Identification and characterization of protein degradation substrates and their receptors

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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.