Abstract
The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1) catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1) and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1Δ and overexpression of TIF51A (eIF5A) or DYS1 is toxic for an asc1Δ strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1Δ mutants in comparison with the pkc1Δ mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of mRNAs associated with cell integrity.
Highlights
Purified from the ribosomes of reticulocyte lysates, the putative eukaryotic translation initiation factor 5A was shown to stimulate the synthesis of methionyl-puromycin in vitro, indicating a role in the formation of the first peptide bond [1]
The Osmotic Stabilizer Requirement for dys1-1 Growth is not Associated with Cell Lysis To generate a conditional DYS1 mutant for the characterization of the role of the hypusine modification in eukaryotic translation initiation factor 5A (eIF5A), we used sitedirected mutagenesis to target conserved residues of deoxyhypusine synthase and selected for mutations that would impair the capacity of the DYS1 gene to complement a dys1D yeast strain
The polysome profile analysis of the dys1-1 mutant demonstrated a significant increase in the polysome to monosome (P/M) ratio (Figure 3B, upper panels; Figure S4). These results are consistent with defects in translation elongation and support the idea that eIF5A plays a role in this step of protein synthesis. These findings reveal that the reduced levels of hypusine formation in the dys1-1 mutant results in translation elongation defects similar to those observed for eIF5A mutants [6,17,19,26]
Summary
Purified from the ribosomes of reticulocyte lysates, the putative eukaryotic translation initiation factor 5A (eIF5A) was shown to stimulate the synthesis of methionyl-puromycin in vitro, indicating a role in the formation of the first peptide bond [1]. The depletion of eIF5A in yeast only showed a small decrease in the overall rate of protein synthesis, arguing against an essential role for eIF5A in general translation [2,3]. There might be a role for eIF5A in translation initiation [15], it has been demonstrated that eIF5A physically interacts with the 80S ribosome and translation elongation factors [16,17] and functionally interacts with elongation factor 2 [18]. The accumulation of polysomes and an increase in the average ribosomal transit time has been observed in yeast eIF5A mutants, supporting a role for eIF5A in translation elongation instead of translation initiation [6,19]
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