Identification and characterization of protein degradation substrates and their receptors

含有F-box结构域的蛋白(FBPs)是E3泛素连接酶的底物受体，在蛋白质底物降解前参与蛋白质的泛素化。对拟南芥基因组的分析表明，超过500种蛋白质，即2%的拟南芥蛋白质组，含有F-box结构域。然而，目前还不可能根据序列相似性来预测FBPs目标降解的蛋白质和蛋白质家族的身份。在这里，有人建议在含有荧光素酶基因陷阱T-DNA结构的转基因拟南芥植物中，在内源基因和荧光素酶报告基因之间产生随机的翻译融合。在这些品系中，荧光素酶活性将被用作蛋白质丰度的间接指示器，以识别在特定刺激下容易蛋白质降解的荧光素酶融合蛋白。在一种互补的方法中，作为一个小FBP家族的目标的降解底物将使用酵母双杂交系统进行鉴定，然后在植物中对这些蛋白质进行表征。这些结合的方法有望识别受蛋白质降解调控的新蛋白质家族，以及赋予蛋白质不稳定性的保守蛋白质结构域。

F-box domain containing proteins (FBPs) are the substrate-receptors of E3 ubiquitin ligases and mediate the ubiquitylation of protein substrates prior to their degradation. Analysis of the Arabidopsis genome has revealed that over 500 proteins, thus 2% of the Arabidopsis proteome, contain an F-box domain. However, it is currently not possible to predict the identity of proteins and protein families that are targeted for degradation by FBPs based on sequence similarities. Here, it is proposed to generate random translational fusions between endogenous genes and the luciferase reporter in transgenic Arabidopsis plants that harbor a luciferase bearing gene trap T-DNA construct. In these lines, luciferase activity will be used as an indirect indicator of protein abundance to identify luciferase fusion proteins that are subject to protein degradation upon exposure to specific stimuli. In a complementary approach, degradation substrates that are the targets of a small FBP family will be identified using the yeast two-hybrid system these proteins will then be characterized in planta. The combined approaches are expected to result in the identification of novel protein families that are regulated by protein degradation and of conserved protein domains that confer protein instability.