title: The deoxyhypusine synthase mutant dys1-1 reveals the association of eIF5A and Asc1 with cell wall integrity.
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.
authors.affiliation: Department of Biological Sciences, Univ Estadual Paulista - UNESP, Araraquara-Saõ Paulo, Brazil.
authors.forename: Fabio Carrilho
authors.initials: FC
authors.lastname: Galvão
authors.forename: Danuza
authors.initials: D
authors.lastname: Rossi
authors.forename: Wagner da Silva
authors.initials: Wda S
authors.lastname: Silveira
authors.forename: Sandro Roberto
authors.initials: SR
authors.lastname: Valentini
authors.forename: Cleslei Fernando
authors.initials: CF
authors.lastname: Zanelli
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D002473:Cell Wall; D004843:Epistasis, Genetic; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D020125:Mutation, Missense; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics* / Q000378:metabolism; D010441:Peptide Chain Elongation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011132:Polyribosomes / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D054682:Ribosome Subunits, Small, Eukaryotic / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D000097574:Eukaryotic Translation Initiation Factor 5A
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D019204:GTP-Binding Proteins; D008239:Lysine; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010448:Peptide Initiation Factors; D011493:Protein Kinase C; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A; C111825:ASC1 protein, S cerevisiae; C100028:hypusine; C063507:deoxyhypusine synthase; C490702:PKC1 protein, S cerevisiae