Abstract
Recent studies have revealed various functions for the small ubiquitin-related modifier (SUMO) in diverse biological phenomena, such as regulation of cell division, DNA repair, and transcription, in yeast and animals. In contrast, the characterization of SUMO target proteins in plants is limited, although plant SUMO proteins are involved in many physiological processes, such as stress responses, regulation of flowering time, and defense reactions to pathogen attack.
Here, we reconstituted the Arabidopsis thaliana SUMOylation cascade in E. coli. This system is a rapid and effective method to evaluate the SUMOylation of potential SUMO target proteins. We tested the capability of this system to conjugate the Arabidopsis SUMO isoforms, AtSUMO1, 2, 3, and 5, to a model substrate, AtMYB30, which is an Arabidopsis transcription factor. All four SUMO isoforms tested were able to SUMOylate AtMYB30. Furthermore, SUMOylation sites of AtMYB30 were characterized by LC-MS/MS followed by mutational analysis in combination with this system. The identification of SUMOylation sites enables us to investigate the direct effects of SUMOylation using SUMOylation-defective mutants.
