Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel.
Nucleic Acids Res. 2011 Oct;39(19):8404-15. doi: 10.1093/nar/gkr555. Epub 2011 Jul 14.
In eukaryotes, exposure to stress conditions causes a shift from cap-dependent to cap-independent translation. In trypanosomatids, environmental switches are the driving force of a developmental program of gene expression, but it is yet unclear how their translation machinery copes with their constantly changing environment. Trypanosomatids have a unique cap structure (cap-4) and encode four highly diverged paralogs of the cap-binding protein, eIF4E; none were found to genetically complement a yeast mutant failing to express eIF4E. Here we show that in promastigotes, a typical cap-binding complex is anchored through LeishIF4E-4, which associates with components of the cap-binding pre-initiation complex. In axenic amastigotes, expression of LeishIF4E-4 decreases and the protein does not bind the cap, whereas LeishIF4E-1 maintains its expression level and associates with the cap structure and with translation initiation factors. However, LeishIF4E-1 does not interact with eIF4G-like proteins in both life stages, excluding its involvement in cap-dependent translation. Using pull-down assays and mass-spectrometry, we identified a novel, non-conserved 4E-Interacting Protein (Leish4E-IP), which binds to LeishIF4E-1 in promastigotes, but not in amastigotes. Yeast two-hybrid and NMR spectroscopy confirmed the specificity of this interaction. We propose that Leish4E-IP is a translation regulator that is involved in switching between cap-dependent and alternative translation pathways.
在真核生物中，暴露于应激条件下会导致从依赖帽结构的翻译向非依赖帽结构的翻译转变。在动基体生物中，环境变化是基因表达发育程序的驱动力，但目前尚不清楚它们的翻译机制如何应对不断变化的环境。动基体生物具有独特的帽结构（帽-4），并编码四个高度分化的帽结合蛋白 eIF4E 的同源物；没有发现它们在遗传上能互补表达 eIF4E 的酵母突变体。在这里，我们发现在前鞭毛体中，通过 LeishIF4E-4 锚定了一个典型的帽结合复合物，该复合物与帽结合起始复合物的成分相关联。在无细胞内阿米巴滋养体中，LeishIF4E-4 的表达减少，并且该蛋白不与帽结合，而 LeishIF4E-1 保持其表达水平并与帽结构和翻译起始因子结合。然而，LeishIF4E-1 在前鞭毛体和无细胞内阿米巴滋养体两个阶段都不与 eIF4G 样蛋白相互作用，排除了其参与依赖帽结构的翻译。通过下拉测定和质谱分析，我们鉴定出一种新型的、非保守的 4E 相互作用蛋白（Leish4E-IP），它在前鞭毛体中与 LeishIF4E-1 结合，但在无细胞内阿米巴滋养体中不结合。酵母双杂交和 NMR 光谱学证实了这种相互作用的特异性。我们提出 Leish4E-IP 是一种翻译调节因子，它参与了依赖帽结构的翻译和替代翻译途径之间的转换。
