 proteins can induce in vitro phosphorylation of the initiation factor eIF2 by stimulating the autophosphorylation and activation of GCN2 kinase. Indeed, these ribosomal proteins do not stimulate other eIF2α kinases, such as PKR and HRI. P1/P2 and the known GCN2 activator deacylated tRNA compete for stimulating the eIF2α kinase activity of GCN2, although the P1/P2 proteins are considerably more active. These findings reveal a capacity of free cytoplasmic ribosomal stalk components to stimulate eIF2α phosphorylation, which in turn would modulate translation in response to specific forms of stress that may be linked with the previously described regulatory function of the ribosomal stalk.

Figure 1. eIF2α phosphorylation at stationary phase in wild-type and in a mutant strain lacking P1/P2 proteins.

S. cerevisiae W303-1b (WT) and mutant strain D4567 were grown up to stationary phase (A₆₀₀ = 4.0) and the amount of phosphorylated and total eIF2α was estimated in total cell extracts resolved by SDS-PAGE. Phosphorylated and total eIF2α (eIF2α-P and eIF2α) were analyzed in Western blots probed with specific antibodies. Similar results were obtained from duplicate experiments. The values under Western blot panels represent the intensities of phosphorylated eIF2α in each lane normalized respect to the corresponding total eIF2α; for comparison, the value obtained in the first lane (WT A₆₀₀ = 0.6) was set as 1. Similar results were obtained from duplicate experiments.

Figure 2. Response of S. cerevisiae stalk mutants to different stresses.

Yeast D45, D67 and D4567 and the parental W303-1b (WT) strains were grown in the presence (+) or absence (–) of 0.5 M NaCl (A); in the presence of 2% (+) or 0.5% (–) glucose (C); or in the presence (+) or absence (–) of amino acids (E), as described in the Materials and methods section. After the appropriate period, the cells were collected, the total cell extracts were resolved by SDS-PAGE and the amount of phosphorylated and total eIF2α, ribosomal acidic proteins P0 and P1/P2, was analyzed as described in Fig. 1. (B, D, F) Quantification of the levels of phosphorylated eIF2α in response to stress. Values represent the ratio eIF2α-P/eIF2α in each case, referred to the values obtained in WT unstressed cells, which were set as 1. The results show the means of two independent experiments plus the standard deviations.

Figure 3. Ectopic expression of ribosomal stalk acidic proteins restores basal levels of eIF2α phosphorylation.

(A) Cells of yeast strains W303-1b (WT), D45, D45p (transformed with pFL36), D45p2α (transformed with pFL36 P2α) and D45p2β (transformed with pFL36 P2β) growing in the mid-exponential phase (A₆₀₀ = 0.6) were harvested and the levels of eIF2α-P, eIF2α and ribosomal acidic proteins P1/P2 in the cell extracts were analyzed as described in Fig. 1 (B) Quantification of the basal levels of phosphorylated eIF2α in each strain. Values represent the ratio eIF2α-P/eIF2α in each case, referred to the values obtained in WT cells, which were set as 1. The results show the means of two independent experiments plus the standard deviations.

Figure 4. Ribosomal stalk acidic P1/P2 proteins stimulate the phosphorylation of eIF2α in a yeast cell-free in vitro translation system.

Yeast cell-free translation extracts from W303-1b strain (WT) and D4567 were assayed as described in Materials and methods, in the presence (+) or absence (–) of purified P1/P2 proteins (SP). (A) Equivalent aliquots of all the assays were analysed by Western blot in order to detect phosphorylated (eIF2α-P) and total eIF2α, GCN2, and ribosomal acidic proteins (P0, P1/P2). (B) Quantification of the levels of phosphorylated eIF2α in response to the presence of P1/P2 acidic proteins. Values represent the ratio eIF2α-P/eIF2α in each case, referred to the values obtained in WT extract without added P1/P2 proteins, which were set as 1. The results show the means of two independent experiments plus the standard deviations.

Figure 5. Ribosomal stalk proteins stimulate the phosphorylation of eIF2α by GCN2 kinase in vitro.

(A) Increasing amounts of a P1/P2 ribosomal extract (SP fraction) from either wild-type W303-1b (WT) or mutant D4567 were added to a phosphorylation assay containing purified eIF2α and GCN2 kinase. (B) The indicated amounts of P1α and P2β recombinant proteins, and equimolecular amounts of the P2β NTD and CTD polypeptides, were added to a GCN2-dependent eIF2α phosphorylation assay. In a parallel assay, a mixed equimolecular total amount of both proteins (0.05 µg of each protein, P1α and P2β) were also tested. The same amount of SP extract (0.1 µg) was used as a control. In both cases, following kinase assay, the samples were resolved by SDS-PAGE, and the amount of phosphorylated (eIF2α-P) and total eIF2α, phosphorylated GCN2 (GCN2-P) and total GCN2, and ribosomal acidic proteins (P1/P2) were estimated by Western blot. Similar results were obtained from duplicate experiments.

Figure 6. Effect of RNA on stimulation of eIF2α phosphorylation by P1/P2 proteins.

(A) Stimulation of eIF2α phosphorylation by P1/P2 proteins is blocked by tRNA. The effect of increasing amounts of tRNA (0.02 µg ≈ 1 pmol, 0.2 µg ≈ 10 pmol, 2 µg ≈ 100 pmol, 5.8 µg ≈ 230 pmol) on GCN2-dependent eIF2α phosphorylation was tested in a kinase assay in the presence or absence of the SP fraction (0.1 µg ≈ 10 pmol), as described in Fig. 5. (B) RNase abolishes the inhibitory effect of RNA on P1/P2-mediated eIF2α phosphorylation. Polysomal RNA (pRNA, 11 µg ≈ 11 pmol), P1/P2 proteins (SP fraction, 0.1 µg) and a RNase mix (2 µg/ml RNase A, 0.1 µg/ml RNase T1, 12.5 µg/ml Micrococcal Nuclease S7, 0.125 mM CaCl₂) were added to the in vitro GCN2-dependent eIF2α phosphorylation assay as indicated, and the samples were processed as described in A. The level of GCN2 autophosphorylation was also estimated using specific antibodies. The values under Western blot panels represent the intensities of phosphorylated proteins in each line normalized respect to the corresponding total proteins; for comparison, the value obtained in the first line (negative controls) was set as 1. Shown are the results of a representative experiment of other with similar results.

Figure 7. Effect of P1/P2 proteins and tRNA on the activity of the GCN2 mutants.

(A) Schematic representation of the structural domains of GCN2 and mouse GCN2 mutants tested in this assay, indicating the punctual mutations or the deletions for each mutant. The full length GCN2 sequence is illustrated by a larger box. The figure is drawn to scale. Highlighted domains include the N-terminal (black box); the ‘Pseudokinase’ (grey box) that is related to subdomains I–XI of eukaryotic protein kinases; the conserved two lobes of the eIF2α kinase domain (black), separated by a large insert (white box); the HisRS-like domain (dark grey box) that includes the three motifs (m1, m2 and m3) conserved among the class II aminoacyl-tRNA synthetases; and a C-terminal domain (clear grey box). (B) Phosphorylation of eIF2α by GCN2 and the indicated GCN2 mutants was tested in the presence or absence of either tRNA (2 µg) or the SP fraction (0.1 µg) as previously described. Similar results were obtained from duplicate experiments.

Figure 8. Effect of P1/P2 proteins on eIF2α phosphorylation by the kinases GCN2, PKR and HRI.

Affinity-purified protein kinases were subjected to eIF2α kinase assay in the presence or absence of P1/P2 (SP fraction, 0.1 µg) and SV RNA (0.1 µg ≈ 0.03 pmol). The samples were analyzed after incubation by electrophoresis and Western blot as described in the previous figures. Similar results were obtained from duplicate experiments.