Reconstitution of Arabidopsis thaliana SUMO pathways in E. coli: functional evaluation of SUMO machinery proteins and mapping of SUMOylation sites by mass spectrometry.
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, only a limited number of proteins have been characterized in plants, 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 Escherichia coli. This system is rapid and effective for the evaluation of the SUMOylation of potential SUMO target proteins. We tested the ability 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 liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by mutational analysis in combination with this system. Using this reconstituted SUMOylation system, comparisons of SUMOylation patterns among SUMO isoforms can be made, and will provide insights into the SUMO isoform specificity of target modification. The identification of SUMOylation sites enables us to investigate the direct effects of SUMOylation using SUMOylation-defective mutants. This system will be a powerful tool for elucidation of the role of SUMOylation and of the biochemical and structural features of SUMOylated proteins in plants.
Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Japan.
Sachiko
S
Okada
Mio
M
Nagabuchi
Yusuke
Y
Takamura
Tsuyoshi
T
Nakagawa
Kaori
K
Shinmyozu
Jun-ichi
J
Nakayama
Katsunori
K
Tanaka
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
D029681:Arabidopsis Proteins; C464659:MYB30 protein, Arabidopsis; D020033:Protein Isoforms; D025841:Small Ubiquitin-Related Modifier Proteins; D014157:Transcription Factors