Repurposing an endogenous degradation system for rapid and targeted depletion of C. elegans proteins
Repurposing an endogenous degradation system for rapid and targeted depletion of C. elegans proteins
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DOI：
10.1242/dev.115048
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发表时间：
2014-12-01
期刊：
影响因子：
4.6
通讯作者：
Nance, Jeremy
中科院分区：
文献类型：
--
作者：
Armenti, Stephen T.;Lohmer, Lauren L.;Nance, Jeremy
有条件地灭活基因功能的能力对于理解发育的分子基础至关重要。在基因和 mRNA 靶向方法中，蛋白质产物可能会持续存在，从而使遗传分析变得复杂。目前选择性蛋白质降解的方法需要药物处理或需要数小时才能去除蛋白质，这限制了它们在研究体内快速发育过程中的效用。在这里，我们重新利用内源蛋白质降解系统来快速去除目标秀丽隐杆线虫蛋白质。我们发现，在 E3 泛素连接酶底物识别亚基 ZIF-1 表达后，用 ZF1 锌指结构域标记的蛋白质可以在通过时间和空间控制检查的所有体细胞类型中快速降解。我们证明，通过将编码 ZF1 标签的序列引入内源基因座，可以将基因改造为条件性功能丧失等位基因。最后，我们使用 ZF1 标记来确定细胞侵袭事件期间 cdc-42 基因功能的位点。 ZF1 标记为动态发育事件的分析提供了一个强大的新工具。
The capability to conditionally inactivate gene function is essential for understanding the molecular basis of development. In gene and mRNA targeting approaches, protein products can perdure, complicating genetic analysis. Current methods for selective protein degradation require drug treatment or take hours for protein removal, limiting their utility in studying rapid developmental processes in vivo. Here, we repurpose an endogenous protein degradation system to rapidly remove targeted C. elegans proteins. We show that upon expression of the E3 ubiquitin ligase substrate-recognition subunit ZIF-1, proteins tagged with the ZF1 zinc-finger domain can be quickly degraded in all somatic cell types examined with temporal and spatial control. We demonstrate that genes can be engineered to become conditional loss-of-function alleles by introducing sequences encoding the ZF1 tag into endogenous loci. Finally, we use ZF1 tagging to establish the site of cdc-42 gene function during a cell invasion event. ZF1 tagging provides a powerful new tool for the analysis of dynamic developmental events.