De Michele Roberto, Ast Cindy, Loqué Dominique, Ho Cheng-Hsun, Andrade Susana LA, Lanquar Viviane, Grossmann Guido, Gehne Sören, Kumke Michael U, Frommer Wolf B
Department of Plant Biology, Carnegie Institution for Science, Stanford, United States.
Institute of Plant Genetics, Italian National Research Council (CNR-IGV), Palermo, Italy.
Elife. 2013 Jul 2;2:e00800. doi: 10.7554/eLife.00800.
Ammonium serves as key nitrogen source and metabolic intermediate, yet excess causes toxicity. Ammonium uptake is mediated by ammonium transporters, whose regulation is poorly understood. While transport can easily be characterized in heterologous systems, measuring transporter activity in vivo remains challenging. Here we developed a simple assay for monitoring activity in vivo by inserting circularly-permutated GFP into conformation-sensitive positions of two plant and one yeast ammonium transceptors ('AmTrac' and 'MepTrac'). Addition of ammonium to yeast cells expressing the sensors triggered concentration-dependent fluorescence intensity (FI) changes that strictly correlated with the activity of the transporter. Fluorescence-based activity sensors present a novel technology for monitoring the interaction of the transporters with their substrates, the activity of transporters and their regulation in vivo, which is particularly valuable in the context of analytes for which no radiotracers exist, as well as for cell-specific and subcellular transport processes that are otherwise difficult to track. DOI:http://dx.doi.org/10.7554/eLife.00800.001.
铵是关键的氮源和代谢中间体，但过量会导致毒性。铵的摄取由铵转运体介导，其调节机制尚不清楚。虽然在异源系统中可以很容易地表征转运，但在体内测量转运体活性仍然具有挑战性。在这里，我们通过将环状排列的绿色荧光蛋白插入两种植物和一种酵母铵转运受体（“AmTrac”和“MepTrac”）的构象敏感位置，开发了一种简单的体内监测活性的测定方法。向表达传感器的酵母细胞中添加铵会触发浓度依赖性荧光强度（FI）变化，这与转运体的活性严格相关。基于荧光的活性传感器是一种监测转运体与其底物相互作用、转运体活性及其体内调节的新技术，这在不存在放射性示踪剂的分析物背景下尤其有价值，以及对于难以追踪的细胞特异性和亚细胞转运过程也是如此。DOI:http://dx.doi.org/10.7554/eLife.00800.001。
