Skip Navigation


Plant and Cell Physiology Advance Access originally published online on April 17, 2009
Plant and Cell Physiology 2009 50(6):1049-1061; doi:10.1093/pcp/pcp056
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow All Versions of this Article:
50/6/1049    most recent
pcp056v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Okada, S.
Right arrow Articles by Tanaka, K.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Okada, S.
Right arrow Articles by Tanaka, K.
Agricola
Right arrow Articles by Okada, S.
Right arrow Articles by Tanaka, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: 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, 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: E-mail, katsunori{at}kwansei.ac.jp; Fax, +81-79-565-7769.


  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 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

Abbreviations: EST, expressed sequence tag; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; IPTG, isopropyl β-D-thiogalactopyranoside; LB, Luria–Bertani; LC-MS/MS, liquid chromatography–tandem mass spectrometry; MCS, multicloning site; ORF, open reading frame; SBM, SUMO-binding motif; SIM, SUMO interaction motif; SUMO, small ubiquitin-related modifier

(Received February 1, 2009; Accepted April 13, 2009)
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.