Ratiometric fluorescence-imaging assays of plant membrane traffic using polyproteins
Ratiometric fluorescence-imaging assays of plant membrane traffic using polyproteins
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DOI：
10.1111/j.1600-0854.2006.00502.x
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发表时间：
2006-12-01
期刊：
影响因子：
4.5
通讯作者：
Moore, Ian
中科院分区：
文献类型：
--
作者：
Samalova, Marketa;Fricker, Mark;Moore, Ian
荧光蛋白标记物被广泛用于报告植物膜交通，然而，缺乏有效的协议来量化荧光或标记物的表达。在这里，来自口蹄疫病毒的20个残基自切割2A肽被用于构建多聚蛋白，这些多聚蛋白与不同细胞区室中的参考蛋白以固定化学计量表达被贩运的标记物。同时瞄准了多对隔室。连同一个定制的图像分析工具，这些结构允许生物合成膜流量进行测定，显着提高灵敏度，动态范围和统计学意义，使用与常见的植物转染和转基因系统兼容的协议。由于可以在群体或单个细胞中监测标记物和效应物表达，因此可以避免饱和现象，并且可以控制随机或表观遗传影响。令人惊讶的是，比率测定构建体的突变分析显示，2A肽对于携带单个内部信号肽的多聚蛋白的有效切割是必需的，而信号肽是必需的。相比之下，携带两个信号肽/锚的构建体需要2A用于有效分离和稳定性，但2A导致这种融合物的氨基末端部分错误分选到液泡中。一个模型来解释2A在这些和其他研究中的植物的行为。
Fluorescent protein markers are widely used to report plant membrane traffic; however, effective protocols to quantify fluorescence or marker expression are lacking. Here the 20 residue self-cleaving 2A peptide from Foot and Mouth Disease Virus was used to construct polyproteins that expressed a trafficked marker in fixed stoichiometry with a reference protein in a different cellular compartment. Various pairs of compartments were simultaneously targeted. Together with a bespoke image analysis tool, these constructs allowed biosynthetic membrane traffic to be assayed with markedly improved sensitivity, dynamic range and statistical significance using protocols compatible with the common plant transfection and transgenic systems. As marker and effector expression could be monitored in populations or individual cells, saturation phenomena could be avoided and stochastic or epigenetic influences could be controlled. Surprisingly, mutational analysis of the ratiometric assay constructs revealed that the 2A peptide was dispensable for efficient cleavage of polyproteins carrying a single internal signal peptide, whereas the signal peptide was essential. In contrast, a construct bearing two signal peptide/anchors required 2A for efficient separation and stability, but 2A caused the aminoterminal moiety of such fusions to be mis-sorted to the vacuole. A model to account for the behaviour of 2A in these and other studies in plants is proposed.