Identification and characterization of NEDD8-modified proteins from plants

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NEDD8 is a ubiquitin-related protein modifier in eukaryotic cells. Neddylation regulates the assembly and activity of cullin-RING type E3 ubiquitin ligases in animals, plants, and fungi. Besides cullins, only a very limited number of NEDD8-modified proteins are known, but no such proteins have as yet been reported from plants. Using a tagged version of Arabidopsis NEDD8 (RUB1), we have purified a number of putative NEDD8-conjugates from the plant model species Arabidopsis thaliana. Here, we propose to confirm the neddylation of these proteins in vivo, to map the neddylation sites, and to examine the relevance of neddylation for protein function. We also propose to establish a more refined purification scheme for NEDD8 conjugates to get a more complete overview of the plant NEDD8ylome and to examine the differential accumulation of NEDD8-modified proteins in mutants deficient in NEDD8 conjugation and deconjugation.