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Plant and Cell Physiology Advance Access published online on April 17, 2009
Plant and Cell Physiology, doi:10.1093/pcp/pcp056
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Reconstitution of Arabidopsis thaliana SUMO pathways in E. coli: functional evaluation of SUMO machinery proteins and mapping of SUMOylation sites by mass spectrometry

Sachiko Okada1,2, Mio Nagabuchi1,3, Yusuke Takamura1, Tsuyoshi Nakagawa3, Kaori Shinmyozu4, Jun-ichi Nakayama5 and Katsunori Tanaka1,2

1Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, 669-1337, Japan
2Nanobiotechnology Research Center, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan
3Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, Matsue 690-8504, Japan
4Proteomics Support Unit, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan
5Laboratory for Chromatin Dynamics, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan

Corresponding Author : Prof. Katsunori Tanaka; Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, 669-1337, Japan; Phone/FAX: +81-79-565-7769; E-mail address: katsunori{at}kwansei.ac.jp


  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, only a limited number of proteins is 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 E. coli. This system is rapid and effective for the evaluation of 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.

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.

Keywords: Arabidopsis - Posttranslational modification - Reconstituted SUMOylation system - SUMO

(Received February 1, 2009; Accepted April 13, 2009)
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