UPL1 and 2, two 405 kDa ubiquitin-protein ligases from Arabidopsis thaliana related to the HECT-domain protein family

The ubiquitin/26S proteasome pathway is a major route for degrading abnorma l and important short-lived regulatory proteins in eukaryotes. Covalent att achment of ubiquitin, which triggers entry of target proteins into the path way, is accomplished by an ATP-dependent reaction cascade involving the seq uential action of three enzymes, E1s, E2s and E3s. Although much of the sub strate specificity of the pathway is determined by E3s (or ubiquitin-protei n ligases, UPLs), little is known about these enzymes in plants and how the y choose appropriate targets for ubiquitination. Here, we describe two 405 kDa E3s (UPL1 and 2) from Arabidopsis thaliana related to the HECT-E3 famil y that is essential in yeast and animals. UPL1 and 2 are encoded by 13 kbp genes 26 cM apart on chromosome I, that are over 95% identical within both the introns and exons, suggesting that the two loci arose from a recent gen e duplication. The C-terminal HECT domain of UPL1 is necessary and sufficie nt to conjugate ubiquitin in vitro in a reaction that requires the position ally conserved cysteine within the HECT domain, E1, and an E2 of the UBC8 f amily. Given that HECT E3s help define target specificity of the ubiquitin conjugation, a continued characterization of UPL1 and 2 should be instrumen tal in understanding the functions of ubiquitin-dependent protein turnover in plants and for identifying pathway substrates.