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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Chloroplast Outer Envelope Protein CHUP1 Is Essential for Chloroplast Anchorage to the Plasma Membrane and Chloroplast Movement^1,[W],[OA]

National Institute for Basic Biology, Okazaki, Aichi 444–8585, Japan (K.O., A.Y., S.-G.K., M.K., F.T., Y.O., T.K., M.W.); Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Nojihigashi, Kusatsu, Shiga 525–8577, Japan (M.K.); and Institute for Protein Research, Osaka University, Suita, Osaka 565–0871, Japan (M.N.)

Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.

² Present address: Graduate School of Life Sciences, Tohoku University, Sendai 980–8577, Japan.

³ Present address: Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812–8581, Japan.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Masamitsu Wada (wada{at}nibb.ac.jp).

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.123075

^* Corresponding author; e-mail wada{at}nibb.ac.jp.

Received May 19, 2008; accepted August 4, 2008; published August 20, 2008.

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