Article |
Address correspondence to Felix Kessler, Institute of Plant Sciences, Plant Physiology and Biochemistry Group, ETH Zürich, Universitätstrasse 2, 8092 Zürich, Switzerland. Tel.: 41-1-632-60-15. Fax: 41-1-632-10-84. E-mail: felix.kessler{at}ipw.biol.ethz.ch
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Abstract |
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Key Words: chloroplasts; protein import; translocon; complex assembly; soluble receptor
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Introduction |
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Toc159 has a tripartite structure, consisting of the NH2-terminal acidic domain (A domain), the G domain, and the COOH-terminal membrane domain (M domain) (Bauer et al., 2000). The G domain binds specifically to GTP (Kessler et al., 1994), but its function and that of the A domain are unknown. The M domain anchors Toc159 in the outer chloroplast membrane (Hirsch et al., 1994), but lacks conventional hydrophobic membrane-spanning sequences. In contrast, Toc34 is inserted into the outer membrane by a COOH-terminal hydrophobic transmembrane stub (Chen and Schnell, 1997; Li and Chen, 1997; May and Soll, 1998; Tsai et al., 1999).
In this study we have analyzed the Arabidopsis homologue of Toc159, atToc159. Analysis of the ppi2 mutant lacking atToc159 has demonstrated an essential function of this protein in chloroplast biogenesis (Bauer et al., 2000), but it has not yet been characterized biochemically. We show that at the chloroplast surface, atToc159 is in a complex with atToc33 and atToc75, homologues of pea Toc34 and Toc75, respectively. Additionally, we demonstrate that in contrast to atToc34 and Toc75, a large soluble pool of atToc159 exists. In a pull-down assay, synthetically synthesized, soluble atToc159 was shown to bind to a hexahistidinyl-tagged, soluble form of atToc33, atToc331265, lacking the COOH-terminal transmembrane stub. Furthermore, the addition of atToc331265 inhibited outer membrane insertion of atToc159, likely in a competitive manner. Thus, the results indicate that atToc33 may function in the assembly of soluble atToc159 into the outer membrane and the Toc complex. Given its known function as an import receptor, these findings suggest that atToc159 may play a role in targeting cytosolic precursors to the chloroplast surface.
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Results |
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Immunolocalization of atToc159
Immunofluorescence experiments using antibodies against atToc1591740 on isolated Arabidopsis protoplasts followed by incubation with FITC-coupled secondary antibodies were done to determine the localization of atToc159 at the cellular level (Fig. 2
A). By confocal laser scanning microscopy, the atToc1591740 antibodies gave a circular, rim-like fluorescence pattern consistent with a localization at the chloroplast periphery (Fig. 2 A, panel 3). A merge (Fig. 2 A, panel 2) between the transmission microscopic image (Fig. 2 A, panel 1) and the fluorescence microscopic image (Fig. 2 A, panel 3) confirmed that the rim-like fluorescence indeed coincided with the chloroplast periphery. In addition, fluorescence occurred in areas between chloroplasts (Fig. 2, arrows), suggesting that atToc159 may also be present in the cytosol. In comparison, antibodies against atToc75141397 gave a rim-like fluorescence (Fig. 2 B, panels 18) comparable to that of the atToc1591740 antibodies, but fluorescence was absent from areas intervening between chloroplasts (Fig. 2 B, panels 4 and 8), consistent with the expected, exclusive localization of atToc75 at the chloroplast surface. Control experiments using only the FITC-coupled secondary antibodies gave only low levels of background fluorescence (Fig. 2 C, panel 2).
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Discussion |
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Toc159 has been biochemically characterized in pea and shown to be essential for chloroplast biogenesis in vivo, but questions regarding its subcellular distribution and association with the Toc complex in Arabidopsis remained open. Here we show that atToc159 exists in an integral membrane, as well as a soluble form. Protease sensitivity and resistance to alkaline extraction confirm that atToc159 is an integral, surface-exposed component of the chloroplast outer membrane (Fig. 1 A). An Arabidopsis Toc complex was identified and isolated in immunopurification experiments using solubilized chloroplast membranes. The complex contained atToc159, atToc33, and atToc75 (Fig. 1 B). Therefore, an Arabidopsis Toc complex exists, homologous in composition to that of pea (Schnell et al., 1994; Ma et al., 1996). However, it remains to be seen whether the Arabidopsis complex is trimeric, as has been demonstrated for the pea complex (Ma et al., 1996). Whereas in pea Toc75, Toc34, and Toc159 appear to be unique, the Arabidopsis homologues of Toc159 and Toc34 are encoded by a small gene family: three genes (atToc159, atToc132, and atToc120) encoding homologues of Toc159 (Bauer et al., 2000), and two genes (atToc34 and atToc33) encoding homologues of Toc34 (Jarvis et al., 1998). Future studies will reveal whether the members of this family act concertedly in a large supercomplex or whether they form a variety of trimeric Toc complexes. Taking into account that atToc75 is likely unique also in Arabidopsis, a combinatorial scope of six different Toc complexes may function in various tissues and plastid types (Cline, 2000).
Previous subcellular localization studies relied entirely on isolated pea chloroplasts (Hirsch et al., 1994; Kessler et al., 1994). In this study, we assessed the cellular distribution of atToc159 using specific antibodies in immunofluorescence (Fig. 2) and Western blotting experiments (Figs. 1 and 3). AtToc159 clearly located to the chloroplast surface by immunofluorescence (Fig. 2), but fluorescence also occasionally occurred in spaces intervening between chloroplasts, suggesting that atToc159 may be present in the cytosol (Fig. 2 A). Such cytosolic fluorescence was noticeably absent in immunofluorescence experiments using specific antibodies against atToc75 (Fig. 2 B). Western blot analysis of soluble and chloroplast proteins revealed partitioning of atToc159, but not of atToc75, between the two fractions (Fig. 3 A); thus, atToc159 exists in an unanticipated soluble form. Identical findings were made for pea Toc159 (Fig. 3 B). The identification of a soluble form of the well-characterized pea Toc159 underscores the significance of the finding.
Earlier targeting experiments indicated that targeting and insertion of atToc159 rely on protease-sensitive receptor components at the chloroplast surface (Muckel and Soll, 1996). This protease sensitivity, as well as the association of atToc159 with atToc33 in the Arabidopsis complex (Fig. 1 B) and with Toc34 upon insertion into isolated pea chloroplasts (Bauer et al., 2000), suggests that atToc33 may contribute to the proposed receptor. Therefore, we tested the ability of atToc159 to bind to atToc33 using a soluble phase binding assay (Fig. 4). In the assay, [35S] atToc159 bound to atToc331265, a soluble version of the protein lacking the COOH-terminal transmembrane stub (Fig. 4 A), in a concentration-dependent fashion. The result suggests that the soluble phase binding assay may have reconstituted a surface recognition step in the membrane assembly of atToc159. Therefore, we tested the ability of [35S] atToc159 to insert into the outer chloroplast membrane in the presence of atToc331265 using resistance to alkaline extraction as the criterion of insertion (Fig. 5). Increasing concentrations of atToc331265 inhibited insertion of [35S]atToc159 by up to 60%. It is likely that the added soluble atToc331265 competed with endogenous atToc33 at the chloroplast surface for binding of [35S] atToc159, thereby interfering with its subsequent insertion. Thus, binding to atToc33 at the cytosolic face of the chloroplast may represent a step in atToc159 recognition. It remains to be determined whether insertion of atToc159 requires the activity of additional outer membrane components. Nevertheless, we conclude that atToc33 may serve at least as part of a receptor complex in the recognition and insertion of atToc159. As both atToc159 and atToc33 are GTP-binding proteins, the possibility of GTP-dependent regulation of their interactions arises. GTP-regulated, homotypic interactions between GTP-binding proteins are not unprecedented. Notably, targeting of SRP to the SRP receptor involves such a mechanism (Walter and Johnson, 1994).
The data available on pea Toc159 and atToc159 summarily suggest that the proteins may function as mobile import receptors partitioning between the cytosol and the Toc complex. However, in vitro studies using isolated chloroplasts do not indicate a requirement for an added plant-specific soluble receptor, with the possible exception of a guidance complex (May and Soll, 2000) stimulating the import of unconventional phosphorylated precursor proteins (Waegemann and Soll, 1996). Moreover, isolated chloroplasts import a chemically denatured precursor in the complete absence of added soluble proteins (Pilon et al., 1992). Conceivably, atToc159 or other proteins present in isolated chloroplasts may serve as soluble import receptors upon release from the chloroplast surface. However, import of precursors into isolated chloroplasts still functions after proteolytic degradation of Toc159 to its 52-kD M domain (Chen et al., 2000), but precursor binding to the chloroplast surface was markedly reduced after degradation of Toc159 to the M domain (Chen et al., 2000). Although Toc159 can be bypassed in vitro, it appears unlikely that the M domain of atToc159 alone would be functional in vivo. Therefore, the strict requirement for atToc159 as a soluble and/or membrane-bound import receptor may be limited to the in vivo system.
This study has uncovered the existence of a soluble form of atToc159 and its targeting to the chloroplast surface. The occurrence of atToc159 in both a soluble and integral membrane form suggests that it may be able to switch. The switch would require that atToc159 be able not only to insert into, but also to dissociate from the outer membrane. Such a scenario implies conformational changes in atToc159, possibly induced by GTP-binding and hydrolysis. However, regulatory factors affecting membrane association of atToc159 are not presently known. AtToc159 may serve as a soluble import receptor, activated and targeted to the chloroplast surface upon precursor binding. Another possibility is that functional import machineries are assembled from pools of soluble components (i.e., atToc159, atToc120, and atToc132) in a regulated fashion. In certain aspects either scenario resembles the SRP system, but also the Sec pathway at the bacterial plasma membrane. SecA energizes protein translocation at the bacterial plasma membrane through ATP-dependent cycles of membrane insertion and deinsertion, thereby driving a precursor across the membrane (Driessen et al., 1998). The similarities between the SRP system and the Toc complex pertaining to the targeting mechanism have been discussed above. Assembly of the Toc complex may use a hybrid mechanism involving functional principles of both the SRP and Sec pathways.
In conclusion, the results of our study outline a possible function of atToc159 as a mobile transit sequence receptor in chloroplast protein import. In the future, precursor interaction and the role of GTP in the targeting of atToc159 to the outer membrane and the assembly of the Toc complex remain to be determined.
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Materials and methods |
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To produce antibodies against atToc75, a PCR product corresponding to amino acids 141397 was amplified from Arabidopsis genomic DNA using primers incorporating an NcoI site at the 5' end and six histidine codons, and a SacI site at the 3' end. The resulting DNA fragment was ligated into the NcoI-SacI sites of pET21d, resulting in pET21d-atToc75141397. The plasmid pET21d-atToc75141397 was transformed into E. coli BL21(DE3) cells, and atToc75141397 was overexpressed and purified under denaturing conditions using Ni-NTA.
For in vitro synthesis of proteins in the presence of [35S]methionine, a reticulocyte-based coupled transcription/translation system (Promega) was used.
Antibodies
Purified atToc75141397 was used to produce rabbit antibodies (Eurogentec). Antibodies were affinity purified using the antigen coupled to Affi-Gel 10 (Bio-Rad Laboratories) according to the supplier's recommendation. The antibodies against atToc159 have been described previously (Bauer et al., 2000). Antibodies against atToc33 and atToc34, as well as psToc159, psToc75, and psToc34 (Ma et al., 1996), were a gift from Dr. D.J. Schnell (University of Massachusetts, Amherst, MA). Antibodies against cytosolic Arabidopsis geranylgeranyl transferase were a gift from Dr. W. Gruissem (Institute of Plant Sciences, ETH, Zurich, Switzerland). Antibodies against chlorophyll a/b binding protein were a gift from the Dr. K. Apel (Institute of Plant Sciences, ETH).
Isolation of proteins from total plant extracts, soluble fractions, and chloroplasts
Seeds of plants (A. thaliana ecotype Columbia 2) used for extraction of soluble proteins or isolation of chloroplasts were vernalized at 4°C. Subsequently, plants were grown on 0.8% agar plates (0.5x Murashige-Skoog medium, 1% wt/vol sucrose) under long-day conditions (16 h light, 8 h dark) or on soil under short-day conditions (8 h light, 16 h dark). Total soluble protein and intact chloroplasts were isolated from leaves from 34-wk-old Arabidopsis plants. Leaves were harvested and floated on ice-cold tap water for 2030 min before homogenization in HB buffer (450 mM sorbitol, 20 mM Tricine/KOH, pH 8.4, 10 mM EDTA, 10 mM NaHCO3, 5 mM Na-ascorbate, 1 mM MnCl2, 1 mM PMSF) using a Waring blender. The homogenized plant material was filtered through two layers of cheese cloth and one layer of Miracloth (Calbiochem) and centrifuged for 2 min at 500 g at 4°C. The supernatant was removed and used for isolation of total soluble protein as described below. The pellet was resuspended in 1 ml of RB buffer (300 mM sorbitol, 20 mM Tricine/KOH, pH 7.6, 5 mM MgCl2, 2.5 mM EDTA), loaded on a Percoll step gradient (40% vol/vol and 85% vol/vol; Amersham Pharmacia Biotech) in RB buffer, and centrifuged for 10 min at 2,500 g at 4°C in a swing-out rotor. Intact chloroplasts were removed from the 40/85% Percoll interphase, washed with 10 vol RB buffer, and used for immunoblot analysis. The supernatant of the initial homogenate (see above) was supplemented with 0.1% vol/vol protease inhibitor cocktail (Sigma-Aldrich) and was cleared by centrifugation for 2 h at 100,000 g at 4°C. Protein was extracted from the supernatant by CHCl3/MeOH precipitation (Wessel and Flügge, 1984). Cellular equivalents were calculated from the amounts of protein and/or chlorophyll contained in the total extracts, soluble and chloroplast fractions, respectively (unpublished data).
Immunopurification of the Arabidopsis Toc complex
To immunopurify the Arabidopsis Toc complex, chloroplasts were lysed under hypertonic conditions (0.6 M sucrose, TES [50 mM Tricine, pH 7.5, 10 mM EDTA containing 0.1% vol/vol protease inhibitor cocktail]) using a Dounce homogenizer. The lysate was separated into membrane and soluble fractions by centrifugation at 40,000 g for 20 min. The total membranes were solubilized in TES, 2% Triton X-100, and cleared by centrifugation at 100,000 g for 10 min. The cleared solubilisate was applied to a column containing an anti-atToc33 IgG fraction coupled to Sepharose. Subsequently, the column was washed with TES, 0.5% Triton X-100, and eluted with 0.2 M glycine, pH 2.2, 0.5% Triton X-100.
Western blot analysis was done according to standard protocols. Horseradish peroxidasecoupled secondary antibodies and enhanced chemiluminescence reaction were used for immunodetection as recommended by the supplier (Roche).
Immunolocalization in protoplasts and chloroplast protein insertion experiments
Immunolocalization using Arabidopsis protoplasts was done according to protocols at http://www.arabidopsis.org/cshl-course/7-gene_expression.html. Affinity-purified antibodies against atToc1591740 and atToc75141397 were used at 5 µg/ml. FITC-labeled secondary antibodies (Pierce Chemical Co.) were used at a dilution of 1:50. Protoplasts were then analyzed by confocal laser scanning microscopy (Leica DM IRBE microscope and Leica TCS SP laser) at excitation and emission wavelengths of 488 and 520 nm, respectively.
Insertion experiments (using isolated chloroplasts corresponding to 15 µg of chlorophyll) and alkaline carbonate extractions were done as described previously (Chen and Schnell, 1997).
Soluble phase binding assay
Hexahistidinyl-tagged atToc331265 was purified as described above and incubated, at the concentrations indicated, with 20 µl of reticulocyte lysate containing [35S] atToc159 in import buffer (final concentrations: 50 mM Hepes, pH 7.7, 330 mM sorbitol, 2 mM Mg[OAc]2, 40 mM KOAc, 0.4 mM GTP, 4 mM ATP, 0.1% Triton X-100 wt/vol) in a final volume of 50 µl and incubated on ice for 5 min. 10 µl of packed Ni-NTA was added, and the incubation continued for 30 min at 4°C under constant mixing to reisolate the hexahistidinyl-tagged atToc331265. The resin was washed three times with 400 µl of import buffer, once with import buffer containing 40 mM imidazole, and then eluted with import buffer containing 200 mM imidazole. Eluates were analyzed by SDS-PAGE and Coomassie blue staining followed by autoradiography.
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Footnotes |
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* Abbreviations used in this paper: A domain, acidic domain; G domain, GTP-binding domain; M domain, membrane domain; Ni-NTA, nickel-nitrilotriacetic acidagarose; SRP, signal recognition particle; Tic, translocon at the inner chloroplast membrane; Toc, translocon at the outer chloroplast membrane.
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Acknowledgments |
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The research was supported by grants from the Swiss National Science Foundation and the Swiss Federal Institute of Technology to F. Kessler. M. Hohwy was supported by a long term fellowship from the Human Frontier Science Program Organization.
Submitted: 5 April 2001
Revised: 23 May 2001
Accepted: 4 June 2001
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References |
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