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Plant and Cell Physiology Advance Access originally published online on July 18, 2006
Plant and Cell Physiology 2006 47(8):1095-1101; doi:10.1093/pcp/pcj080
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© The Author 2006. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Fluorescence Cross-Correlation Analyses of the Molecular Interaction between an Aux/IAA Protein, MSG2/IAA19, and Protein–Protein Interaction Domains of Auxin Response Factors of Arabidopsis Expressed in HeLa Cells

Hideki Muto1, Issei Nagao2, Taku Demura3, Hiroo Fukuda3,4, Masataka Kinjo2 and Kotaro T. Yamamoto1,*

1 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060-0810 Japan
2 Laboratory of Supramolecular Biophysics, Research Institute for Electronic Science, Hokkaido University, Sapporo, 060-0812 Japan
3 Plant Science Center, RIKEN, 1-7-22 Suehiro, Tsurumi-ku, Yokohama, 230-0043 Japan
4 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Tokyo, 113-0033 Japan

* Corresponding author: E-mail, kty{at}sci.hokudai.ac.jp; Fax, +81-11-706-2739.

Since auxin may elicit numerous developmental responses by the use of a combination of auxin response factors (ARFs) and their Aux/IAA repressors, it is important to determine the interaction between the two protein families in a quantitative manner. We transiently expressed the C-terminal protein–protein interaction domains (CTDs) of Arabidopsis ARFs, MP/ARF5 and NPH4/ARF7, and MSG2/IAA19, fused to fluorescent proteins in HeLa cells, and determined their molecular interactions with fluorescence cross-correlation spectroscopy (FCCS). Almost complete association was found between MSG2 and MP-CTD and between MSG2 and NPH4-CTD. Approximately 20% association was found for MSG2 homodimers, NPH4-CTD homodimers and MP-CTD/NPH4-CTD heterodimers. Homotypic binding of MP-CTD may be weaker than that of MSG2. MSG2 was localized in cytoplasmic compartments in HeLa cells, whereas it was localized in the nuclei in plant cells. The fact that the heterotypic interaction between MSG2 and ARF-CTDs is stronger than each of the homotypic interactions appears to be the molecular basis for tight control of the transcriptional activity of ARFs by auxin. These results also show that FCCS is useful to examine protein–protein interactions especially for transcriptional regulators.

(Received April 1, 2006; Accepted June 20, 2006)
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