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      The Deoxyhypusine Synthase Mutant dys1-1 Reveals the Association of eIF5A and Asc1 with Cell Wall Integrity

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          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.

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          Author and article information

          Contributors
          Role: Editor
          Journal
          PLoS One
          PLoS ONE
          plos
          plosone
          PLoS ONE
          Public Library of Science (San Francisco, USA )
          1932-6203
          2013
          1 April 2013
          : 8
          : 4
          : e60140
          Affiliations
          [1]Department of Biological Sciences, Univ Estadual Paulista – UNESP, Araraquara-Saõ Paulo, Brazil
          Universidade de Sao Paulo, Brazil
          Author notes

          Competing Interests: The authors have declared that no competing interests exist.

          Conceived and designed the experiments: CFZ SRV. Performed the experiments: FCG DR WdSS. Analyzed the data: FCG DR CFZ. Contributed reagents/materials/analysis tools: FCG DR WdSS. Wrote the paper: FCG DR CFZ SRV.

          Article
          PONE-D-12-34885
          10.1371/journal.pone.0060140
          3613415
          23573236
          9fad1807-bafb-4bd2-a196-754f7f4203b8
          Copyright @ 2013

          This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

          History
          : 7 November 2012
          : 21 February 2013
          Page count
          Pages: 12
          Funding
          This work was supported by grants to SRV and CFZ from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and PADC from Faculdade de Ciências Farmacêuticas, UNESP. The authors also thank FAPESP for fellowships awarded to the authors (FCG and DR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
          Categories
          Research Article
          Biology
          Genetics
          Gene Expression
          Protein Translation
          Gene Function
          Gene Networks
          Molecular Genetics
          Model Organisms
          Yeast and Fungal Models
          Saccharomyces Cerevisiae
          Molecular Cell Biology
          Gene Expression
          Protein Translation
          Cell Growth
          Cellular Stress Responses

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          Uncategorized

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