Journal Article

Reconstitution of <i>Arabidopsis thaliana</i> SUMO Pathways in <i> E. coli</i>: Functional Evaluation of SUMO Machinery Proteins and Mapping of SUMOylation Sites by Mass Spectrometry

Sachiko Okada, Mio Nagabuchi, Yusuke Takamura, Tsuyoshi Nakagawa, Kaori Shinmyozu, Jun-ichi Nakayama and Katsunori Tanaka

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 50, issue 6, pages 1049-1061
Published in print June 2009 | ISSN: 0032-0781
Published online April 2009 | e-ISSN: 1471-9053 | DOI: https://dx.doi.org/10.1093/pcp/pcp056
Reconstitution of Arabidopsis thaliana SUMO Pathways in  E. coli: Functional Evaluation of SUMO Machinery Proteins and Mapping of SUMOylation Sites by Mass Spectrometry

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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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, SUMOy-lation 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.

Keywords: Arabidopsis; Post-translational modification; Reconstituted SUMOylation system; SUMO

Journal Article.  6751 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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