Ptushkina M, Vasilescu S, Fierro-Monti I, Rohde M, McCarthy J E
Department of Gene Expression, National Biotechnology Research Centre, Braunschweig, Germany.
Biochim Biophys Acta. 1996 Aug 14;1308(2):142-50. doi: 10.1016/0167-4781(96)00096-6.
The 5' cap structure of eukaryotic mRNAs is believed to play a role in a number of cellular processes, including pre-mRNA splicing, nuclear export and translation. An essential cap-binding protein that is likely to mediate the participation of the cap in at least one of these processes is the eukaryotic translation initiation factor eIF4E. This protein is thought to facilitate the initial ribosomal interaction with the 5' end of the mRNA, involving the binding of eIF4E to the cap in the cytoplasm. Yet the subcellular distribution and mechanism of targeting of eIF4E has been an unresolved issue. We have therefore examined whether eIF4E in the yeast Saccharomyces cerevisiae is directed to the nucleus by virtue of a nuclear localization sequence (NLS) in its amino acid sequence. eIF4E was fused with the "marker proteins' yeast invertase and jellyfish green fluorescent protein. The distribution of these fusions could be followed using immunofluorescence and confocal microscopy of protoplasts and whole cells. These and other fusions were used to show that while yeast eIF4E does not possess an efficiently functioning NLS, it can be transported into the nucleus if provided with a known active NLS. However, an NLS-eIF4E fusion of this type cannot be stably supported by the cell, most likely because of its inhibitory effects when present in large quantities in the nucleus, whereas an NLS fusion with a mutant form of eIF4E that has reduced cap-affinity is tolerated.
真核生物信使核糖核酸（mRNA）的5' 帽结构被认为在许多细胞过程中发挥作用，包括前体mRNA剪接、核输出和翻译。一种可能介导帽结构参与这些过程中至少一个过程的重要帽结合蛋白是真核生物翻译起始因子eIF4E。这种蛋白质被认为有助于核糖体与mRNA的5' 端进行初始相互作用，这涉及eIF4E在细胞质中与帽结构的结合。然而，eIF4E的亚细胞分布和靶向机制一直是一个未解决的问题。因此，我们研究了酿酒酵母中的eIF4E是否因其氨基酸序列中的核定位序列（NLS）而被导向细胞核。eIF4E与“标记蛋白”酵母转化酶和水母绿色荧光蛋白融合。这些融合蛋白的分布可以通过原生质体和全细胞的免疫荧光和共聚焦显微镜来追踪。这些以及其他融合蛋白被用于表明，虽然酵母eIF4E不具有高效运作的NLS，但如果提供已知的活性NLS，它可以被转运到细胞核中。然而，细胞不能稳定支持这种类型的NLS-eIF4E融合蛋白，最有可能的原因是当它大量存在于细胞核中时具有抑制作用，而与帽亲和力降低的eIF4E突变形式的NLS融合蛋白则可被耐受。
