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JXB Advance Access originally published online on August 13, 2004
Journal of Experimental Botany 2004 55(405):2015-2027; doi:10.1093/jxb/erh226
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Journal of Experimental Botany, Vol. 55, No. 405, © Society for Experimental Biology 2004; all rights reserved

RESEARCH PAPER

Identification of ASK and clock-associated proteins as molecular partners of LKP2 (LOV kelch protein 2) in Arabidopsis

Masahiro Yasuhara2 *, Shunya Mitsui2 *, Hiroshi Hirano1, Rieko Takanabe2, Yoko Tokioka1, Norihisa Ihara2, Akihiro Komatsu2, Motoaki Seki3, Kazuo Shinozaki3 and Tomohiro Kiyosue1,{dagger}

1Gene Research Center, Kagawa University, 2393 Ikenobe, Miki-cho Kita-gun, Kagawa 761-0795, Japan
2Department of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho Kita-gun, Kagawa 761-0795, Japan
3Laboratory of Plant Molecular Biology, RIKEN (Institute of Physical and Chemical Research), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan

{dagger} To whom correspondence should be addressed. Fax: +81 87 891 3405. E-mail: tkiyosue{at}ag.kagawa-u.ac.jp

The ADO/FKF/LKP/ZTL family of proteins of Arabidopsis thaliana Heynh. have a LOV domain, an F-box motif, and a kelch repeat region. LKP2 is a member of this family and functions either within or very close to the circadian oscillator in Arabidopsis. Promoter–GUS fusion studies revealed that the LKP2 gene was highly active in rosette leaves. In CaMV 35S:LKP2-GFP plants, GFP-associated fluorescence was detected in nuclei, suggesting that LKP2 is a nuclear protein. Yeast two-hybrid analysis demonstrated that LKP2 interacted with some Arabidopsis Skp1-like proteins (ASK), as do other ADO/FKF/LKP/ZTL family proteins, suggesting that LKP2 can form an SCF (Skp1-Cullin-F-box protein) complex that functions as a ubiquitin E3 ligase. LKP2 interacted not only with itself but also with other members of the family, LKP1 and FKF1. The two-hybrid analysis also demonstrated that LKP2 interacted with TOC1, a clock component, but not with CCA1 or LHY, negative regulators of TOC1 gene expression. The LOV domain of LKP2 was shown to be necessary and sufficient for the interaction with TOC1. An interaction between LKP2 and APRR5, a paralogue of TOC1, was also observed, but LKP2 did not interact with APRR3, APRR7, or APRR9, other paralogues of TOC1.

Key words: Circadian clock, F-box, kelch repeat, LOV domain, yeast two-hybrid analysis


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