Interaction of Actin and the Chloroplast Protein Import Apparatus*

Actin filaments are major components of the cytoskeleton and play numerous essential roles, including chloroplast positioning and plastid stromule movement, in plant cells. Actin is present in pea chloroplast envelope membrane preparations and is localized at the surface of the chloroplasts, as shown by agglutination of intact isolated chloroplasts by antibodies to actin. To identify chloroplast envelope proteins involved in actin binding, we have carried out actin co-immunoprecipitation and co-sedimentation experiments on detergent-solubilized pea chloroplast envelope membranes. Proteins co-immunoprecipitated with actin were identified by mass spectrometry and by Western blotting and included the Toc159, Toc75, Toc34, and Tic110 components of the TOC-TIC protein import apparatus. A direct interaction of actin with Escherichia coli-expressed Toc159, but not Toc33, was shown by co-sedimentation experiments, suggesting that Toc159 is the component of the TOC complex that interacts with actin on the cytosolic side of the outer envelope membrane. The physiological significance of this interaction is unknown, but it may play a role in the import of nuclear-encoded photosynthesis proteins.

Actin filaments are major components of the cytoskeleton and play numerous essential roles, including chloroplast positioning and plastid stromule movement, in plant cells. Actin is present in pea chloroplast envelope membrane preparations and is localized at the surface of the chloroplasts, as shown by agglutination of intact isolated chloroplasts by antibodies to actin. To identify chloroplast envelope proteins involved in actin binding, we have carried out actin co-immunoprecipitation and co-sedimentation experiments on detergent-solubilized pea chloroplast envelope membranes. Proteins co-immunoprecipitated with actin were identified by mass spectrometry and by Western blotting and included the Toc159, Toc75, Toc34, and Tic110 components of the TOC-TIC protein import apparatus. A direct interaction of actin with Escherichia coliexpressed Toc159, but not Toc33, was shown by co-sedimentation experiments, suggesting that Toc159 is the component of the TOC complex that interacts with actin on the cytosolic side of the outer envelope membrane. The physiological significance of this interaction is unknown, but it may play a role in the import of nuclear-encoded photosynthesis proteins.
Actin is a ubiquitous protein of eukaryotic cells. Actin microfilaments are formed from polymerization of actin monomers and are a major component of the cytoskeleton. In plant cells, actin microfilaments are arranged in longitudinal arrays of thick actin bundles with randomly oriented thin actin filaments extending from the bundles (1). Chloroplasts are either aligned along the actin bundles or closely associated with the fine filaments and are surrounded by baskets of actin microfilaments (1,2). A direct interaction of chloroplasts with the actin cytoskeleton has been postulated to anchor chloroplasts at appropriate intracellular positions (3). Chloroplast movement depends on cytosolic actin filaments and is stimulated by high light intensity (4). A chloroplast envelope protein involved in blue light-dependent chloroplast repositioning has been identified by the analysis of the Arabidopsis chup1 (chloroplast unusual positioning 1) mutant, which was unable to relocate its chloroplasts under high light stimulation (5). CHUP1 is a protein exclusively targeted to the chloroplast outer envelope membrane that is essential for chloroplast anchorage to the plasma membrane (6). CHUP1 interacts with actin and profilin, a modulator of actin polymerization, and it may play a regula-tory role in actin polymerization during chloroplast photo-relocation (7).
The interaction of amyloplasts with the actin cytoskeleton has been implicated in gravity perception and signal transduction. Several models for the role of the actin cytoskeleton have been proposed (8), but the nature of the interaction is not known. However, disruption of the actin cytoskeleton enhanced sedimentation of amyloplasts and promoted gravitropism (9,10), and a role for myosin has been proposed on the basis of inhibitor experiments (11).
The actin cytoskeleton and myosin have also been implicated in plastid stromule movement. Stromules (stroma-filled tubules) are highly dynamic tubular structures extending from the surface of all plastid types (12,13). Stromules are delimited by the inner and outer plastid envelope membranes, which are closely associated (for a review, see Refs. 12 and 13). Experiments with inhibitors of microfilament-and microtubulebased movement suggested that stromules move along actin microfilaments powered by the ATPase activity of myosin motors (14). Physical connection between the envelope membranes seems likely to be required to provide a means of coordinating the movement of the inner envelope membrane with the microfilament-associated outer envelope membrane. There is evidence for direct connection of the inner and outer envelope membranes at contact sites, which support protein translocation through the protein import apparatus (15,16). This apparatus consists of two membrane protein complexes that associate to allow translocation of nucleus-encoded proteins from the cytoplasm to the interior stromal compartment (for a review, see 17). The translocon at the outer envelope membrane of chloroplasts (TOC complex) 2 mediates the initial recognition of preproteins and their translocation across the outer membrane (18). The translocon at the inner envelope membrane of chloroplasts (TIC complex) physically associates with the TOC complex and provides the membrane translocation channel for the inner membrane. In addition, the TOC and TIC complexes interact with a set of molecular chaperones, which assist the transfer of imported proteins (19 -21).
With the aim of identifying components involved in the interaction of the chloroplast envelope with the actin cytoskeleton, we have used actin co-immunoprecipitation and co-sedimentation experiments with detergent-solubilized pea chloro-plast envelope membranes. Components of the TOC-TIC protein import apparatus have been identified by mass spectrometry and Western blotting, and a direct interaction of Escherichia coli-expressed Toc159 with actin was demonstrated by co-sedimentation. This interaction may have a so far unrecognized physiological role in chloroplast protein import.

EXPERIMENTAL PROCEDURES
Materials-Pea seeds (Pisum sativum Progress no. 9) were obtained from King and Co. Ltd (Kelvedon, Essex, UK) and grown on vermiculite for 13 days at 22°C in a greenhouse with supplemental lighting giving 275 mol of photons m Ϫ2 s Ϫ1 . Human platelet non-muscle actin was obtained from Cytoskeleton, Inc. (Denver, CO), and filaments were prepared by incubation for 1 h in 5 mM Tris-HCl, pH 7.5, 2 mM MgCl 2 , 50 mM KCl, 1 mM ATP. Mouse monoclonal antibodies raised against chicken actin (clone C4) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and secondary horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies were obtained from GE Healthcare.
Agglutination Experiments-Antibodies raised against chloroplast envelope proteins or actin were used to examine the agglutination of isolated intact chloroplasts. For agglutination assays, 10 l of chloroplast suspension containing 18 g of chlorophyll were incubated for 10 min on a glass slide with 5 l of washing buffer and 5 l of antibodies. The slides were examined at room temperature by confocal laser scanning microscopy using a TCS-SP2 operating system (Leica) using an immersion 40ϫ objective. Chloroplasts were visualized by transmission and chlorophyll fluorescence. Chlorophyll was excited using the 543-nm line of a He-Ne laser, and fluorescence was collected between 630 and 750 nm.
Expression in E. coli-The plasmids encoding AtToc159, AtToc33G, and AtToc159G fused to GST were introduced into E. coli BL21. The strains were cultured overnight in 5 ml of L-broth medium containing carbenicillin (100 g/ml) at 37°C and transferred into 100 ml of L-broth medium containing carbenicillin (100 g/ml) at 37°C. After incubation for 4 h, expression was induced by the addition of isopropyl 1-thio-␤-D-galactopyranoside to 1 mM final concentration. After induction for 4 h at 37°C, the cells were recovered by centrifugation at 5000 ϫ g for 10 min at 4°C and resuspended in 2 ml of Trisbuffered saline (10 mM Tris-HCl, pH 7.5, 155 mM NaCl). The cells were broken by sonication (6 ϫ 10-s pulses at 24 kHz) and centrifuged at 100,000 ϫ g for 1 h. The supernatants were used for actin co-sedimentation assays.
Western Blot Analysis-Proteins were solubilized in SDSloading buffer (0.1 M Tris-HCl, pH 7.8, 10% (v/v) glycerol, 2% (w/v) SDS, 25 mM dithiothreitol, 0.1% (w/v) bromphenol blue). Samples were analyzed by SDS-PAGE (23). After electrophoresis, proteins were fixed in isopropanol/acetic acid (3:1, v/v) for 30 min before staining with colloidal Coomassie Blue (24) or silver (25). Proteins were stained overnight in colloidal blue (1.3 M ammonium sulfate, 34% methanol, 0.5% acetic acid, 0.1% (w/v) Coomassie Brilliant Blue G-250; Sigma-Aldrich) and destained in distilled water until the desired contrast was achieved. For silver staining, the gel was washed twice for 10 min in distilled water followed by a 1-min incubation in 1.8 mM sodium hydrosulfite, two 1-min washes in distilled water, 30 -60 min of incubation in 12 mM silver nitrate, a 10-s wash in distilled water, incubation in 0.19 M sodium carbonate, 40 mM sodium thiosulfate, and 0.25% (v/v) formaldehyde until the desired contrast was achieved, and fixing for 30 min in 0.33 M Tris-HCl, 2% (v/v) acetic acid. For Western blotting, proteins were electrophoretically transferred to nitrocellulose membrane (Amersham Biosciences) (26). The proteins were visualized with rabbit or mouse primary antibody against a specific protein and anti-rabbit or anti-mouse IgG-horseradish peroxidase conjugate and the ECL Plus Western blotting detection system (PerkinElmer Life Sciences). Protein quantification was done by Bradford assay (Bio-Rad).
Immunoprecipitation Assays-Pea chloroplast envelope membranes (5 g of proteins per l) were solubilized in 0.5% (w/v) n-dodecylmaltoside by mixing for 30 min on a rotating wheel at 4°C. Insoluble material was removed by centrifugation at 3000 ϫ g for 10 min. The protein concentration of the supernatant was determined by Bradford protein assay. An aliquot with 1 mg of protein was incubated overnight with 40 l of a 50% slurry of protein G PLUS-agarose beads (Santa Cruz Biotechnology) conjugated with monoclonal actin antibodies (Santa Cruz Biotechnology) in 500 l of Tris-buffered saline, 0.5% n-dodecylmaltoside at 4°C on a rotating wheel. Beads were pelleted by a 30-s centrifugation at 3000 ϫ g and washed 5 times with 1 ml of Tris-buffered saline, 0.1% (w/v) Tween 20. Proteins remaining on the beads were solubilized in 20 l of SDS-loading buffer and separated by SDS-PAGE for colloidal blue staining and mass spectrometry analysis, silver staining, or Western blot analysis.
Actin Co-sedimentation Assays-The protein samples (lysed bacterial suspension or 1% (w/v) Lubrol (Sigma-Aldrich)-solubilized pea chloroplast envelope membranes) were precleared by centrifugation at 100,000 ϫ g for 1 h, and the supernatant was used for the actin co-sedimentation assay. The proteins (30 or 50 g) were mixed with various amounts (0 -1.6 g) of human non-muscle actin (Cytoskeleton Inc.) in a total volume of 100 l of Tris-buffered saline. The samples were incubated on a rotating wheel for 1 h at room temperature and centrifuged at 100,000 ϫ g for 45 min. The pellets were suspended in 40 l of SDS-loading buffer, vortexed, and heated at 95°C for 5 min, and 15-l samples were loaded on two SDS-12% polyacrylamide gels for silver staining and Western blot analysis.
Mass Spectrometry Analysis-After SDS-PAGE and colloidal blue staining of chloroplast envelope proteins co-immunoprecipitated with actin, the gel was cut into 17 bands between the top of the gel and the immunoglobulin heavy chain and 15 bands between the immunoglobulin heavy and light chains. Proteins present in the two bands containing the immunoglobulin heavy and light chains and proteins with a higher mobility than the light chain, i.e. with molecular mass Ͻ26 kDa, were not analyzed. Each gel slice was processed individually to allow comparison of the identified proteins with their electrophoretic mobilities. All analyses were carried out at the Cambridge Centre for Proteomics (Department of Biochemistry, University of Cambridge). Proteins within the gel slices were reduced, carboxyamidomethylated, and then digested with trypsin on a Mass-PrepStation (Waters, Manchester, UK). The resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry. Reverse-phase liquid chromatographic separation of peptides was achieved with a PepMap C18 reverse phase, 75-m inner diameter, 15-cm column (LC Packings, Amsterdam) on a capillary liquid chromatography system (Waters) attached to a Dionex Dual Gradient liquid chromatography system attached to a QSTAR XL mass spectrometer (Applied Biosystems, Framingham, MA). The tandem mass spectrometry fragmentation data were used to search the National Center for Biotechnology Information (NCBI) Viridiplantae protein data base and a pea cDNA data base containing 70,007 contigs generated by massively parallel pyrosequencing (27) using the MASCOT search engine. Probability-based MAS-COT scores were used to evaluate identifications. Only matches with p Ͻ 0.05 for random occurrence were considered significant.

RESULTS
Actin Is Localized at the Chloroplast Surface-Preliminary experiments were carried out to examine whether actin was present at the surface of isolated intact chloroplasts by agglutination with antibodies to actin. Chloroplasts were isolated under low salt conditions to decrease contamination by cytosolic actin filaments and carefully washed to remove unbound actin monomers. The chloroplast suspension was incubated on a glass slide with antibodies to actin, to the chloroplast outer envelope protein OEP21 (28), or to the chloroplast inner envelope protein IEP37 (29) and examined by bright field and fluorescence microscopy (Fig. 1A). In the absence of antibodies, the chloroplasts were uniformly distributed on the slide, and incubation with antibodies to IEP37 had little effect on the distribution of the chloroplasts, confirming that the chloroplasts were intact. However, incubations with antibodies to OEP21 and to actin resulted in marked aggregation and clumping of the chloroplasts, suggesting that these proteins were accessible at the chloroplast surface.
Western blot analysis was then carried out to determine whether actin was detectable in purified pea chloroplast envelope membranes. Samples of pea leaf extract, isolated intact chloroplasts, and purified chloroplast envelope membranes were analyzed by SDS-PAGE (Fig. 1B). The protein profile of the envelope membrane was similar to those previously published, with three bands at 97, 86, and 75 kDa, a predominant band at 29 kDa corresponding to the phosphate translocator, FIGURE 1. Interaction of actin with the pea chloroplast envelope. A, immunoagglutination assays of isolated intact chloroplasts prepared from pea leaves. Chloroplasts were incubated with antibodies as specified. Control antibodies were directed against the chloroplast inner and outer membrane proteins IEP37 and OEP21, respectively. B, Coomassie Blue-stained SDS-PAGE gel of protein markers, total cell extract (CE), chloroplast (Chl), and chloroplast envelope (Env)-enriched fractions. C, Western blot of the SDS-PAGE gel in B using antibodies specific for outer envelope membrane protein OEP21, inner envelope membrane protein IEP37, and actin. and some contamination with the large subunit of the Rubisco (30). Western blot analysis with antibodies against IEP37 and OEP21 demonstrated a strong enrichment of these chloroplast inner and outer membrane proteins in the envelope fraction (Fig. 1C). The presence of actin in the pea leaf fractions was examined with a monoclonal antibody against chicken actin. A strong reaction to a 42-kDa protein was obtained with the pea leaf extract, indicating that the monoclonal antibody recognized pea actin. This protein was barely detectable in the intact chloroplast fraction, although it was present at a detectable level in the chloroplast envelope fraction. This represents a clear enrichment of actin in the envelope fraction by comparison with the chloroplast fraction, confirming an association of actin with the envelope membrane.
Co-immunoprecipitation of Actin-associated Envelope Proteins-In an attempt to identify envelope membrane proteins interacting with actin, antibodies to actin were incubated with detergent-solubilized envelope membranes, and the coimmunoprecipitated proteins were analyzed by SDS-PAGE. Pea chloroplast envelope membranes were solubilized with 0.5% (w/v) dodecylmaltoside, a mild non-ionic detergent, and insoluble material was removed by centrifugation. The solubilized envelope membrane proteins were incubated with protein G-agarose beads in the presence or absence of antibodies to chicken actin, and proteins bound to the beads were separated by SDS-PAGE. Silver staining was used to visualize the immunoprecipitated polypeptides, because colloidal blue staining was not sufficiently sensitive to detect polypeptides other than the immunoglobulin chains. A lane containing proteins eluted from protein G-agarose beads incubated in the presence of anti-actin antibodies but in the absence of envelope proteins was used to detect proteins from the antibody preparation. Major bands of 50 and 25 kDa, corresponding to immunoglobulin heavy and light chains, were present in the lanes from incubations with actin antibodies ( Fig. 2A). Several discrete envelope membrane proteins, particularly those of 110, 86, 75, and 33-34 kDa, were specifically immunoprecipitated by the antiactin antibodies and were not present in the lane of envelope proteins bound to protein G-agarose beads in the absence of anti-actin antibodies. Furthermore, Western blot analysis of this gel with anti-actin antibodies detected a strong band at 42 kDa in the lane containing immunoprecipitated envelope membrane proteins (marked by a red arrow in Fig. 2B). The immunoglobulin heavy and light chains and a contaminating band at 41 kDa, seen as stained bands in Fig. 2A, were also detected by the secondary anti-mouse IgG antibodies in lanes from incubations containing anti-actin antibodies. The band at 41 kDa, which has a slightly faster electrophoretic mobility than the 42-kDa actin protein, is dependent on the presence of the anti-actin antibodies and not on the presence of the envelope proteins. This suggests it is a contaminant from the anti-actin antibodies retained on the agarose beads. Because this band is detected by the anti-actin antibodies (Fig. 2B), it is probably an actin contaminant present within the antibody preparation. This experiment provides further evidence for the presence of actin in the chloroplast envelope preparation and indicates that specific envelope proteins were co-immunoprecipitated by anti-actin antibodies.
Identification of the Chloroplast Envelope Proteins Co-immunoprecipitated with Actin-In an attempt to identify envelope proteins co-immunoprecipitated with actin, the experiment described above was repeated, and the SDS-polyacrylamide gel was stained with colloidal blue to allow mass spectrometric analysis of immunoprecipitated proteins. The gel was cut into 17 slices above the immunoglobulin heavy chain and into 15 slices between the heavy and light chains, and the proteins were digested in the gel with trypsin. Each gel slice was processed individually to allow comparison of the identified proteins with their electrophoretic mobilities. Proteins migrating with the same mobility as the two immunoglobulin chains or migrating more rapidly than the light chain, i.e. with a molecular mass Ͻ26 kDa, were not analyzed. The tryptic peptides were analyzed by liquid chromatographytandem mass spectrometry, and the mass data were compared with the NCBI Viridiplantae protein data base using MASCOT software. Only proteins with a MASCOT score above 100 with more than 2 distinct peptides identified and with a molecular mass corresponding to the migration position on the SDS-PAGE gel are shown in Table 1. All the proteins identified showed matches with previously known pea sequences. None of the unidentified peptides matched with plant proteins from other species in the NCBI Viridiplantae data base. The importance of species-specific comparisons for stringent identification of proteins from peptide mass data has recently been emphasized (27), so the mass data were compared with a six-frame translation of a pea cDNA data base generated by massively parallel pyrosequencing (27). This confirmed the identification of all the proteins in Table 1 but failed to identify any additional proteins. The presence of actin was confirmed by the mass identification of 10 different peptides and a high MASCOT score of 585. Many of the other proteins identified as co-immunoprecipitating with actin are components of the TOC-TIC complex. Three components of the TOC complex (Toc159, Toc75, and Toc34) and five components of the TIC complex (Tic110, Tic55, Tic40, Tic32, and ClpC) were identified. Ferredoxin NADP ϩ reductase, which has been reported to associate with the TIC complex (31), was also detected, but Tic62, with which ferredoxin NADP ϩ reductase is reported to interact (31), was not identified in the immunoprecipitated material.
Many of the proteins identified as migrating in the gel at the expected size were also detected in material running at the top of the gel (bands 1 and 2). These proteins may represent aggregated material precipitated on the agarose beads rather than proteins specifically immunoprecipitated with actin. They include cytochrome f, the D2 subunit of photosystem II, the ␣ subunit of ATP synthase, malate dehydrogenase, glycolate oxidase, glyceraldehyde 3-phosphate dehydrogenase, ferredoxinsulfite reductase, and the phosphate translocator. All of the other proteins, including the components of the TOC-TIC complex, were found at the expected size and were not detected in the top bands of the gel. Toc159 was detected in band 2, but it is reported to migrate at 200 kDa (32), and peptides from its degradation product Toc86 were detected at the expected size in band 10 ( Table 1). The identification of components of the TOC-TIC complex at their expected sizes and not in the material at the top of the gel suggests specific co-immunoprecipitation with actin.
Two inner envelope proteins unrelated to the TOC-TIC complex were associated with the immunoprecipitated material; that is, a chloroplast lipoxygenase, involved in oxylipin synthesis (for review, see Ref. 33), and VIPP1, involved in thylakoid formation (34). These proteins were identified at the expected size in the gel and not in the material at the top of the gel.
TOC-TIC Proteins Are Co-immunoprecipitated with Antibodies to Actin-To confirm the identification of TOC-TIC proteins co-immunoprecipitated with actin, the immunoprecipitated proteins were analyzed by Western blotting using antibodies against individual envelope membrane proteins. The whole process of immunoprecipitation with anti-actin antibodies, SDS-PAGE, and Western blot analysis of the proteins bound to the beads was carried out as previously shown in Fig. 2B. The results for Western blotting of the same immunoprecipitation experiment with antibodies to Toc159, Toc75, Toc34 (35), Tic110 (19), and VIPP1 (34) are shown in Fig. 3. The whole experiment was repeated three times, establishing the reproducibility of the immunoprecipitation protocol and the results obtained. Probing the Western blot with anti-actin antibodies confirmed that a 42-kDa protein was enriched in the immunoprecipitate compared with the solubilized membranes. Antibodies to IEP37 and OEP21 were used as negative controls to confirm the specificity of the co-immunoprecipitation.
The anti-Toc159 antibodies identified two bands corresponding to the 159-kDa intact protein and the 86-kDa degra- Immunoprecipitation of actin-associated proteins from 1 mg dodecylmaltoside-solubilized chloroplast envelope membrane proteins was carried out as for Fig. 2. Env, 10 g of dodecylmaltoside-solubilized chloroplast envelope membrane proteins (positive control); Beads ϩ env, eluate of protein G-agarose beads incubated with 1 mg of envelope proteins; Beads ϩ antibodies, eluate of protein G-agarose beads conjugated with the actin antibody in the absence of envelope membrane proteins; Beads ϩ antibodies ϩ env, eluate of protein G-agarose beads conjugated with the actin antibody incubated with 1 mg of envelope proteins. Antibodies against proteins identified by mass spectrometry were anti-Toc159, anti-Toc75, anti-Toc34, anti-Tic110, anti-VIPP1, and anti-actin. Antibodies directed against the chloroplast inner and outer membrane proteins IEP37 and OEP21, respectively, were used to check the specificity of the immunoprecipitation reaction.

TABLE 1 Identification of chloroplast envelope proteins co-immunoprecipitated with actin
Immunoprecipitation of dodecylmaltoside-solubilized pea chloroplast envelope proteins with anti-actin antibodies conjugated to protein G-agarose beads was as described in Fig. 2. Bands were cut from the gel, and tryptic peptides were examined by mass spectrometry. Proteins in bold were localized only in a band corresponding to their expected size. All other identified proteins were localized both in a band corresponding to their expected size and in a band at the top of the gel. Mass, expected molecular mass (kDa); Band, the number of the cut gel band (1 is the top band, 17 is the band above the immunoglobulin heavy chain, 18 is the band under the immunoglobulin heavy chain, and 31 is the band above the immunoglobulin light chain); Peptides, the number of distinct peptides identified for the protein; Score, the MASCOT score. Except for actin, the identified proteins have been ranked according to Score. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PSII D2, D2 protein (PsbD) of photosystem II; EF-Tu, chloroplast elongation factor Tu. dation product (32) in the envelope membrane preparation, and these proteins were both detected in the anti-actin-immunoprecipitated material. A similar result was obtained with antibodies to Toc75 and to Tic110, both of which identified a single band of the correct mobility in the anti-actin immunoprecipitated material, confirming their co-immunoprecipitation with actin. Anti-Toc34 antibodies recognized a 34-kDa protein in the immunoprecipitate, but a slightly less intense band at the same position was also detected in material eluted from protein G-agarose beads incubated in the absence of the anti-actin antibodies. This suggests that at least part of the Toc34 associated with the anti-actin protein G-agarose beads may not be dependent on a specific interaction with actin. Although the Toc159, Toc75, Toc34, and Tic110 proteins were all detected in the anti-actin immunoprecipitate, none of these proteins were enriched in the immunoprecipitate compared with the envelope membrane preparation, suggesting that the majority of the proteins in the membrane are not actin-associated. The presence of VIPP1 in the anti-actin immunoprecipitate was also examined using anti-VIPP1 antibodies. The antibodies detected two bands in the envelope membrane protein fraction (Fig. 3). The upper band is reported to be present only in thylakoid membranes, whereas the lower band is present in both thylakoid and envelope inner membranes (34). Both bands of VIPP1were detected in the material eluted from the anti-actin protein G-agarose beads, and they were also present in the material eluted from protein G-agarose beads in the absence of the anti-actin antibodies. However, the lower VIPP1 band was enriched in the anti-actin immunoprecipitate compared with the material associated with the protein G-agarose beads in the absence of anti-actin antibodies, suggesting the possibility that some VIPP1 may be part of a complex specifically associated with actin.

Identified pea protein
TOC-TIC Proteins Co-sediment with Actin Filaments-To confirm the ability of components of the TOC-TIC protein import apparatus to interact with actin, a different approach examining interaction with exogenous actin filaments was used. The principle is to incubate detergent-solubilized envelope membrane proteins with actin filaments for 1 h at room temperature, then collect the filaments by centrifugation and identify interacting proteins by Western blotting. The experiments were repeated three times, establishing the reproducibility, and the results of one experiment are presented Fig. 4. Pea chloroplast envelope membranes were solubilized with 1% Lubrol and centrifuged at 100,000 ϫ g for 1 h to remove insoluble material. Confirmation of the solubilization of envelope proteins by Lubrol was obtained by SDS-PAGE and Western blotting with antibodies to envelope proteins used previously (Fig. 4). This confirmed the presence of Toc159, Toc75, Toc34, Tic110, VIPP1, IEP37, and OEP21 in 2 g of Lubrol-solubilized envelope proteins. The solubilized envelope proteins (30 g protein) were then incubated with various amounts (0 -0.8 g of protein) of preprepared actin filaments for 1 h, and the filaments and associated proteins were collected by centrifugation. After centrifugation, half of the pellets were separated by SDS-PAGE and silver-stained, and the other half was analyzed by Western blot (Fig. 4). The silver-stained gel confirmed that the actin filaments had been pelleted by the centrifugation, whereas the Western blots showed the distribution of the envelope proteins in the different fractions. Toc159, Toc86, Toc75, and Toc34 all co-sedimented with 0.8 g of actin and were not pelleted in the absence of added actin filaments. This suggests a specific interaction of the TOC complex with actin filaments.
Tic110 and the lower band of VIPP1 both pelleted in the absence of actin filaments, possibly because the co-sedimentation experiment was carried out at room temperature, which may have promoted protein aggregation, whereas all the previous experiments had been carried out at 4°C. However, the abundance of Tic110 and the lower band of VIPP1 in the pellet increased in the presence of actin filaments. The upper band of VIPP1 appeared to be less well solubilized by Lubrol than the lower band, and only the lower band was pelleted by centrifugation, supporting the results of the Western blot experiments. This was not observed with the envelope proteins IEP37 and OEP21, which did not pellet in any of the samples, suggesting that the behavior of Tic110 and VIPP1 was specific to these proteins and not because of protein entrapment in a meshwork of actin filaments. However, Tic110 and Vipp1 are both inner envelope membrane proteins that would not be expected to interact directly with actin, and their association with actin fil- . Co-sedimentation of solubilized pea envelope membrane proteins with actin filaments. Envelope (Env) membrane proteins were solubilized with 1% Lubrol for 15 min and centrifuged at 100,000 ϫ g for 1 h. Supernatant (30 g protein) was incubated for 1 h at room temperature with 0 -0.8 g of actin filaments, and the actin was pelleted by centrifugation at 100,000 g for 45 min. Negative control, 0.8 g of actin incubated in the absence of envelope membrane proteins and centrifuged at 100,000 g for 45 min. Pelleted proteins were solubilized with SDS and separated by SDS-PAGE on two identical gels; proteins were detected by silver staining of one gel, and the other was used for Western blotting. Antibodies against proteins identified by mass spectrometry were: anti-Toc159, anti-Toc75, anti-Toc34, anti-Tic110, and anti-VIPP1. Antibodies directed against the chloroplast inner and outer membrane proteins IEP37 and OEP21, respectively, were used to check the specificity of the co-sedimentation.
aments would be through interaction with outer envelope membrane proteins.
Toc159 Interacts Directly with Actin Filaments-The previous experiments have pointed strongly to interaction of components of the TOC complex with actin. In an attempt to identify direct interactions of individual proteins with actin, the actin filament co-sedimentation experiment was repeated with E. coli-expressed proteins. Toc159 and Toc34 are the two subunits of the TOC core complex that have extensive cytosolic domains, whereas Toc75 is largely embedded in the membrane (36). Arabidopsis homologs of pea Toc159 and Toc34, called AtToc159 and AtToc33, respectively, were used for the actin filament co-sedimentation experiments. Three constructs for expression of full-length AtToc159 (37) or the GTPase domains of AtToc159 and AtToc33 were used. AtToc33G consists of residues 1-265 of AtToc33 (38), whereas AtToc159G-GST consists of residues 727-1093 of AtToc159 fused to GST (39). The proteins were produced in E. coli, and cells were lysed by sonication. Broken cell suspensions were centrifuged at 100,000 ϫ g for 1 h, and supernatants (5 g of protein) were examined for the presence of the TOC proteins by SDS-PAGE and silver staining (Fig. 5A). AtToc33G and AtToc159G-GST were produced in sufficient quantities to be seen on the silverstained gel (arrows in Fig. 5A) but AtTocC159 could be detected only by Western blot of the same gel with anti-AtToc159 antibodies (Fig. 5B). The Western blot for AtToc159 detected a range of bands between 160 and 85 kDa that are probably the result of degradation of the protein.
E. coli supernatants (50 g protein) containing the TOC proteins were then incubated for 1 h at room temperature in the presence or absence of actin filaments, and the filaments were collected by centrifugation at 100,000 ϫ g for 45 min. Pelleted proteins were separated by SDS-PAGE and silver-stained or transferred to nitrocellulose membrane for Western blot analysis (Fig. 5, A  and B). AtToc33G and AtToc159G-GST were both present in similar amounts in the pellet with or without actin filaments, suggesting that these proteins did not bind specifically to the actin filaments, and the presence of the proteins in the pellet may be because of aggregation at room temperature. In contrast, the polypeptides from the full-length AtToc159 construct were preferentially pelleted from the incubation containing actin filaments, suggesting a direct interaction of Toc159 with actin. To confirm this, co-sedimentation of AtToc159 with different amounts of actin microfilaments was examined (Fig. 5C). The experiment was repeated twice with the same result. Incubations contained AtToc159 (30 g of protein of E. coli supernatant) and different amounts (0 -1.6 g of protein) of actin filaments. Western blotting showed that increased amounts of AtToc159 polypeptides were pelleted with increased amounts of actin filaments. Although small amounts of the AtToc159 polypeptides were pelleted in the absence of actin, the actin-dependent increase in pelleted AtToc159 polypeptides suggests a specific and direct interaction of Toc159 with actin.

DISCUSSION
This work has identified Toc159 as an outer envelope membrane protein that interacts with actin. This interaction appears to be responsible for the co-immunoprecipitation and co-sedimentation of many of the components of the TOC-TIC protein import apparatus with actin. Using mass spectrometry on tryptic peptides of co-immunoprecipitated proteins, we identified three components of the TOC complex (Toc159, Toc75, and Toc34) and five components of the TIC complex (Tic110, Tic55, Tic40, Tic32, and ClpC). Unfortunately, despite considerable amounts of work on the pea chloroplast protein import apparatus, the exact composition of the TOC-TIC complex is not completely understood (17). The principal components of the TOC complex, Toc159, Toc75, and Toc34, were all coimmunoprecipitated with actin. Three of the accepted components of the TIC core complex, Tic110, Tic40, and ClpC, were also detected in the actin immunoprecipitate. However, Tic20 was not examined because it migrated outside the area of the polyacrylamide gel analyzed by mass spectrometry, and similarly, it was not possible to examine the presence of Toc12 and Tic22, which may be involved in linking the TOC and TIC complexes (13). Additional components, such as Tic32, Tic55, and Tic62, which have been proposed to impart redox control on the import pathway (40), were not all detected by mass spec- FIGURE 5. Interaction of actin filaments with Arabidopsis TOC proteins expressed in E. coli. Expression of AtToc33G, AtToc159G-GST, and AtToc159 was induced by 1 mM isopropyl 1-thio-␤-D-galactopyranoside in E. coli BL21. Bacteria were broken by sonication and centrifuged at 100,000 ϫ g for 1 h, and the supernatant was used for co-sedimentation assays with actin microfilaments. A, silver-stained SDS-PAGE gel of actin co-sedimentation assay with TOC proteins. E. coli supernatants (50 g protein) were incubated for 1 h at room temperature with or without actin filaments and centrifuged at 100,000 ϫ g for 45 min. Pellets were solubilized with SDS and loaded on an SDS-12% PAGE gel. Untreated E. coli supernatants (5 g protein) were loaded as positive controls for AtToc33G, AtToc159G-GST, and AtToc159. Arrows show the location of AtToc33G and AtToc159G-GST. B, Western blot of an identical SDS-PAGE gel using antibodies against the A-domain of AtToc159. C, co-sedimentation of E. coli-expressed AtToc159 with increasing amounts of actin filaments. E. coli supernatant (30 g protein) was incubated for 1 h at room temperature with 0 -1.6 g of actin filaments and centrifuged at 100,000 ϫ g for 45 min. Pelleted proteins were solubilized with SDS and separated by SDS-PAGE on two identical gels; proteins were detected by silver staining of one gel, and the other was used for Western blotting with antibodies to AtToc159. E. coli supernatant (3 g of protein) was loaded as a positive control and, after the incubation of 1.6 g of actin in the absence of E. coli, loaded as a negative control.
trometry. Tic62 was not detected, although ferredoxin NADP ϩ reductase, which is proposed to interact with Tic62, was present in the immunoprecipitated material. It is, therefore, difficult to draw definitive conclusions concerning the presence of the extrinsic components of the TIC complex. However, it is possible that these components are recruited by the core TIC complex only under specific conditions and are not always part of the TIC complex.
A direct interaction of Toc159 with actin was demonstrated using co-sedimentation of actin filaments with E. coli-expressed AtToc159. AtToc159 is composed of three different domains; that is, the A, or acidic, domain (amino acid residues 1-758), the G, or GTPase, domain (residues 759 -1092), and the M, or membrane, domain (residues 1093-1503) (37). The interaction with actin appears likely to be through the A-domain; the M-domain is embedded in the chloroplast envelope outer membrane, and AtToc159G-GST, which contains only the G-domain, did not co-sediment with actin filaments. Amino acid residues 139 -828 of AtToc159 are predicted to have weak similarity to the myosin tail domain (Interpro IPR002928) using the SUBA data base (41), and amino acid residues 156 -298 are similar to ProDom domain PD005034, which might interact with actin domain PDA033L7 (42). However, the A-domain of the pea protein exhibits only 20% identity with the Arabidopsis sequence. Although the A-domains of both proteins are acidic, the pea protein has more pronounced repetitive motifs than AtToc159 (32), and the ProDom domain PD005034 is not conserved in pea. Toc159 is particularly susceptible to proteolysis, which removes a large part of the A-domain, resulting in the formation of Toc86, which consists of amino acid residues 638 -1503 of Toc159 (32). Toc86 was detected in the complex immunoprecipitated by anti-actin antibodies, and degraded forms of AtToc159 were co-sedimented with actin filaments, but it is not clear if these truncated forms were able to interact with actin, or whether interactions were with the complete Toc159 protein, which was subsequently degraded during sample preparation. Further experiments will be needed to define more precisely which part of Toc159 interacts with actin.
The identification of the part of Toc159 that interacts with actin is important to establish whether any of the other Toc159 isoforms (such as Toc132 and Toc120) are likely to interact with actin. Toc132 and Toc120 have shorter A-domains than Toc159, and this may affect their ability to bind actin. Although all the Toc159 isoforms are implicated in chloroplast protein import, Toc132 and Toc120 are involved in the import of chloroplast housekeeping proteins, and Toc159 is specialized for the import of photosynthesis proteins (37). For import of photosynthesis proteins, two models have been proposed for preprotein recognition by the TOC complex; that is the "targeting model," where the newly synthesized preprotein is first bound by a free cytosolic form of Toc159, and the "motor model," where the transit peptide is first phosphorylated and then bound to Toc34 associated with the other TOC subunits (17). In support of the first model, Toc159 has been reported to exist in cytosolic and membrane-bound forms (38,43) and is proposed to be the major point of contact for preproteins during the early stages of protein import (35). The soluble form of Toc159 was able to bind preproteins (43,44), and a link to actin might provide a route to favor exchange between the soluble and membrane forms of Toc159. In this way actin might play a role in facilitating chloroplast protein import. Interaction with Toc159 may indicate a hitherto unsuspected role for actin in chloroplast protein import.
Previously, actin has been implicated in plastid movement (45), including light-dependent chloroplast positioning (3,5,46), amyloplast sedimentation in gravitropism (9, 10) and chloroplast inheritance in cell division (47), and in stromule formation and movement (10,48,49). CHUP1 has been identified as an actin and profilin-binding protein required for blue light-dependent chloroplast positioning (5-7), but CHUP1 homologs were not identified among the pea chloroplast envelope membrane proteins co-immunoprecipitated by actin in the work presented here. The failure to identify tryptic peptides of pea CHUP1 by mass spectrometry may be because of a failure to preserve the actin-CHUP1 interaction during chloroplast envelope membrane isolation in low ionic strength media or possibly because the actin epitopes recognized by the antibodies were hidden by the interaction with CHUP1.
A role for the TOC complex in gravitropism has recently been suggested following the isolation of mutants of Toc75-III and Toc132 that modify the effects of the arg1 (altered response to gravity1) mutant in Arabidopsis (50). These two mar (modifiers of arg1) mutants affect gravitropic behavior of Arabidopsis roots only when in the presence of arg1. The mechanism of action of the TOC complex in gravitropism is not known, but it appears unlikely that it involves alteration of the structure or density of the amyloplasts as amyloplasts in the double mutant lines showed wild-type sedimentation behavior (50). The role of the actin cytoskeleton in the behavior of the amyloplasts in the arg1 mutant is currently controversial (50,51), but the possibility of an interaction of the TOC complex with the actin cytoskeleton should not be overlooked.
Myosin has been implicated in gravitropism (11) and in the movement of stromules (14), and myosin XI has been reported to associate with chloroplasts in maize (52). However, tryptic peptides of myosin were not identified in the present study. This may be because of the removal of myosin during the low ionic strength washes of isolated chloroplasts to depolymerize actin filaments. Currently nothing is known of the way in which myosin interacts with the chloroplast envelope membrane.
The interaction of the TOC-TIC complex with actin through Toc159 potentially provides a means of linking the inner and outer envelope membranes during stromule movement. However, it is probable that the TOC-TIC complex exists as such only during the translocation of imported proteins. If the TOC-TIC complex is the only means of linking the inner and outer envelope membranes, then a relationship between the abundance of stromules and rates of plastid protein import might be expected. Currently, there appears to be no evidence for such a relationship. In addition, Toc159 is found predominantly in chloroplast envelope membranes, whereas stromules and stromule movement are more prevalent in non-green plastids (12,13), which are likely to contain Toc132 and Toc120 that possess a shorter A-domain than Toc159. Evidence of a role for the TOC-TIC complex in stromule formation might be obtained by examining stromule formation and movement in mutants of TOC components (17).
VIPP1 was associated with actin in the co-immunoprecipitation and co-sedimentation experiments, although in both experiments a smaller amount of VIPP1 was present in the control incubations in the absence of actin. Two forms of VIPP1 are present in chloroplasts; one localized only in the thylakoid membrane and the other localized in the inner envelope and thylakoid membranes (34). The latter form is enriched in the actin-associated material from the envelope membrane. It is, therefore, improbable that this form of VIPP1 interacts directly with actin. VIPP1 is known to interact with Hsp70B chaperones (53,54), and these chaperones may associate with the stromal face of the TIC complex to support protein folding (19). However, no Hsp70 tryptic peptides were identified by mass spectrometry, so the nature of a possible interaction with the actinassociated TOC-TIC complex is unclear. VIPP1 is involved in thylakoid membrane formation by vesicle formation from the chloroplast inner envelope membrane (34), and the quantity of thylakoid membrane proteins is closely correlated to the amount of VIPP1 in chloroplasts (55), so an interaction with an actin-TOC-TIC complex might play a role in thylakoid formation by helping the import and folding of thylakoid proteins and the budding of thylakoid vesicles from the chloroplast envelope inner membrane.
In summary, this work demonstrates an interaction between actin and Toc159, apparently resulting in the formation of an actin-TOC-TIC complex. The physiological significance of this interaction is unknown, but it may play a role in the import of nuclear-encoded photosynthesis proteins or in different processes such as stromule movement, chloroplast positioning, or gravitropism.