Barrass J David, Mendoza-Ochoa Gonzalo I, Maudlin Isabella E, Sani Emanuela, Beggs Jean D
Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh;
Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh; Department of Plant Sciences, University of Cambridge.
J Vis Exp. 2019 Jul 20(149). doi: 10.3791/59874.
The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A β-estradiol-inducible AID system was created, with expression of TIR1 under the control of a β-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with β-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate β-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.
植物生长素结合受体TIR1在生长素存在的情况下识别含有特定生长素诱导降解基序（AID）的蛋白质，并将其靶向降解。该系统已在许多非植物真核生物中得到应用，使得带有AID基序标签的靶蛋白在添加生长素后被降解。TIR1的表达水平至关重要；过度表达会导致即使在没有生长素的情况下，AID标签蛋白也会被降解，而低表达则会导致耗尽缓慢。构建了一种β-雌二醇诱导的AID系统，TIR1的表达受β-雌二醇诱导型启动子的控制。通过在添加生长素之前改变与β-雌二醇孵育的时间，可以调节TIR1的水平。本方案描述了如何使用AID系统快速耗尽靶蛋白。合适的β-雌二醇孵育时间取决于靶蛋白的丰度。因此，有效的耗尽取决于最佳时机，同时也能使非生长素依赖性耗尽最小化。
