Caracterização funcional de eIF5A: análise genética e molecular utilizando o modelo de Saccharomyces cerevisiae
Carregando...
Data
2006-03-08
Autores
Zanelli, Cleslei Fernando [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
O fator de início de tradução de eucariotos 5A (eIF5A) é uma proteína altamente conservada desde arquebactérias a mamíferos e sofre uma modificação póstraducional única, necessária para sua maturação funcional, chamada de hipusinação. Apesar do grau de conservação de eIF5A, e da essencialidade de sua função nos organismos estudados, seu papel no metabolismo celular ainda se encontra indeterminado. Vários mutantes condicionais de eIF5A, sensíveis ao aumento de temperatura, têm sido isolados e caracterizados na levedura Saccharomyces cerevisiae. Utilizando um desses mutantes de eIF5A, o alelo tif51A-1, foi isolado o gene PKC1 como um supressor em alto número de cópias do fenótipo de sensibilidade a temperatura deste mutante. O entendimento de como se dá esta interação genética foi um dos enfoques deste trabalho. Foi mostrado que a via de MAP quinases que atua abaixo de Pkc1 não é responsável pela supressão deste mutante e a identificação dos novos supressores do mutante tif51A-1, GIC1 e ZDS1, levou à sugestão de uma nova via de sinalização a partir de Pkc1. Com a realização de experimentos subsequentes, foi confirmado que a nova via Pkc1-Zds1-Gic1 é responsável pela supressão do mutante tif51A-1 promovida por PKC1. Além disso, estes três supressores são importantes para a polaridade celular em S. cerevisiae, um processo essencial para a progressão no ciclo celular deste organismo, e, interessantemente, os mutantes tif51A-1 e tif51A-3 de eIF5A evidenciaram defeitos na polarização do citoesqueleto de actina na temperatura não permissiva. Esses dados evidenciam uma correlação de eIF5A com progressão no ciclo celular de S. cerevisiae.
The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein from archaebacteria to mammals and undergoes hypusination, an essential unique post-translational modification. Despite the high degree of conservation of eIF5A and its essential function in the studied organisms, its cellular role remains unclear. Several temperature-sensitive eIF5A mutants have been isolated and characterized in the yeast Saccharomyces cerevisiae. Using one of these mutants, the tif51A-1 allele, PKC1 was identified as a high-copy suppressor of the temperature-sensitive phenotype shown by this mutant. The understanding of this genetic interaction was one of the aims of this work. It was shown that the MAP kinase cascade downstream Pkc1 is not responsible for this phenotypic suppression and the identification of the new tif51A-1 suppressors, GIC1 and ZDS1, suggested a new signaling pathway branching from Pkc1. Further analysis confirmed that Pkc1-Zds1-Gic1 constitute a new pathway that is responsible for tif51A-1 mutant suppression promoted by PKC1. Moreover, these three suppressors are important for cell polarity in S. cerevisiae, an essential process for cell cycle progression in yeast, and, interestingly, the eIF5A mutants tif51A-1 and tif51A-3 showed defects in actin cytoskeleton polarization at the restrictive temperature. These data supported a connection between eIF5A and cell cycle progression in S. cerevisiae. As eIF5A was originally implicated in the process of translation, in order to better investigate the specific function of this factor, polysomal profiling analysis was performed and it was demonstrated that eIF5A interacts with monosomes in a tranlation dependent manner and, besides that, eIF5A mutants show altered polysomal distribution suggesting a possible defect in the elongation step of translation.
The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein from archaebacteria to mammals and undergoes hypusination, an essential unique post-translational modification. Despite the high degree of conservation of eIF5A and its essential function in the studied organisms, its cellular role remains unclear. Several temperature-sensitive eIF5A mutants have been isolated and characterized in the yeast Saccharomyces cerevisiae. Using one of these mutants, the tif51A-1 allele, PKC1 was identified as a high-copy suppressor of the temperature-sensitive phenotype shown by this mutant. The understanding of this genetic interaction was one of the aims of this work. It was shown that the MAP kinase cascade downstream Pkc1 is not responsible for this phenotypic suppression and the identification of the new tif51A-1 suppressors, GIC1 and ZDS1, suggested a new signaling pathway branching from Pkc1. Further analysis confirmed that Pkc1-Zds1-Gic1 constitute a new pathway that is responsible for tif51A-1 mutant suppression promoted by PKC1. Moreover, these three suppressors are important for cell polarity in S. cerevisiae, an essential process for cell cycle progression in yeast, and, interestingly, the eIF5A mutants tif51A-1 and tif51A-3 showed defects in actin cytoskeleton polarization at the restrictive temperature. These data supported a connection between eIF5A and cell cycle progression in S. cerevisiae. As eIF5A was originally implicated in the process of translation, in order to better investigate the specific function of this factor, polysomal profiling analysis was performed and it was demonstrated that eIF5A interacts with monosomes in a tranlation dependent manner and, besides that, eIF5A mutants show altered polysomal distribution suggesting a possible defect in the elongation step of translation.
Descrição
Palavras-chave
Biologia molecular, Leveduras (Fungos) - Genética, Clonagem - Biotecnologia, Saccharomyces cerevisiae - Genética, Eukaryotic translation
Como citar
ZANELLI, Cleslei Fernando. Caracterização funcional de eIF5A: análise genética e molecular utilizando o modelo de Saccharomyces cerevisiae. 2006. 167 f. Tese (doutorado) - Universidade Estadual Paulista, Instituto de Química, 2006.