Interaction of AtHMGB15, an ARID-HMG family protein, with RING-H2 type E3 ubiquitin ligase AtATL79

Recent studies have shown the importance of Ub/proteasome pathway in regulating transcription for proper synchronization of gene expression. Using yeast two-hybrid screening, we have identified an Arabidopsis RING-H2 type of E3 ubiquitin ligase, AtATL79 that interacts with ARID-HMG protein AtHMGB15 mainly through the ARID domain. Sequence analysis of the RING domain of AtATL79 indicates the presence of conserved six Cys and two His residue that coordinate two Zn+2 ions. AtATL79 is a membrane-bound protein that colocalizes with AtHMGB15 in the nucleus. AtATL79 is an E3 ubiquitin ligase that self-ubiquitinates at Lys 76 residue in vitro. Moreover, AtHMGB15 was found to be polyubiquitinated by AtATL79 both in vitro and in vivo at lysine residue 420. Interestingly, polyubiquitination of AtHMGB15 was observed in various tissues of Arabidopsis, and its stability significantly increases in the presence of 26S proteasome inhibitor MG132. Our results suggest that the Ub/26S proteasome system regulates cellular AtHMGB15 protein level in different tissues of Arabidopsis to regulate the spatiotemporal activity of the protein.