Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement.

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.

The intracellular distribution of organelles is essential for optimizing metabolic activities in plant cells; hence, the mechanisms by which organelles move to their proper positions have long been investigated (Wada and Suetsugu, 2004). Chloroplast movement for efficient light absorption is the most precisely studied of these phenomena, because of the importance of photosynthesis (Zurzycki, 1955;Takemiya et al., 2005). Chloroplasts change their position dynamically ac-cording to the ambient light intensity. Under weak light conditions, chloroplasts gather at the plasma membrane along the periclinal cell wall in palisade cells (the accumulation response) in order to receive optimal sunlight exposure for efficient photosynthesis. In contrast, under strong light conditions, chloroplasts are positioned at the plasma membrane along the anticlinal cell walls (the avoidance response) to avoid photodamage to the photosynthetic machinery (Kagawa and Wada, 2000;Kasahara et al., 2002;Wada et al., 2003). Hence, chloroplast movement is essential for plants to get energy safely and efficiently under various light conditions. Chloroplast positioning in the dark is also known, but the patterns vary with plant species and tissues (Suetsugu et al., 2005).
The cytoskeleton has also been implicated in chloroplast relocation movement (for review, see Takagi, 2003;Wada et al., 2003). Pharmacological approaches have shown that actin filaments are the main mediators of chloroplast relocation movement (Kadota and Wada 1992a;Tlalka and Gabrys, 1993;Sato et al., 2001). Several different actin filament structures were observed around chloroplasts in some plants tested by indirect fluorescence methods (Kadota and Wada, 1992b;Dong et al., 1996Dong et al., , 1998Kandasamy and Meagher, 1999;Sakurai et al., 2005;Kumatani et al., 2006). Although these findings suggest that the specific actin filament structures around chloroplasts are important for chloroplast relocation movement, it is not well understood when and how the actin structures function. Cytoskeletal myosin has also been suggested to contribute to chloroplast relocation movement (Haupt and Scheuerlein, 1990). When Arabidopsis palisade mesophyll cells are treated with inhibitors of myosin, such as 2,3-butanedione monoxime (BDM), N-ethylmaleimide, and 1-(5-iodonapthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride, chloroplasts are defective in the accumulation response but not in the avoidance response (Paves and Truve, 2007). This suggests that an actomyosin system is at least involved in the chloroplast accumulation response.
Receptors on the surface of each organelle are essential regulators of the organelle response to stimuli and are linked to the cellular components that mediate organelle transport (Bretscher, 2003). In budding yeast, for example, a complex made of mitochondrial membrane proteins such as Mmm1p, Mdm10p, and Mdm12p is located on the mitochondrial outer membrane and links the mitochondria to the actin cytoskeleton during fission (Boldogh et al., 2003). Mitochondria Figure 1. Immunoblot analysis of CHUP1. A, Diagram of the CHUP1 protein representing the regions used as antigens for the production of polyclonal antibodies. Anti-CHUP1 antibodies targeting the N terminus (aH) and C terminus (aT) were produced against the amino acid sequences between positions 200 to 320 and positions 700 to 1,004, respectively. Both regions are shown as black lines. FABR, F-actinbinding region; PRR, Pro-rich region; CtR, C-terminal region. B and C, Total protein (20 mg each) extracted from leaves (B) and isolated chloroplasts (C) of wild-type (WT) and chup1 plants were analyzed by immunoblotting with the two anti-CHUP1 antibodies. The asterisks indicate the major CHUP1 bands (approximately 150 kD). D, The protease sensitivity of CHUP1 shows that its localization is similar to that of Toc159, which was used as a control chloroplast outer envelope protein. Toc75 is also an outer envelope protein deeply embedded in the membrane. Tic110 is an inner envelope protein. cpHsp70 is abundant in the stroma. use the Arp2/3 complex, which polymerizes actin monomers at the rear side of mitochondria, as a force generator to move in a manner similar to the intracellular movement of bacteria such as Listeria monocytogenes and Shigella flexneri in animal cells (Gouin et al., 2005). Rab27 on the melanosome surface regulates a motor protein for melanosome movement (Wu et al., 2002). These examples suggest that the key proteins for chloroplast relocation movement may also exist on the chloroplast surface. Previously, we isolated the chup1 mutant (Oikawa et al., 2003), which shows aggregation of chloroplasts at the bottom of cells and lacks chloroplast relocation responses to any light conditions. The CHUP1 gene encodes a protein with several putative functional regions that are related to actin polymerization and might be involved in chloroplast relocation movement. CHUP1 is thought to be the only protein among the recently found proteins related to chloroplast movement (such as JAC1, PMI1, PMI2, and PMI15) that localizes on the chloroplast envelope (Oikawa et al., 2003;Schmidt von Braun and Schleiff, 2008). However, the actual localization of full-length CHUP1 remains unclear. Furthermore, it is also not clear whether these predicted functional regions of CHUP1 actually function physiologically to regulate chloroplast relocation downstream of the photoreceptor signal cascade.
In this study, we focused on CHUP1 function from the viewpoint of its localization. We found that fulllength CHUP1 localizes on the outer envelope of chloroplasts and that this localization is essential for CHUP1 function. Furthermore, we found that the CHUP1 protein consists of three functional regions: a chloroplast translocation signal at the N terminus, a region that anchors the chloroplast to the plasma membrane and has a coiled-coil character, and a cytoskeleton-associated region. Here, we report that CHUP1 is targeted to chloroplasts and has the novel physiological function of regulating chloroplast localization by anchoring chloroplasts to the plasma membrane and forming a bridge to the actin cytoskeleton. GFP-CHUP1 and CHUP1-GFP genes under the control of the CaMV 35S promoter were introduced into chup1 mutant plants for expression. For each construct, a representative line is shown among multiple lines examined. B, Immunoblot analysis of CHUP1 proteins in wild-type (WT), chup1, GFP-CHUP1, and CHUP1-GFP lines with aH (anti-CHUP1 antibody against CHUP1  ). The top gel shows total leaf results, and the bottom gel shows results from isolated chloroplasts. C, Distribution patterns of chloroplasts in wild-type, chup1, GFP-CHUP1, and CHUP1-GFP lines after adaptation to different light conditions. Detached leaves of these plants were put on an agar surface in a petri dish and incubated in the dark (D), under weak white light (W), or under strong white light (S) for 3 h before observation by light microscopy. Bar = 10 mm. D, Fluorescence images of GFP-CHUP1 and CHUP1-GFP in transgenic plants. Subcellular localization of GFP fluorescence from GFP-CHUP1 and CHUP1-GFP in palisade mesophyll cells was observed with a confocal laser scanning microscope. Green indicates fluorescence from GFP, and red indicates fluorescence from chlorophyll in chloroplasts. Bar = 10 mm.

Detection of CHUP1 in an Isolated Chloroplast Fraction
To determine the subcellular localization of the fulllength CHUP1 biochemically, we performed immunoblot analyses of whole leaves and isolated chloroplasts using two different polyclonal antibodies, one against the N-terminal (head) 200 to 320 amino acids (aH) and the other against the C-terminal (tail) 700 to 1,004 amino acids (aT) of CHUP1 (Fig. 1A). A specific CHUP1 band of approximately 150 kD was detected with both anti-CHUP1 antibodies in whole leaf extracts of wild-type plants but not in extracts of chup1 plants (Fig. 1B). The CHUP1 signal was also detected in the purified chloroplast fraction from wild-type plants (Fig. 1C). Interestingly, CHUP1 protein was not detected after treatment of isolated chloroplasts with the protease thermolysin (Fig. 1D). The transport protein Toc159, which is also sensitive to thermolysin, is localized on the chloroplast outer envelope and known to project into the cytoplasm (Soll and Schleiff, 2004), suggesting that CHUP1 may also be located on the chloroplast surface.

Transgenic Plants Expressing Fusion Proteins of CHUP1 with GFP
To confirm the subcellular localization of CHUP1, we generated transgenic plants stably expressing GFP fused to the N terminus (GFP-CHUP1) or C terminus (CHUP1-GFP) of CHUP1 under the control of the cauliflower mosaic virus (CaMV) 35S promoter on the chup1 background ( Fig. 2A). An immunoblot analysis using whole leaf extracts of transgenic plants confirmed that CHUP1-GFP and GFP-CHUP1 accumulated to the levels of endogenous CHUP1 in the wild type, although the GFP-CHUP1 and CHUP1-GFP were detected as different sizes (Fig. 2B, top). GFP-CHUP1 was not detected in an isolated chloroplast fraction (Fig. 2B, bottom). Analyses of intracellular chloroplast distribution under various light conditions revealed that the transgenic lines expressing CHUP1-GFP showed normal chloroplast relocation, but GFP-CHUP1 could not complement the chup1 phenotype (Fig. 2C). Although the GFP fluorescence of CHUP1-GFP and GFP-CHUP1 was faint, CHUP1-GFP was observed at the periphery of chloroplasts and GFP-CHUP1 was not (Fig. 2D). The localization observed is consistent with the results of the immunoblot analyses (Figs. 1 and 2, B and D).

Correlation between the N-Terminal Hydrophobic Region of CHUP1 and Localization of CHUP1 at the Periphery of Chloroplasts
To investigate the chloroplast targeting region of CHUP1, we used particle bombardment to transiently express various fragments of CHUP1 fused to GFP in leaf cells of wild-type plants. Two types of fluorescence patterns were observed, one surrounding chloroplasts and the other cytosolic (Fig. 3A). CHUP1 fragments containing the N-terminal hydrophobic region (NtHR) Figure 3. Transient expression analysis of GFP fusion proteins with truncated CHUP1 and the N-terminal region of CHUP1. A, Each fragment obtained from various parts of CHUP1 was fused to the N terminus of GFP (CHUP1 n -GFP, where n = 1-25, 1-100, 1-300, or 1-500) or to the C terminus of GFP (GFP-CHUP1 n , where n = 1-25, 500-1,004, 750-1,004, or 950-1,004) and expressed transiently under the control of the CaMV 35S promoter. B, Various parts of NtHR were fused to the N terminus of GFP (NtHR n -GFP, where n = 1-20, 1-15, 1-10, 1-5, 5-25, 10-25, 15-25, or 20-25) and expressed transiently under the control of the CaMV 35S promoter. GFP fluorescence of each construct was observed and analyzed using five cells in each experiment. Green indicates fluorescence from GFP, and red indicates fluorescence from chlorophyll in chloroplasts. Numbers at top and bottom of the photographs are the amino acid positions within CHUP1 n . Bars = 20 mm.
were fused to the N terminus of GFP to form CHUP1 1-25 -GFP, CHUP1 1-100 -GFP, CHUP1 1-300 -GFP, and CHUP1 1-500 -GFP. All were detected at the periphery of the chloroplasts, consistent with the distribution of CHUP1-GFP in Figure 2D. CHUP1 fragments were then fused to the C terminus of GFP to form GFP-CHUP1 1-25 , GFP-CHUP1 500-1004 , GFP-CHUP1 750-1004 , and GFP-CHUP1 950-1004 ; all were detected in cytosol (Fig. 3A). This result was consistent with the distribution of GFP-CHUP1 in stably expressed transgenic lines shown in Figure 2D, but neither GFP-CHUP1 nor CHUP1-GFP fluorescence was observed in the transiently expressing plants (data not shown). To clarify which parts or amino acids among 25 amino acids in NtHR were necessary for the localization, we constructed various deletion constructs deleted from either the N-terminal or C-terminal side of NtHR fused with GFP and transiently expressed them in leaf palisade mesophyll cells. Almost all 25 amino acids tested were necessary for targeting NtHR-GFP fragments to chloroplasts (Fig. 3B).
Next, we compared the amino acid sequence of the Arabidopsis NtHR with those of CHUP1 orthologs from rice (Oryza sativa), fern (Adiantum capillus-veneris), and moss (Physcomitrella patens). We found that the NtHR of all of the orthologs contained large hydrophobic residues and some charged and polar amino acids, such as Arg and Ser, as consensus amino acids ( Fig. 4A). When NtHR-GFPs of the CHUP1 orthologs were expressed transiently in Arabidopsis leaf cells, GFP fluorescence was detected at the periphery of chloroplasts for OsCHUP1 1-25 (rice CHUP1) and PpCHUP1A 1-25 (moss CHUP1) but not AcCHUP1A 1-25 (fern CHUP1; Fig. 4B). To investigate the importance of the consensus amino acids Arg-4, Ser-12, and Arg-20, each modified NtHR sequence fused to GFP was transiently expressed in Arabidopsis leaf cells. GFP fluorescence was found at the periphery of chloroplasts when one of these amino acids in the NtHR was changed to Ala, whereas the fluorescence was found in the cytosol when two or all three of these amino acids were changed (Fig. 4B).

Transgenic Lines Expressing NtHR-GFP Show Abnormal Distribution of Chloroplasts
To address the correlation between NtHR and chloroplast distribution, NtHR-GFP was expressed stably under the control of the CaMV 35S promoter in wildtype cells of Arabidopsis (Fig. 5A). Transgenic lines were classified into three groups according to the expression level of NtHR-GFP (Fig. 5B). In the highly expressing lines, endogenous CHUP1 was detected as the same level as in wild-type plants (Fig. 5C). GFP fluorescence was found at the periphery of chloroplasts in all transgenic lines (Fig. 5D); however, chloroplasts in these transgenic lines showed abnormal positioning and aggregation even under weak light conditions (Fig. 5D). The chloroplast distribution became more abnormal as the NtHR-GFP expression level increased, and chloroplast positioning in the highly expressing line was similar to that in the chup1 mutant (Fig. 5D). These results suggest that excessively expressed NtHR-GFP disturbs the function of CHUP1 by competing with authentic CHUP1 to target chloroplasts; that is, it has a dominant-negative effect. To confirm this hypothesis, we performed a similar experiment with CHLOROPLAST OUTER ENVELOPE PROTEIN7 (AtOEP7), which inserts its N terminus into the outer envelope and exposes its C . Hydrophilic amino acids in NtHR are critical for targeting CHUP1 to the chloroplast periphery. A, Multiple alignment of CHUP1like proteins. The sequences were aligned using the ClustalW program at the European Bioinformatics Institute Web site. AtCHUP1, Arabidopsis CHUP1 (AB087408); OsCHUP1, Oryza sativa CHUP1 (NM001072463.1); AcCHUP1A and AcCHUP1B, Adiantum capillusveneris CHUP1A (AB444611) and CHUP1B (AB444612); PpCHUP1A and PpCHUP1B, Physcomitrella patens CHUP1A (AB292414) and CHUP1B (AB292415). Blue shading indicates hydrophobic amino acids, and red shading indicates hydrophilic amino acids replaced with Ala for the experiments shown in B. B, Transient expression of GFP fusions of NtHR from AtCHUP1 and CHUP1-like proteins such as OsCHUP1, AcCHUP1A, and PpCHUP1A. C, Effects of amino acid substitutions in the NtHR of AtCHUP1 on the chloroplast targeting signal. Amino acids Arg-4, Ser-12, and Arg-20, shown in red in A, were replaced with Ala (red letters). GFP fluorescence patterns of each construct were observed and analyzed using more than five cells in each experiment. Green indicates fluorescence from GFP, and red indicates fluorescence from chlorophyll in chloroplasts. Bars = 20 mm. terminus to the cytosol (Lee et al., 2001). A construct of the OEP7 targeting signal fused to GFP (OEP7 1-50 -GFP) was introduced into wild-type plants. The sequence of the targeting signal region of OEP7 (OEP7 1-50 ) shows very low similarity to the NtHR sequence, although both sequences have hydrophobic regions (Fig. 5E). Nevertheless, in the transgenic plants, chloroplasts aggregated and GFP fluorescence was observed at the periphery of chloroplasts (Fig. 5F), consistent with the pattern seen with NtHR-GFP.

Requirement of the N-Terminal Targeting Signal for CHUP1 Function
We next examined whether the amino acid sequence of NtHR is essential for CHUP1 function by replacing the NtHR of CHUP1 with OEP7 1-50 . Genes encoding the full-length CHUP1 or two CHUP1s modified at the N terminus (CHUP1 DNtHR and OEP7 1-50 -CHUP1 DNtHR ) were expressed in chup1 mutant plants under the control of the CaMV 35S promoter (Fig. 6A). The transcript of each construct in each transgenic line was confirmed by reverse transcription (RT)-PCR with specific primers (Fig. 6B). Endogenous CHUP1, exogenous CHUP1, and OEP7 1-50 -CHUP1 DNtHR were expressed at their predicted sizes, as confirmed by immunoblot analysis, but CHUP1 DNtHR was not (Fig.  6C). It is possible that CHUP1 DNtHR was degraded because it was not localized to the correct position, the chloroplast outer envelope. We then tested whether the mutant phenotypes were complemented by these constructs under various light conditions. Chloroplasts in transgenic lines expressing CHUP1 or OEP7 1-50 -CHUP1 DNtHR showed normal chloroplast relocation similar to that of the wild-type plants under all light conditions tested, but chloroplasts in the  Figure 2D. Bar = 20 mm. CHUP1 DNtHR transgenic lines did not (Fig. 6D). These results indicate that the presence of an N-terminal region is essential for CHUP1 function, although the wild-type sequence can be replaced with OEP7 1-50 . Thus, the localization of full-length CHUP1 at the chloroplast envelope is essential to its function in the chloroplast relocation machinery.
The Coiled-Coil Region of CHUP1 Anchors Chloroplasts to the Plasma Membrane CHUP1 has various putative functional regions, including an N-terminal chloroplast targeting signal region (N), a coiled-coil region (C), a filamentous actin binding region (A), a Pro-rich region (P), and a C-terminal highly conserved region (Ct; Fig. 7A). However, the roles of these regions in chloroplast relocation movement remain unknown. To examine whether these regions participate in the physiological regulation of chloroplast relocation, we conjugated various types of truncated CHUP1 to GFP and stably expressed these genes in chup1 plants under the control of the CaMV 35S promoter (Fig. 7A). An immunoblot analysis showed that these transgenes were expressed in each transgenic plant (Fig. 7B). However, the calculated sizes of the accumulated CHUP1-GFP proteins in the transgenic lines expressing CHUP1 1-300 (N-C), CHUP1 1-500 (N-C-A), and CHUP1 1-700 (N-C-A-P) were larger than the sizes estimated from the gene con-structs (Fig. 7B). In contrast, the transgenic line expressing CHUP1 1-50,500-1004 (N-P-Ct) showed a specific signal at the predicted size (Fig. 7B).
Next, we investigated the physiological effects of these constructs on chloroplast relocation under various light conditions by observing chloroplast positioning, focusing from top to bottom of the palisade mesophyll cells, with a microscope. In wild-type palisade mesophyll cells, a few chloroplasts were localized to the surfaces of cells, but mostly at the anticlinal position if they had been kept in the dark for 3 h. After 12 h in the dark, most chloroplasts were localized to the cell bottom, as reported previously (Suetsugu et al., 2005). In contrast, chloroplasts accumulated along the periclinal and anticlinal sides of palisade mesophyll cells under weak and strong light conditions, respectively (Fig. 7C). On the other hand, at all light intensities, and even in the dark, the chloroplasts of N-C, N-C-A, and N-C-A-P transgenic plants were positioned along the whole part of anticlinal sides of palisade mesophyll cells, as observed for chloroplast positioning in the wild-type plants under strong light conditions (Fig. 7C). The N-P-Ct transgenic plant still showed the chup1 phenotype (Fig. 7C). Since all of these constructs except for N-P-Ct contain the coiled-coil region of CHUP1, the function of the coiled-coil region must be responsible for the chloroplast distribution along the anticlinal cell walls.
To further examine whether chloroplasts in these transgenic lines respond to light, we analyzed chloro-  . Numbers under each construct are the amino acid numbers from the N terminus. Transgenic lines expressing each construct are represented as N-C for CHUP1 1-300 , N-C-A for CHUP1 1-500 , N-C-A-P for CHUP1 1-720 , and N-P-Ct for CHUP1 1-50,500-1004 . B, Immunoblot analysis of transgenic lines. Immunoblotting was performed using total protein from the transgenic lines and anti-CHUP1 antibodies (aH and aT). Asterisks show the specific band of CHUP1-GFP detected by each antibody. C, Distribution patterns of chloroplasts in wild-type (WT), chup1, and transgenic lines. Leaves of wild-type, chup1, and transgenic plants were adapted to the dark (D), weak white light (W), or strong white light (S) for 3 h and observed with a microscope. Bar = 20 mm. D, Time-lapse analyses of chloroplast relocation movement induced by microbeam irradiation in the cells of N-C-A and wild-type plants. A middle part of the cell surface was irradiated with a microbeam of weak blue light (3 mmol m 22 s 21 ) for 60 min and then with strong blue light (30 mmol m 22 s 21 ) for 30 min. Images were taken every minute and stacked for a movie file (Supplemental Movies S1 and S2). Bar = 10 mm. E, Distribution patterns of chloroplasts in each transgenic line in the presence of LatB and BDM. Leaves of transgenic lines were treated with 100 mM LatB or 50 mM BDM under white light for 3 h. Bar = 20 mm. These experiments were repeated at least three times. CHUP1 1-300 (N-C) does not contain the filamentous actin-binding region raises the question of whether cytoskeletal components are involved in this chloroplast distribution. We tested whether treatment with cytoskeletal inhibitors would change the chloroplast distribution pattern under weak light in the transgenic lines (Fig. 7E). The chloroplast distribution pattern was not affected at all by latrunculin B (LatB) and BDM in any line tested (Fig. 7E).
To address the function of the coiled-coil region for attaching chloroplasts to the plasma membrane, we transiently expressed three truncated N-terminal regions of CHUP1 fused with GFP in wild-type cells (Fig. 8A). GFP fluorescence from CHUP1 25-322 -GFP (i.e. the coiled-coil region with GFP) was detected at the plasma membrane in a pattern identical to that of the fluorescence of N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (Fig. 8B), which was used as a plasma membrane marker (Bolte et al., 2004). However, the fluorescence of CHUP1 25-166 -GFP (a truncated coiled-coil region with GFP) was detected in the cytosol, similar to the pattern of GFP as a control (Fig. 8B). GFP fluorescence from CHUP1 25-500 -GFP in palisade mesophyll cells was too weak to determine its subcellular localization (Fig. 8B). These results indicate that the coiled-coil region of CHUP1 interacts with the plasma membrane, enabling CHUP1 to bridge the chloroplast and the plasma membrane to anchor chloroplasts on the anticlinal side.

Effects of Cytoskeletal Inhibitors on Chloroplast Accumulation Movement
To investigate the role of the coiled-coil region of endogenous CHUP1 in light-dependent chloroplast relocation, we observed the distribution patterns of chloroplasts in wild-type, chup1, phot1phot2, and N-C-A lines under weak light conditions after treatment with the cytoskeleton inhibitors LatB and BDM (Fig.  9). When these plants were kept in the dark for 3 h, chloroplasts gathered at the cell bottom in chup1 cells but were located along the anticlinal plasma membrane in the wild-type, phot1phot2, and transgenic N-C-A plants (Fig. 9A). In the wild type, chloroplast accumulation at the periclinal plasma membrane was usually observed after transferring the plants to weak light, but the relocation movement was not observed after treatment with LatB or BDM (Fig. 9A). No chloroplast relocalization was observed in phot1phot2, N-C-A, and chup1 plants irrespective of the presence of cytoskeletal inhibitors and even under weak light conditions (Fig. 9A). Finally, we observed chloroplast distribution under strong light in these plants. We observed chloroplast relocation from the avoidance to the accumulation positions only in wild-type plants, and this movement was prevented by the cytoskeletal inhibitors (Fig. 9B). These results indicate that chloroplast distributions in phot1phot2 and N-C-A leaves are determined by the coiled-coil structure of endogenous  CHUP1 that is located along the anticlinal plasma membrane.

DISCUSSION
Proteins located on an organelle envelope, such as Mmm1p, Mdm10p, and Mdm12p on mitochondria (Boldogh et al., 2003), Vac8 on vacuoles (Tang et al., 2003), Inp2 on peroxisomes (Fagarasanu et al., 2006), and Rab27 on melanosomes (Wu et al., 2002), have been reported to play a role in the intracellular localization of the organelles. We propose that CHUP1 has similarly an important function in chloroplast relocation movement because it was found to localize on the outer envelope and also at the peripheral region of chloroplasts in this work.
We detected full-length CHUP1 in the isolated chloroplast fraction by immunoblotting. Expression of CHUP1-GFP in chup1 mutant plants complemented the chup1 phenotype, and the GFP fluorescence was detected at the chloroplast periphery. In contrast, GFP-CHUP1 did not complement the chup1 phenotype, and its GFP fluorescence was mostly detected in the cytosol, probably because the NtHR of CHUP1 was masked by the GFP fused to the N terminus so that CHUP1 could not be properly targeted to chloroplasts. To better define the targeting region of AtCHUP1, we investigated the targeting sequence of CHUP1 by using GFP-fused constructs possessing deletions or substitutions within the NtHRs of CHUP1s from Arabidopsis and other species, such as rice, fern, and moss. Our results showed that almost 25 amino acids and two of the three consensus amino acids, Arg-4, Ser-12, and Arg-20, were important for targeting. However, the fluorescence of AcCHUP1A 1-25 -GFP was not detected around the chloroplast periphery. AcCHUP1A 1-25 lacks only one of the three consensus amino acids in the conserved hydrophobic region. This suggests that either the seven amino acids in length between these consensus amino acids in the targeting sequence is critical for the targeting mechanism or that the targeting sequence of AcCHUP1A is longer than that of CHUP1 from other species. Future work will be designed to test these possibilities. The importance of NtHR for the distribution and function of CHUP1 was further demonstrated in transgenic plants expressing CHUP1 without the NtHR (CHUP1 DNtHR ), which did not complement the chup1 phenotype. However, transgenic plants expressing OEP7 1-50 -CHUP1 DNtHR were able to restore the wildtype phenotype in chup1 mutants, suggesting that OEP7 1-50 can substitute for NtHR. This finding means that the specific amino acid sequence of NtHR itself is not critical for CHUP1 function, but the presence of an N-terminal targeting sequence and the subsequent localization of CHUP1 on the chloroplast outer envelope are essential. The OEP7 signal peptide inserts its N terminus into the chloroplast outer envelope by recognizing the specific lipid composition in the outer envelope (Schleiff et al., 2001); therefore, NtHR might also be directly inserted into the chloroplast outer envelope. Stable transgenic lines overexpressing OEP7 1-50 -GFP or NtHR-GFP in wild-type plants (Fig.   Figure 9. Distribution pattern of chloroplasts under cytoskeletal inhibitor treatments. A, Plants of wild-type (WT), chup1, phot1phot2, and CHUP1 1-500 -GFP lines were kept in the dark for 3 h (D). The plants were transferred to weak light (D-W) with or without LatB (+LatB) or BDM (+BDM) for 3 h. B, The plants were adapted with strong light (S) and then moved to weak light (S-W) with or without LatB or BDM for 3 h. Other details are the same as in Figure 2C. Bars = 20 mm. 5, D and E) showed chloroplast aggregation similar to the chup1 mutant phenotype (Lee et al., 2001;Oikawa et al., 2003). These results indicate that NtHR and the targeting sequence of OEP7 occupy the same position on the chloroplast outer envelope.
The interpretation of the dominant-negative effect of overexpressed NtHR-GFP or OEP7 1-50 -GFP in wildtype cells is not clear. Because the structures of CHUP1 and GFP are very different from each other, competition between CHUP1 and GFP for the same binding partners is not plausible. One possible interpretation is that CHUP1 competes with NtHR-GFP or OEP7 1-50 -GFP for a nonspecific, three-dimensional space on the outer envelope. If the overexpressed NtHR-GFP or OEP7 1-50 -GFP occupies the surface of the outer envelope more rapidly than newly synthesized CHUP1, the existing CHUP1 proteins might gradually be replaced by the GFP proteins, resulting in the release of CHUP1 from the outer envelope and chloroplast aggregation.
The sequences that target many chloroplast proteins to the chloroplast outer envelope have been well studied (Hofmann and Theg, 2005;Inoue, 2007). However, it has been difficult to predict the consensus pattern for the chloroplast outer envelope targeting sequence because of its variety and complexity (Inoue, 2007). In fact, CHUP1 has not been recognized as a chloroplast outer envelope protein in recent databases and proteome analyses (Schleiff et al., 2003;van Wijk, 2004). The amino acid sequence of NtHR, therefore, is a novel type of signal sequence for targeting a protein to the chloroplast outer envelope, and as such, it provides additional information for research on chloroplast outer envelope proteins.
In this work, we studied the roles of the predicted functional regions or domains of CHUP1 on chloroplast relocation movement using deletion constructs of CHUP1 fused to GFP. In the transgenic lines expressing CHUP1 1-300 -GFP (abbreviated N-C), which lacks an actin-binding region, chloroplasts did not aggregate but localized along the anticlinal plasma membrane under any light intensity without a cytoskeletal system. These results suggested that the N-C region of CHUP1 functions as a bridge connecting a chloroplast and the plasma membrane, with NtHR inserting into the chloroplast outer membrane and the coiled-coil region binding to plasma membrane proteins.
One possible explanation for why chloroplasts in the N-C line plants show an anticlinal but not a periclinal distribution is that protein(s) interacting with the coiled-coil region exists only in the anticlinal plasma membrane of palisade mesophyll cells and not in the periclinal membrane. However, a similar distribution pattern of chloroplasts was observed in the cells treated with inhibitors of actin polymerization or myosin function (Fig. 7). Furthermore, a chloroplast distribution similar to that in N-C lines was also reported in transgenic plants overexpressing the kinase domain of phot2 (Kong et al., 2007) and in a chloroplast relocation mutant, jac1, which is defective in the accumulation response (Suetsugu et al., 2005).
These findings suggest that chloroplast positioning along the anticlinal plasma membrane is the default position for chloroplasts when chloroplast relocation movement becomes defective.
The F-actin-binding region and the C-terminal Prorich region of CHUP1 might be involved in actin polymerization (Oikawa et al., 2003). It was reported recently that CHUP1 has the ability to bind to profilin and G-actin (Schmidt von Braun and Schleiff, 2008). These functional regions might be essential for chloroplasts to move from their primary position at the anticlinal to the periclinal plasma membrane for the accumulation response or to the bottom of the cell for dark positioning. In this work, we identified two other functional regions in CHUP1, the NtHR, which targets CHUP1 to the chloroplast outer envelope, and the coiled-coil region, which anchors the chloroplast to the plasma membrane. These four functional regions of CHUP1 mediate the interaction between chloroplasts and the plasma membrane and are involved in the polymerization of or interaction with actin filaments involved in chloroplast relocation movement. The overall function of CHUP1 enables chloroplasts to spread over the cell surface to form a single layer without aggregation or to move in any direction to maximize efficient photosynthesis.

Plant Materials and Growth Conditions
Seeds of wild-type (accession Columbia, gl-1), chup1-2 (Oikawa et al., 2003), and transgenic lines of Arabidopsis (Arabidopsis thaliana) were sown on the surface of one-third-strength Murashige and Skoog medium solidified with 0.8% (w/v) agar containing 1% (w/v) Suc on plastic culture plates as described previously (Kagawa and Wada, 2000), incubated at 4°C for 2 d, and then grown under light conditions of 16 h of light (white fluorescent light at 70 mmol m 22 s 21 ) and 8 h of dark at 22°C for 3 to 4 weeks in an incubator (Biotron LH300-RPSMP; Nippon Medical and Chemical Instruments).

CHUP1 Antisera Preparations
Two different regions of the CHUP1 gene, corresponding to H1 (121 amino acids of CHUP1 at the coiled-coil region; CHUP1 200-320 ) and T1 (the 305 amino acids of CHUP1 at the C-terminal region; CHUP1 700-1004 ), were cloned into a pET21d vector for expression in Escherichia coli BL21 (DE3) cells (Novagen) as the His-tagged fusion proteins CHUP1-H1 and CHUP1-T1, respectively. Expression was induced by the addition of 1 mM isopropyl-1-thio-b-Dgalactopyranoside for 4 h at 37°C. The cells were harvested and broken in a commercial bacterial cell lysis buffer (CelLytic; Sigma-Aldrich). CHUP1-H1 was recovered in the soluble fraction, whereas CHUP1-T1 became insoluble. CHUP1-T1 was solubilized with a buffer containing 7 M urea, 50 mM Tris-HCl (pH 7.5), and 300 mM NaCl. The solubilized protein was purified with a Talon metal affinity column (Clontech). The purified proteins were desalted with NAP-10 columns (GE Healthcare) containing 0.01% Triton X-100 and used for the generation of antibodies in rabbits, as described previously (Asakura et al., 2004).

Immunoblotting
Rosette leaves of Arabidopsis were homogenized with phosphate-buffered saline (137 mM NaCl, 8.1 mM Na 2 HPO 4 , 2.68 mM KCl, and 1.47 mM KH 2 PO 4 ) containing 10% (w/v) glycerol. The homogenized samples were centrifuged at 10,000g for 5 min. The supernatants (20 mg of protein) were mixed with an equal volume of 23 sample buffer (50 mM Tris-HCl [pH 6.8], 2% [w/v] SDS, 6% [v/v] 2-mercaptoethanol, and 10% [w/v] glycerol). The solubilized samples were subjected to SDS-PAGE separation on a 7.5% (w/v) acrylamide gel. The resolved proteins were blotted onto a Hybond-P membrane (GE Healthcare). The anti-CHUP1-H1 (aH) and anti-CHUP1-T1 (aT) antibodies were diluted 1:2,000 and used to detect the CHUP1 proteins. An anti-rabbit IgG conjugated with alkaline phosphatase (GE Healthcare) was used as a secondary antibody for both the aH and aT primary antibodies. Finally, crossreactive protein bands were detected using a commercial alkaline phosphatase development kit (ProtoBlot II AP System with Stabilized Substrate Kit; Promega).
To determine the CHUP1 localization in chloroplasts, immunoblot analysis was performed with isolated chloroplasts treated with thermolysin, 100 or 200 mg mL 21 for 20 min on ice. Toc159, Toc75, Tic110, and cpHsp70 were used as known controls of chloroplast proteins (Asakura et al., 2004;Kikuchi et al., 2006).

Chloroplast Preparation
Rosette leaves of Arabidopsis (10 g fresh weight) were homogenized in 200 mL of a homogenizing medium (50 mM HEPES-KOH [pH 7.8], 330 mM sorbitol, 2 mM EDTA, 1 mM MnCl 2 , 1 mM MgCl 2 , 1 mM phenylmethylsulfonyl fluoride, and 5 mM sodium ascorbate) using a modified mixer equipped with razors. The homogenate was filtered through a doubled rayon-polyester filter (Miracloth; Calbiochem). The filtered samples were centrifuged at 1,500g for 5 min. The precipitate was gently resuspended in 10 mL of a washing medium containing 50 mM HEPES-KOH (pH 7.8), 330 mM sorbitol, and a protease inhibitor cocktail (Complete EDTA-free; Roche Diagnostics). This fraction was loaded on layers of 40% and 80% Percoll medium (40% or 80% [v/v] Percoll [GE Healthcare], 50 mM HEPES-KOH [pH 7.8], 330 mM sorbitol, and the protease inhibitor cocktail). After centrifugation at 1,500g for 15 min, intact chloroplasts banded at the interface between the 40% and 80% Percoll media were carefully recovered. The isolated chloroplasts were washed with the washing medium by centrifugation at 1,500g for 5 min. The precipitate was resuspended in the washing medium and used as intact chloroplast samples. All procedures were carried out at 4°C.

Plasmid Construction
To generate plasmids for transiently expressing fusion proteins of each DNA fragment and GFP, the corresponding regions were amplified by PCR using specific primers and a CHUP1 cDNA or OEP7 cDNA as a template (Supplemental Table S1). The PCR products were cloned into the CaMV35-sGFP(S65T) plasmid (Chiu et al., 1996;Niwa et al., 1999) at the SalI-NcoI site for N-terminal GFP fusion and at the BsrGI-NotI site for C-terminal GFP fusion. The resulting plasmids were named as described in Supplemental Table S1. To generate plasmids for transformation of Arabidopsis, each DNA fragment that had been cloned in the CaMV35-sGFP(S65T) plasmid for transient expression of GFP fusion proteins as described above was introduced into a binary vector, pPZP211, at the XbaI-SmaI site (Supplemental Table S1).

Transformation of Arabidopsis
Transformation was performed according to the Agrobacterium tumefaciensmediated floral dip method (Clough and Bent, 1998). T1 plants were selected on a medium containing one-third-strength Murashige and Skoog salts and 1% Suc with 0.8% agar and 25 mg mL 21 kanamycin. On the basis of segregation of kanamycin resistance, T2 lines that contained a single transgene locus were selected, and homozygous T3 seeds were maintained for further experiments. At least three homozygous lines were used for each transgenic plant in this study. All of the transgenic plants were isolated independently.

Transient Expression
Arabidopsis leaves were bombarded with gold particles (1 mm in diameter) coated with plasmid DNA using a gene delivery system (PDS-1000/He particle delivery system; Bio-Rad) as described previously (Oikawa et al., 2003). The samples were incubated at 22°C for 12 h before observation with a microscope.

Observation of Chloroplast Relocation
Chloroplast relocation in palisade mesophyll cells of transformants was analyzed with detached leaves incubated on agar plates under weak light (10 mmol m 22 s 21 ), strong light (100 mmol m 22 s 21 ), or in the dark for 3 h after adaptation in the dark for 6 h. Chloroplast positioning of each palisade mesophyll cell was visually determined with a microscope (Axioplan2; Zeiss) while adjusting focuses throughout the cell from top to bottom.

Fluorescence Microscopic Analysis
The subcellular localization of CHUP1-GFP, GFP-CHUP1, and NtHR-GFP was determined using a fluorescence microscope (Axioplan2; Zeiss) or a laser scanning microscope (LSM Meta 510; Zeiss). Before observation, detached leaves were adapted to weak light for 3 h.

Time-Lapse Imaging of Transformed Cells
Chloroplast relocation movement of wild-type and transgenic lines was observed as described previously (Kagawa and Wada, 2000). The individual cell was irradiated with a microbeam of blue light with a spot size of 19 mm in diameter. Images of observed cells were obtained at 30-s intervals. The intensity of the blue light was 3 mmol m 22 s 21 for the weak light response and 30 mmol m 22 s 21 for the strong light response. Cells were incubated in the dark for at least 12 h before use.

Measurement of Relative Fluorescence Intensity for NtHR-GFP Accumulation
Fluorescence intensity was measured as a gray value with the public domain software ImageJ (http://rsb.info.nih.gov/ij/). The average fluorescence intensities of GFP were obtained by measuring the fluorescence intensity at the chloroplast periphery of 20 individual chloroplasts in both the GFP and NtHR-GFP lines. The fluorescence intensity of each NtHR-GFP line was expressed as fluorescence percentage normalized to the value of the GFP line with SD.

Inhibitor Assay
LatB (Biomol) and BDM (Sigma-Aldrich) were used to disrupt the actin filament and inhibit myosin function, respectively. LatB and BDM were dissolved in dimethyl sulfoxide as stock solutions of 2 mM and 1 M, respectively. Final concentrations of 100 mM LatB and 50 mM BDM were used in the experiments. Detached leaves placed on agar plates were adapted to either strong light or dark for 3 h. Then the leaves were submerged in medium containing either inhibitor and incubated under weak light for 3 h. Chloroplast distribution was observed with a microscope as described previously (Oikawa et al., 2003).

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Table S1. Primer sets for the plasmids used in these experiments.
Supplemental Movie S1. The accumulation response and avoidance response of chloroplasts in wild-type cells.
Supplemental Movie S2. The response does not occur in N-C-A transgenic plants. For details, see the legend of Figure 7D.