Levins Elizabeth, Tseng Ching-Ying, Patrick Ryan M, Mayberry Laura K, Cole Nicola, Browning Karen S
Department of Molecular Biosciences and Institute for Cell and Molecular Biology, University of Texas at Austin , Austin, TX, USA.
Translation (Austin). 2016 Nov 29;4(2):e1257408. doi: 10.1080/21690731.2016.1257408. eCollection 2016.
The use of fluorescent proteins fused to other proteins has been very useful in revealing the location and function of many proteins. However, it is very important to show that the fusion of these reporter proteins does not impact the function of the protein of interest. Plants have 2 forms of the cap-binding protein that function in initiation of translation, eIF4E and a plant specific form, eIFiso4E. In an attempt to determine the cellular localization of eIFiso4E, fusions to GFP were made, but were found to not be competent to rescue the lethal phenotype of plants lacking eIF4E and eIFiso4E. This suggested that the GFP fusions at either the N- or C-terminus of eIFiso4E were not functional. Biochemical analysis of the fusions revealed that eIFiso4E•GFP fusions were not able to bind to mGTP Sepharose indicating that they were not functional as cap-binding proteins. Analysis of eIF4E•GFP fusions, both in yeast and , showed that the N-terminal fusion may be functional, whereas the C-terminal fusion bound mGTP Sepharose very poorly and functioned poorly in yeast. These results highlight the importance of verification both biochemically and that reporter fusions of proteins maintain activity and are stable in order to prevent observations that may result in artifacts.
将荧光蛋白与其他蛋白融合使用，在揭示许多蛋白的定位和功能方面非常有用。然而，证明这些报告蛋白的融合不会影响目标蛋白的功能非常重要。植物有两种在翻译起始中起作用的帽结合蛋白形式，即eIF4E和一种植物特有的形式eIFiso4E。为了确定eIFiso4E的细胞定位，构建了与绿色荧光蛋白（GFP）的融合体，但发现其无法挽救缺乏eIF4E和eIFiso4E的植物的致死表型。这表明在eIFiso4E的N端或C端的GFP融合体没有功能。对融合体的生化分析表明，eIFiso4E•GFP融合体不能与mGTP琼脂糖结合，这表明它们作为帽结合蛋白没有功能。对酵母中的eIF4E•GFP融合体的分析表明，N端融合体可能有功能，而C端融合体与mGTP琼脂糖的结合非常差，在酵母中的功能也很差。这些结果突出了在生化方面进行验证的重要性，并且表明蛋白的报告融合体要保持活性且稳定，以防止可能导致假象的观察结果。
