Estudo do Fator de Início de Tradução de Eucariotos 5A (eIF5A) na tradução específica e na ligação direta com ribossomo

O fator de início de tradução 5A (eIF5A) é altamente conservado em arqueas e em eucariotos e sofre uma modificação pós-traducional única e essencial chamada hipusinação. Este fator já foi relacionado com início de tradução, transporte nucleocitoplasmático, decaimento de mRNA e proliferação celular. Resultados recentes colocam essa proteína novamente no cenário da síntese proteica, mais especificamente na etapa de elongação da tradução. Estudos que relacionam eIF5A com proliferação celular e transição G1/S do ciclo celular e via secretória sugerem seu envolvimento com tradução específica de proteínas que atuam na progressão do ciclo celular. No intuito de avaliar a hipótese da participação de eIF5A na tradução de um subgrupo específico de mRNAs, envolvidos na via secretória e proliferação celular, este trabalho estudou inicialmente, o envolvimento de eIF5A na translocação de proteínas para o RE. Os resultados revelam que eIF5A não atua na via pós-traducional, mas sugere que desempenhe um papel na translocação pela via co-traducional. A análise proteômica realizada com um mutante de Dys1, que apresenta redução de eIF5A ativa, revelou diversas proteínas diferencialmente presentes, as quais participam dos processos de biogênese de ribossomo e regulação da tradução, ciclo celular, organização de membrana e via secretória. A proteína Asc1 foi selecionada para análises adicionais por ser uma proteína amplamente envolvida no controle traducional, por diversos mecanismos. Foram realizados ensaios de interação genética entre Asc1, Dys1 e eIF5A, e os resultados obtidos sugerem que essas proteínas participam, conjuntamente, da regulação do processo de tradução por afetar a tradução de grupos de...The translation initiation factor 5A (eIF5A) is highly conserved in archaea and eukaryotes and undergoes an unique and essential posttranslational modification named hypusination. This factor has been associated with translation initiation, nucleocytoplasmic transport, mRNA decay and cell proliferation. Recent results place this protein back in the protein synthesis scenario, acting specifically at the elongation step of translation. Studies that correlate eIF5A with cell proliferation, cell cycle and the secretory pathway suggest its involvement in the translation of specific mRNAs that act on cell cycle progression. In order to evaluate this hypothesis, this study has investigated the involvement of eIF5A in protein translocation into the ER. The results have shown that eIF5A does not act in the post-translational pathway, but suggest that it plays a role in protein translocation via the co-translational pathway. The proteomic analysis performed with a mutant of Dys1, which shows a reduction of active eIF5A, revealed many proteins differentially present, that participate in the process of ribosome biogenesis and regulation of translation, cell cycle, organization of membrane and secretory pathway. The protein Asc1 was chosen for futher analysis due to its involvemet in several mechanisms at translational control. We performed genetic interaction assays with Asc1, Dys1 and eIF5A, and the results suggest that these proteins coordinately... (Complete abstract click electronic access below)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

eIF5A and EF-P: two unique translation factors are now traveling the same road

Translational control is extremely important in all organisms, and some of its aspects are highly conserved among all primary kingdoms, such as those related to the translation elongation step. The previously classified translation initiation factor 5A (eIF5A) and its bacterial homologue elongation factor P (EF-P) were discovered in the late 70's and have recently been the object of many studies. eIF5A and EF-P are the only cellular proteins that undergo hypusination and lysinylation, respectively, both of which are unique posttranslational modifications. Herein, we review all the important discoveries related to the biochemical and functional characterization of these factors, highlighting the implication of eIF5A in translation elongation instead of initiation. The findings that eIF5A and EF-P are important for specific cellular processes and play a role in the relief of ribosome stalling caused by specific amino acid sequences, such as those containing prolines reinforce the hypothesis that these factors are involved in specialized translation. Although there are some divergences between these unique factors, recent studies have clarified that they act similarly during protein synthesis. Further studies may reveal their precise mechanism of ribosome activity modulation as well as the mRNA targets that require eIF5A and EF-P for their proper translation. (C) 2014 John Wiley & Sons, Ltd.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

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

<div><p>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 <i>Saccharomyces cerevisiae</i> 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 <i>dys1-1</i> 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 <i>dys1-1</i> 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 <i>dys1-1</i> 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 <i>dys1-1</i> is not associated with cell lysis. We observed that the <i>dys1-1</i> genetically interacts with the sole yeast protein kinase C (Pkc1) and Asc1, a component of the 40S ribosomal subunit. The <i>dys1-1</i> mutant was synthetically lethal in combination with <i>asc1Δ</i> and overexpression of <i>TIF51A</i> (eIF5A) or <i>DYS1</i> is toxic for an <i>asc1Δ</i> 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 <i>dys1-1</i> and <i>asc1Δ</i> mutants in comparison with the <i>pkc1Δ</i> 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.</p> </div

Genetic interaction between Dys1 and Asc1 is related to Asc1 binding to the 40S ribosome subunit.

<p>The indicated strains were plated onto medium not containing or containing 5-FOA and grown at 25°C for 3 days for plasmid shuffle.</p

The Dys1, Asc1 and Pkc1 mutants showed a distinguished sensitivity to compounds affecting cytoplasmic membrane and cell wall integrity.

<p>(A) The strains were plated onto medium supplemented with the indicated drugs and grown at 25°C for 3 days. (B) The growth was measured relative to each respective isogenic wild type strain (100%).</p

The <i>dys1-1</i> mutant reveals a drastic reduction in Dys1 protein levels resulting in a reduction in hypusine-containing eIF5A.

<p>(A) Determination of mutant Dys1 protein levels. Wild type and <i>dys1-1</i> mutant strains were grown to mid-log phase at the permissive temperature in YPD medium containing 1 M sorbitol. The cells were lysed and 10 µg of total protein were blotted with the indicated antibodies. Samples were probed for eEF2 as a loading control. (B) Detection of hypusine-containing eIF5A of wild type and <i>dys1-1</i> mutant strains. Total eIF5A was immunoprecipitated and subjected to SDS-PAGE. Hypusine-containing eIF5A was revealed by autoradiography. (C) Quantification of relative hypusination levels after analysis of hypusine-containing versus total eIF5A, comparing the <i>dys1-1</i> mutant with the wild type (<i>DYS1</i>) and expressed the quantification as percent of wild type.</p

The reduction in hypusine formation in <i>dys1-1</i> mutant results in a reduction in total protein synthesis, and the polysome profile is characteristic of translation elongation defects.

<p>(A) The indicated strains were grown to mid-log phase, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060140#pone-0060140-g001" target="_blank">Figure 1B</a> and radiolabeled [³H]leucine was added to the medium. The incorporation of [³H]leucine into total proteins was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060140#s4" target="_blank">Materials and Methods</a>. (B) Whole cell extracts (WCE) of the indicated strains were fractionated through centrifugation in a sucrose density gradient. Optical scans (OD_254nm) of the gradients are shown. The areas of the 80S and polysome peaks were compared to calculate the P/M ratio. The polysome profile fractions and the WCE were collected and blotted against the indicated antibodies. (C) Quantification of the ribosome-bound eIF5A relative to the amount of ribosomes (normalized by ribosomal protein L5) in the polysome profile fractions. The values obtained with the wild type strain were considered as 100% and those obtained with mutant strains were expressed as percentages of the wild type in the bar graphs.</p