A modified GFP facilitates counting membrane protein subunits by step-wise photobleaching in Arabidopsis
A modified GFP facilitates counting membrane protein subunits by step-wise photobleaching in Arabidopsis
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DOI：
10.1016/j.jplph.2017.03.009
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发表时间：
2017-06-01
影响因子：
4.3
通讯作者：
Lin, Jinxing
中科院分区：
文献类型：
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作者：
Song, Kai;Xue, Yiqun;Lin, Jinxing
膜蛋白通过形成低聚物或分子复合物发挥功能。目前，分步光漂白已被应用于植物细胞中荧光标记亚基的计数，这需要一个准确可靠的控制来区分单个亚基并定义基础荧光。然而，使用固定化GFP分子的常见方法显然不适用于活植物细胞的分析。利用空间强度分布分析（SpIDA），我们发现A206K突变降低了GFP分子的二聚化。由内源性AtCLC2启动子驱动的肉豆蔻酰gfp （A206K）的进一步异位表达允许在低表达水平下对单个分子进行成像。结果表明，转基因pCLC2::Myristoyl-mGFP（A206K）系中二聚体的百分比比pCLC2::Myristoyl-GFP系明显降低，证实了其在确定GFP基础荧光强度方面的应用。综上所述，我们的研究结果表明，pCLC2::Myristoyl-mGFP（A206K）可以作为单体GFP的标准对照，便于对拟南芥膜蛋白的分步光漂白进行分析。
Membrane proteins exert functions by forming oligomers or molecular complexes. Currently, step-wise photobleaching has been applied to count the fluorescently labelled subunits in plant cells, for which an accurate and reliable control is required to distinguish individual subunits and define the basal fluorescence. However, the common procedure using immobilized GFP molecules is obviously not applicable for analysis in living plant cells. Using the spatial intensity distribution analysis (SpIDA), we found that the A206K mutation reduced the dimerization of GFP molecules. Further ectopic expression of Myristoyl-GFP(A206K) driven by the endogenous AtCLC2 promoter allowed imaging of individual molecules at a low expression level. As a result, the percentage of dimers in the transgenic pCLC2::Myristoyl-mGFP(A206K) line was significantly reduced in comparison to that of the pCLC2::Myristoyl-GFP line, confirming its application in defining the basal fluorescence intensity of GFP. Taken together, our results demonstrated that pCLC2::Myristoyl-mGFP(A206K) can be used as a standard control for monomer GFP, facilitating the analysis of the step-wise photobleaching of membrane proteins in Arabidopsis thaliana.