Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells
Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells
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DOI：
10.1073/pnas.1216880109
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发表时间：
2012-12-04
影响因子：
11.1
通讯作者：
Cleveland, Don W.
中科院分区：
文献类型：
--
作者：
Holland, Andrew J.;Fachinetti, Daniele;Cleveland, Don W.
诱导降解是鉴定特定蛋白质或蛋白质复合物功能的有效方法。最近，植物生长素诱导的降解决定子（AID）系统已被证明可以降解非植物细胞中的AID标记的靶蛋白。在这里，我们证明了一个艾滋病标记的蛋白质可以在功能上取代内源性蛋白质耗尽的RNAi，导致诱导空表型生长素添加后迅速。AID系统被证明能够控制在细胞核或细胞质区室中的AIDS标记蛋白的稳定性，甚至当掺入蛋白质复合物中时。诱导降解发生后，迅速加入生长素与蛋白质半衰期减少到9分钟，并进行一级动力学完成。艾滋病介导的不稳定性被证明是迅速可逆的。诱导降解显示在所有细胞周期阶段（包括有丝分裂）中启动并持续，使得该系统特别适用于鉴定哺乳动物细胞周期中特定点期间感兴趣的蛋白质的功能。
Inducible degradation is a powerful approach for identifying the function of a specific protein or protein complex. Recently, a plant auxin-inducible degron (AID) system has been shown to degrade AID-tagged target proteins in nonplant cells. Here, we demonstrate that an AID-tagged protein can functionally replace an endogenous protein depleted by RNAi, leading to an inducible null phenotype rapidly after auxin addition. The AID system is shown to be capable of controlling the stability of AID-tagged proteins that are in either nuclear or cytoplasmic compartments and even when incorporated into protein complexes. Induced degradation occurs rapidly after addition of auxin with protein half-life reduced to as little as 9 min and proceeding to completion with first-order kinetics. AID-mediated instability is demonstrated to be rapidly reversible. Induced degradation is shown to initiate and continue in all cell cycle phases, including mitosis, making this system especially useful for identifying the function(s) of proteins of interest during specific points in the mammalian cell cycle.