Tandem fluorescent protein timers for in vivo analysis of protein dynamics
Tandem fluorescent protein timers for in vivo analysis of protein dynamics
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DOI：
10.1038/nbt.2281
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发表时间：
2012-07-01
影响因子：
46.9
通讯作者：
Knop, Michael
中科院分区：
文献类型：
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作者：
Khmelinskii, Anton;Keller, Philipp J.;Knop, Michael
细胞的功能状态在很大程度上取决于其蛋白质组的时空组织。目前存在测量蛋白质周转和定位的特定方面的技术，但只有结合几种方法，才能全面分析不同尺度上的蛋白质动力学。在这里，我们描述了串联荧光蛋白定时器(TFT)，两种单色荧光蛋白以不同的动力学方式成熟的融合，我们用它来分析活细胞中的蛋白质周转和流动性。我们将TFT融合到酵母中的蛋白质，以研究细胞成分的寿命、分离和遗传以及蛋白质在亚细胞间的流动性；测量蛋白质降解动力学，而不需要时间进程测量；以及对蛋白质周转调节因子进行高通量筛选。我们的实验揭示了核孔复合体的稳定性质和不对称遗传，并确定了N端规则介导的蛋白质降解的调节因子。
The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule-mediated protein degradation.