A GTP-driven motor moves proteins across the outer envelope of chloroplasts

Abstract

The translocation of proteins across cellular membranes is a key mechanistic problem for every cell. The preprotein translocon at the chloroplast outer envelope is responsible for precursor protein recognition and translocation across the outer envelope. We have reconstituted the translocation process into proteoliposomes from single subunits or by using the purified translocon. Precursor proteins are recognized by the Toc34 receptor in an initial GTP-dependent process. Translocation across the plane of the membrane then occurs through the Toc75 channel in a GTP-dependent process. Correspondingly, GTP hydrolysis of Toc proteoliposomes is 100-fold enhanced in the presence of preprotein. Complete translocation is demonstrated by processing of the precursor form to the mature form by the stromal processing peptidase and by protease resistance of the imported protein. Molecular chaperones are not involved in this translocation event. We show that Toc159 acts as a GTP-driven motor in a sewing-machine-like mechanism.

Chloroplasts are plant-specific organelles that carry out oxygenic photosynthesis. They are of endosymbiotic origin and are thus surrounded by two membranes, the inner and the outer envelope (1). Most of the protein complement of the organelle is imported posttranslationally by the coordinated action of two translocons, the Tic complex in the inner envelope and the Toc complex in the outer envelope (2–4). The Toc core complex is composed of three subunits, a GTP-dependent preprotein receptor Toc34 (5–7), a second GTP-binding protein Toc159 (5, 8), and the translocation channel Toc75 (9, 10). Toc64 (11) is not as tightly associated with the other three components and can be separated from the Toc core complex by application of the appropriate experimental conditions (12).

The function of single Toc subunits has been studied in some detail. Toc75 expressed in a heterologous system and reconstituted into a lipid bilayer forms a voltage-dependent, cation-selective ion channel (13). Addition of precursor proteins or a peptide, which mimics the transit sequence, results in a strong decrease of conductivity (10, 13), indicating that Toc75 has a low-affinity precursor binding site. The channel diameter of the isolated protein was estimated to be between 20 and 25 Å (10). Toc34 recognizes and binds precursor proteins in the presence of GTP (7). Furthermore, the endogenous GTPase activity of Toc34 is strongly stimulated by precursor proteins (14), suggesting that they function as GTPase-activating factors. GTP binding is inhibited by ATP-dependent phosphorylation of Toc34 by an outer-envelope-localized protein kinase (7, 15). Thus, Toc34 is regulated at least at two different levels. A third regulatory circuit was proposed from the data obtained by analyzing the crystal structure of Toc34 (16). Homodimerization might also influence Toc34 function. However, it should be noted that the cytosolic domain of Toc34 shows strong homology to the G domain of the 86-kDa fragment of Toc159 (5, 6). Therefore, in vivo the heterodimerization (17) could actually be needed for precursor translocation (see below). Toc159 is a second GTP-binding protein involved in translocation of preproteins (5, 6). It is very protease sensitive in isolated organelles and is largely present as an 86-kDa fragment. This 86-kDa fragment still contains the nucleotide and precursor binding sites (5, 6, 18, 19). However, Toc159 is one member of a family of related proteins that also includes Toc132, Toc120, and Toc90 (4). All members of this family have strong homology in the C-terminal part of the protein, including the 86-kDa fragment of Toc159, whereas the so-called N-terminal A domain is less conserved and is not functionally characterized (4).

Similar to the mitochondrial translocase of the outer membrane complex, nothing is known about the driving force to move preproteins across the outer organellar membrane. Translocation across the chloroplastic Tic or the mitochondrial translocase of the inner membrane complex is most likely driven by ATP-dependent action of molecular chaperones (20–22). Here we present a reconstituted system of protein import into proteoliposomes by using Toc subunits that allows the elucidation of single steps of the translocation process in a detailed manner.

Materials and Methods

Materials, Antibodies, and General Procedures.

Plant lipids were obtained from Nutfield Nurseries (Surrey, U.K.), n-decyl-β-maltoside was obtained from Glycon Biochemicals (Luckenwalde, Germany), and octyl glucoside and nucleotides were obtained from Roche Molecular Biochemicals. Protein concentration was determined by using the Bio-Rad protein assay. The production of the used antibodies was described earlier: Toc159 in ref. 8, Toc75 in ref. 13, and Toc34 in ref. 6. GTP hydrolysis was performed and quantified following the established protocol (14).

Isolation of the Toc Components and of the Toc Complex.

Toc75 containing a hexa-histidine tag was expressed and purified as described (23). Escherichia coli harboring the pET21d-full-length-Toc34–6xHis expression vector was grown at 37°C in 2YT media containing 50 mg/liter ampicillin, and expression was induced by inoculation with 1 mM isopropyl-β-d-thiogalactopyranoside. Cells were harvested after 3 h and resuspended to a final concentration of 0.2 mg/ml (wet cell pellet) in 100 mM Na-phosphate/300 mM NaCl/5 mM 2-mercaptoethanol/10 mM MgCl₂/10% glycerol/20 mM dodecylmaltoside (pH 7.0). After cell lysis, nonsoluble cell fragments and inclusion bodies were pelleted at 10,000 × g at 4°C for 20 min. The pellet was washed and finally resuspended in 100 mM Na-phosphate/8 M urea/5 mM 2-mercaptoethanol (pH 8) and incubated for at least 10 min at room temperature. The suspension was centrifuged at 15,000 × g at 4°C for 5 min, and supernatant was passed over preequilibrated Ni-nitrilotriacetic acid material (Qiagen, Hilden, Germany). The column was washed with 5 vol of 100 mM Na-phosphate/6 M urea/5 mM 2-mercaptoethanol/20 mM imidazole (pH 7), and protein was eluted with 100 mM Na-phosphate/6 M urea/5 mM 2-mercaptoethanol/250 mM imidazole (pH 7). Before use, protein was dialyzed into the buffer system indicated. To gain the 86-kDa fragment of Toc159 (Toc159f), 300 μl of purified outer envelope was subjected onto 8% SDS/PAGE, and Toc159f was electroeluted. SDS was removed by addition of Serdolit PAD I (Serva) in the presence of 4 M urea. Toc complex was isolated as described (12).

Reconstitution of the Toc Components.

The isolated complex was reconstituted into liposomes composed of the average lipid composition of the outer envelope (24). For that, the lipid mixture (20-mM lipid concentration) was extruded in 20 mM Hepes (pH 7.6)/200 mM sucrose to generate liposomes (25, 26) and was resolved in octyl glucoside at a ratio of 1:0.5 (wt/wt, lipids/detergent). The Toc complex was complemented with 40 mM dodecylmaltoside (final concentration), added to the lipid mixture, incubated for 1 h at 4°C, and finally dialyzed for 16 h at 4°C against the same buffer not containing detergent. The remaining detergent was removed by incubation for 1 h at 22°C with Biobeads SM-II (Bio-Rad; 25:1, wt/wt, biobeads:initial detergent). The mixture was filtered through gauze, extruded again, and pelleted for 30 min at 50,000 × g through a sucrose cushion of 200 mM. Liposomes were resuspended at the indicated lipid concentration. Toc34, Toc75, and Toc34/Toc75 were reconstituted according to the protocol above with the following exception. Toc34 and Toc75 were complemented by 20 mM dodecylmaltoside and 1 M NSDB-256 (final) before addition to the detergent-solubilized liposomes. For coreconstitution, Toc75 was incubated with the liposomes for 60 min at 25°C before addition of Toc34.

Binding and Import Analysis.

In vitro transcription/translation of the mature (mSSU) and precursor (preSSU) forms of the small subunit of Rubisco by using a rabbit reticulocyte lysate system including the labeling with [³⁵S]methionine was described previously (27). Expression of preSSU-His (pET21d) was initiated in the presence of [³⁵S]methionine by using RTS100-system (Roche Molecular Biochemicals). The proteins were loaded onto Talon material (CLONTECH), washed in the presence of 500 mM NaCl, and eluted in the presence of 50 mM imidazole.

The labeled protein was incubated with empty or proteoliposomes in 20 mM Hepes (pH 7.6)/125 mM NaCl/0.5 mM MgCl₂, and indicated amounts of nucleotide for 5 min at 25°C, followed by competition with N-liposomes [10 mM final lipid concentration, extruded in 20 mM Hepes (pH 7.6)/125 mM NaCl; ref. 25] for 10 min at 25°C. Both liposome species were separated by centrifugation through a cushion containing 200 mM sucrose. Further treatment is described in the figure legends. For investigation of protein translocation, liposomes were loaded with stromal extract containing the stromal signal peptidase purified according to ref. 28 and supplemented by 40 units/ml Apyrase.

Visualization and Quantification of Binding and Translocation.

Binding and translocation of the radioactively labeled proteins were visualized by using a Phospho-Image plate (Fuji). For quantification, the Phospho-Image plate was scanned by using Phospho-Image Reader FLA 5000 (Fuji) and quantified by using an Aida-Image analyzer (Raytest Isotopenmessgeräte, Staubenhard, Germany). Data were presented by using SIGMA PLOT 2000 (SPSS, Chicago) or CORELDRAW 10.0 (Corel, Ottawa).

Results

To better understand how preproteins can move across the outer envelope, we developed an in vitro import system into proteoliposomes. Full-length Toc34 and Toc75 were expressed heterologously in E. coli and purified by metal-chelating affinity chromatography to homogeneity (Fig. 1A; refs. 14 and 23). The Toc core complex, consisting of Toc159 mostly present as the proteolytic fragment of 86 kDa (Toc159f), Toc75, and Toc34, was isolated from the purified outer envelope of pea chloroplasts (Fig. 1A). Toc159f still contains the GTP-binding site (5, 8) and can still bind precursor proteins (18). This protein was eluted from SDS/PAGE as described in Materials and Methods (Fig. 1A). Toc159 (17), Toc75 (9), and Toc34 (5, 6) behave as integral membrane proteins. The Toc subunits show a typical and characteristic proteolytic pattern in intact chloroplasts on treatment with proteases (i.e., Toc159 yields a typical 52-kDa fragment), whereas Toc75 is deeply embedded in the membrane and almost inaccessible to proteases (29). Toc34 is also protease sensitive but yields only a very small protease-resistant fragment. The reconstituted Toc subunits showed the same differential behavior to proteolysis (Fig. 1 B and C). This observation indicates an in situ-like topology of the Toc subunits in proteoliposomes, a prerequisite before binding and translocation properties can be experimentally defined.

Figure 1.

Reconstitution of the subunits of the Toc core complex. (A) The purified Toc complex (Toc) was controlled by SDS/PAGE followed by silver staining (lane 1). Purity of heterologously expressed Toc34 (34H) and Toc75 (75H) containing C-terminal hexa-histidine tags and eluted Toc159f (159_f) was controlled by SDS/PAGE followed by Coomassie blue staining (lanes 2, 3, and 4). (B) The isolated Toc complex was reconstituted into liposomes, diluted to a 2-mM final lipid concentration (B, lanes 1 and 4; C, lane 5), and incubated with 25 ng of trypsin for 5 min at 25°C in a volume of 100 μl in the absence (B, lanes 2 and 5; C, lane 6) or presence (B, lanes 3 and 6) of 0.1% Triton X-100. Reconstitution and proteolysis were followed by immunodecoration by using antibodies against Toc159 (B Left; C, αToc159), Toc34 (B Lower Right; C, αToc34) and Toc75 (B Upper Right; C, αToc75). (C) Toc34 (lanes 3 and 4), Toc75 (lanes 9 and 10), Toc34/Toc75 (lanes 7 and 8), Toc159f/Toc75 (lanes 11 and 12), and Toc159f (lanes 13 and 14) were reconstituted (lanes 3, 7, 9, 11, and 13), and protease resistance was tested (lanes 4, 8, 10, 12, and 14) by the procedure used for B. For comparison, outer-envelope vesicles (lane 1) were also treated with trypsin (lane 2).

In general, proteins are targeted to chloroplasts by an N-terminal cleavable transit peptide, which is both essential and sufficient for recognition and translocation (2–4). Therefore, either the precursor or the mature form of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (preSSU and mSSU, respectively) was incubated with empty liposomes or proteoliposomes containing the entire Toc core complex or Toc75 alone. mSSU bound to empty liposomes and proteoliposomes with similar efficiency, indicating a nonspecific interaction. On the other hand, preSSU interacted strongly and exclusively with the Toc complex and not with empty liposomes. These differences in binding of mSSU and preSSU to empty proteoliposomes might be due to different surface properties (charges and hydrophobicity) of the polypeptides. Toc75, which has a low-affinity binding site for preproteins (30), showed some interaction (Fig. 2A). The Toc complex contains two GTP-binding proteins, namely Toc34 (5, 6) and Toc159 (5, 8). Binding of precursor proteins is nucleotide-dependent and can be used as another criterion of specificity in in vitro experiments. In the presence of GTP, preSSU binding to proteoliposomes is strongly stimulated in assays that contain either Toc34 or Toc159 or the Toc complex (Fig. 2B). In contrast, GTP does not stimulate binding of preSSU to empty liposomes or Toc75 proteoliposomes (Fig. 2B), which is in accordance with the in situ data obtained when using intact chloroplasts (30). preSSU binding to Toc complex-containing liposomes is also supported by the nonhydrolyzable GTP analogue guanosine 5′-[β,γ-imido]triphosphate (GMP-PNP), which is in agreement with the proposed model for Toc34, which binds precursor proteins in its GTP- but not in its GDP-bound form (7, 14). In the absence of GTP, other nucleoside triphosphates, such as ATP or xanthosine triphosphate, can support precursor binding to isolated Toc34 (14). In line with this observation, ATP, but not ADP or GDP, can support binding of preSSU to Toc proteoliposomes (Fig. 2C). Toc34 has endogenous GTPase activity, which is greatly stimulated by precursor proteins. Therefore, we tested the capacity of the Toc complex to hydrolyze GTP (Fig. 2D). In the absence of preprotein or in the presence of the mature form of the precursor protein, mSSU, a basal slow hydrolysis rate was observed (Fig. 2E). In the presence of preSSU, the hydrolysis rate was stimulated >100-fold, demonstrating that preSSU functions as a GTPase-activating protein (23). From the results in Figs. 1 and 2, we conclude that reconstituted proteoliposomes are functionally active and can be used as a bona fide system to define single steps in the import process.

Figure 2.

Biological activity of the Toc proteoliposomes. (A) ³⁵S-labeled mSSU (Top) and preSSU (Middle) were incubated with empty liposomes (lanes 2–4) or proteoliposomes (lanes 5–10; 1-mM final lipid concentration; nomenclature as in Fig. 1) in the presence of 1 mM GTP (lanes 2–10) followed by competition with N-liposomes. Proteoliposomes were diluted to 1-mM final lipid concentration and incubated with 10 ng of trypsin for 5 min at 25°C in a volume of 100 μl in the absence (lanes 3, 6, and 9) or presence (lanes 4, 7, and 10) of 0.1% Triton X-100. One hundred percent of the translation product used is shown for comparison (lane 1). The binding of preSSU (gray bar) and mSSU (white bar) of at least three independent experiments was quantified and compared with the amount of translation product used. (B) Binding of [³⁵S]methionine-labeled preSSU to empty (lanes 2, 3, 13, and 14) or proteoliposomes (lanes 4–11 and 15–18; nomenclatures in Fig. 1) was performed in the absence (lanes 2, 4, 6, 8, 10, 13, 15, and 17) or presence (lanes 3, 5, 7, 9, 11, 14, 16, and 18) of 0.5 mM MgCl₂/1 mM GTP. Ten percent of used translation product is shown for comparison (lanes 1 and 12). (C) Binding of ³⁵S-labeled preSSU to Toc proteoliposomes was initiated in the absence (−Mg) or presence of 0.5 mM MgCl₂ and 1 mM of the indicated nucleotide. The SE of at least three independent experiments are indicated. (D) The hydrolysis of 330 nM radiolabeled [α-³²P]GTP in the absence (lane 1) or presence (lane 2) of the purified Toc complex (5-nM final concentration) was performed. (E) The amount of hydrolyzed GTP at time points indicated are means of at least three independent experiments in the absence (E, ●, solid line) or presence of 100 nM purified mSSU (E, ▵, dash-dot-dot line) or preSSU (E, □, dashed line).

The step after recognition and binding is translocation. Complete translocation into chloroplasts is assessed by two criteria: first, protease-resistant appearance in a membrane-bound compartment and, second, processing of the precursor to the mature form (6, 18). When Toc proteoliposomes were treated with the protease trypsin, protease-protected preSSU was detected in the presence of GTP or a mixture of GTP and ATP but neither in the absence of nucleoside triphosphates nor in the presence of the nonhydrolyzable GTP analogue GMP-PNP (Fig. 3A). preSSU is not protease resistant per se, because it becomes mostly degraded after lysis of the liposomes by Triton X-100 (Fig. 3A, lane 7). In addition, preSSU interacting with empty liposomes is also completely protease accessible. The data indicate that preSSU can be imported into proteoliposomes.

Figure 3.

GTP drives protein translocation through Toc complex. (A) ³⁵S-labeled preSSU was incubated with empty liposomes (lanes 2–4) or proteoliposomes (lanes 5–7, 1 mM final lipid concentration; nomenclature as in Fig. 1) in the presence of 1 mM nucleotide. After competition, proteoliposomes were proteolyzed by trypsin in the absence (lanes 3 and 5) or presence (lanes 4, 7, and 10) of 0.1% Triton X-100. Five percent of the translation product used is shown (lane 1). (B) Outer envelope membranes (lane 1), proteoliposomes containing Toc34 and Toc75 (lane 2) or the Toc complex (lane 3), or the purified radiolabeled preSSU (RTS; lane 4) were subjected to SDS/PAGE followed by immunodecoration by using antibodies against HSP100 (Top), HSP70 (Middle), and CPN60 (Bottom). (C) Binding of the purified preSSU (RTS) to the proteoliposomes containing stromal extract and apyrase (lanes 1–12) was initiated in the presence of 1 mM of the indicated nucleotide. After competition with N-liposomes, liposomes were dissolved by 0.1% Triton X-100 (lanes 2, 4, 6, 8, 10, and 12) and further incubated for 30 min at 25°C. (D) Toc proteoliposomes containing stromal extract and apyrase were incubated with preSSU (RTS) in the presence of 1 mM GMP-PNP (lanes 5–8) or 1 mM GTP (lanes 9 and 10; see C for details). After final incubation for 30 min at 25°C in 100 μl, proteolysis by thermolysin (4 μg/ml final) at 4°C was initiated for 5 (lanes 2, 6, and 10), 10 (lanes 3, 7, and 11), and 20 min (lanes 4, 8, and 12) and stopped by excess of EDTA/EGTA. In lanes 1–4, 50% of the used preSSU was treated as described above. (E) Liposomes were loaded with stromal extract, apyrase, and 150 mM poly-l-glutamic acid/poly-l-aspartic acid. The binding of in vitro-translated preSSU to proteoliposomes was performed in the absence (lanes 3, 4, 7, and 8) or presence (lanes 5, 6, 9, and 10) of 1 mM GTP. For comparison, translation product (lane 1) was incubated for 30 min at 25°C with stromal extract (lane 2).

A chloroplast-soluble extract can be used as a source for the stromal processing peptidase, which converts the precursor into the mature form (28). Proteoliposomes were therefore formed in the presence of a chloroplast-soluble extract and simultaneously in the presence of apyrase, a nucleoside triphosphate-hydrolyzing enzyme. This manipulation should eliminate any nucleoside triphosphates from the inside of the liposomes. To exclude an effect from components of the translation mixture such as chaperones, nucleoside phosphates, or ions on translocation, preSSU containing a carboxy-terminal hexa-histidine tag was purified by metal affinity chromatography before use. Chaperones of the HSP70, HSP100, and chaperonin 60 class were absent from purified preSSU as well as from the different proteoliposomes used in this study (Fig. 3B), except those containing the processing extract (data not shown). Processing-competent Toc proteoliposomes were able to import preSSU only in the presence of GTP but not in the presence of ATP or nonhydrolyzable nucleoside triphosphate analogues (Fig. 3C). About 50% of the radiolabeled protein, which was recovered with the Toc proteoliposomes in the presence of GTP, was in its processed mature form. The processing system was functional under all different conditions because, after lysis of the proteoliposomes by detergent, the nonimported preSSU was converted to mSSU (Fig. 3C). Therefore, the absence of processed SSU (e.g., in the presence of ATP) was in no case due to an inactive processing system (Fig. 3D, lanes 5 and 6). Because of the presence of GTP-hydrolyzing apyrase inside the liposomes and a protease-resistant precursor population in the absence of stromal extract (Fig. 2A), it is extremely likely that GTP is used by a protein on the outside of the liposomes (outer surface).

Furthermore, mSSU is resistant to protease treatment in proteoliposomes (Fig. 3D), indicating that the protein is completely imported into the lumen of the liposome. Precursor protein bound to the surface of the Toc proteoliposomes in the absence of GTP or in the presence of GMP-PNP is highly susceptible to proteolysis, whereas in the presence of GTP it is less protease sensitive (Fig. 3D), probably because of a stable interaction with the Toc complex. Import of preSSU into Toc proteoliposomes is previously unreported and indicates the action of a GTP-driven motor and not a translocation by a pulling force or trapping by ATP-dependent chaperones.

Presequences of both mitochondrial and chloroplast precursor proteins carry an overall positive charge. It has been proposed that translocation across the outer membrane is accomplished by an electrophoretic effect or by a high density of negative charges on the inner leaflet of the membrane (acid bristle theory; ref. 31). To mimic such an effect, we included 75 mM of each polyglutamic acid and polyaspartic acid in the liposomes and measured import in the presence or absence of GTP. The presence of the polyanions could neither support nor stimulate import as measured by the appearance of mSSU (Fig. 3E). Together, these results point to Toc159 as a GTP-dependent motor that drives preproteins across the membrane.

Therefore, the import channel Toc75 was coreconstituted either with Toc34 or Toc159f. Toc75/Toc34 proteoliposomes were unable to support preSSU import in the presence of GTP [Fig. 4A, compare before lysis (uneven numbers) with after lysis (even numbers)]. In contrast, Toc75/Toc159f proteoliposomes promoted translocation of preSSU in the presence of GTP but not in the presence of GDP or the nonhydrolyzable analogue GMP-PNP (Fig. 4C, lanes 5, 7, and 9). Translocated preSSU was converted to mSSU by the stromal processing peptidase (Fig. 4C, lane 5), whereas, in the absence of GTP or in the presence of GDP and GMP-PNP, processing occurred only after lysis of the liposomes and release of the processing peptidase into the supernatant [Fig. 4C, compare lanes 3, 7, and 9 (before lysis) with lanes 4, 8, and 10 (after lysis)]. Proteoliposomes containing only Toc159f were unable to promote translocation across the membrane (Fig. 4B). We conclude that the minimal translocon unit consists of the Toc75 import channel and the GTP-driven motor Toc159.

Figure 4.

Toc159f is sufficient to transport a precursor through the Toc75 pore in a GTP-dependent manner. Toc34/Toc75 (A), Toc159f (B), and Toc159f/Toc75 (C) were reconstituted into liposomes. The different proteoliposomes containing stromal extract and apyrase were then incubated with purified preSSU (RTS; lanes 3–10) in the presence of 1 mM of the indicated nucleotide. After competition with N-liposomes, liposomes were either not dissolved (lanes 3, 5, 7, and 9) or dissolved by 0.1% Triton X-100 (lanes 4, 6, 8, and 10) and further incubated for 30 min at 25°C. For control, 10% of the purified protein used (lane 1) was incubated with stromal extract (lane 2).

Discussion

We have established a system to reconstitute protein translocation into liposomes by using purified translocon subunits and an in vitro-synthesized precursor protein. By using either the purified Toc core complex (consisting of Toc159f, Toc75, and Toc34) or a combination of single subunits, it was possible to deduce a clear sequence of events leading to precursor translocation, which was not previously possible by using intact chloroplasts.

Toc75 is thought to form the translocation channel (10). This idea is consistent with our observation that Toc75 proteoliposomes interact only to a low degree with the precursor in a nucleotide-independent manner. Toc34 can function as a GTP-dependent receptor polypeptide (7, 14), which is in a ratio of 1:1 with Toc75 in the isolated complex (12). The ppi1 mutant in Arabidopsis thaliana, which contains a T-DNA (portion of the tumor-inducing plasmid that is transferred to plant cells) insertion in the Toc34 gene, shows a strong delay in greening and growth; however, the mutant remains viable and fertile, and can grow on soil (32). The same was observed for antisense plants of A. thaliana Toc34 (33). When Toc75 and Toc34 were coreconstituted, precursor proteins were recognized and bound to proteoliposomes with high yield in a GTP-dependent mode. ATP could substitute for GTP (Fig. 2C), however, only with a lower yield and only when preSSU was synthesized in a reticulocyte lysate system and added to the proteoliposomes with this complex mixture. The reticulocyte lysate contains both guanosine and adenosine triphosphates, as well as enzymes such as adenylate kinase and nucleoside diphosphate kinase, which will continuously replenish low, but sufficient, levels of GTP to stimulate binding. However, translocation into the liposomes was still not detected. This observation is consistent with the proposed role of Toc34 as a GTP-regulated receptor of high affinity (7).

Toc159 is an unusual large GTP-binding protein consisting of three distinct domains (4). The most N-terminal A domain is highly variable and not present in all members of the family (4). The specific role of the A domain is still elusive. Toc159 is very protease sensitive and present in isolated chloroplasts or envelope membranes as a carboxyl-terminal, 86-kDa fragment (Toc159f). A further treatment of chloroplasts with protease (thermolysin) results in complete degradation of Toc159f to a 52-kDa fragment (8). Thermolysin-treated chloroplasts still exhibit a residual import capacity (34), which could indicate a second translocation pathway across the outer envelope (35) or a slow basal import that functions by diffusion through the Toc75 channel and chaperone action to move the precursor inside (20–22). Toc159f is, however, still active and can function as a precursor-interacting protein (18). The interaction of Toc159f depends on GTP (Fig. 2), indicating again that it has retained some activity. When Toc159f was coreconstituted with Toc75, translocation of preSSU into proteoliposomes occurred in a way that required GTP hydrolysis. These data establish two things: first, Toc159f has endogenous GTPase activity, and, second, it functions as a molecular motor.

How can we reconcile the proposed role of Toc159 as a receptor with the data presented herein indicating that it works as a translocation motor? All studies (5, 8, 18), including the present one, show that Toc159f binds precursor proteins. A further analysis was not previously possible because the studies were performed with intact organelles. A model was proposed that Toc159 functions as a soluble receptor that shuttles from the cytosol to the chloroplasts (17); however, the experimental support for this idea is currently missing, and other ideas are also feasible (e.g., a soluble pool exists because Toc159 might have a high turnover rate). Import of the soluble form of Toc159 depends on guanosine nucleotide binding (36, 37), which might induce a favorable conformation for translocation, similar to the GTP dependence of Toc34 import (26). Furthermore, the interaction between the G domains of Toc34 and Toc159 also stimulates Toc159 insertion into the outer envelope membrane. This heterodimerization could also play a role in handing over a precursor protein from the initial receptor Toc34 to the motor Toc159. Three independent observations support that Toc159f has activities other than those of a precursor binding protein. First, a Toc159 loss-of-function mutant (ppi2; ref. 38) shows a very pronounced growth phenotype (e.g., albino, no thylakoid formation, reduced photosynthesis, unable to grow on soil; ref. 37). Second, the ratio of Toc159:Toc75:Toc34 in the Toc core complex is ≈1:4:4, pointing more to a catalytic than a static function of Toc159 (12). Third, it has been shown that Toc159f remains in contact with mature sequences of the preprotein during translocation (30). The driving force for the unidirectional movement through the translocon seems to be generated on the cytosolic side by the Toc159 GTPase in a pushing mechanism. This mechanism is visualized as a sewing-machine-like process, as explained below and as portrayed in Fig. 5.

Figure 5.

Model of the translocation process at the outer envelope of chloroplasts. Import is initiated by GTP-dependent recognition of the precursor protein by Toc34 (step 1). GTP hydrolysis by Toc34 induces heterodimerization and transfer of the precursor toward Toc159 (step 2). The subsequent hydrolysis of GTP induces a structural change within the receptor, pushing the precursor into or across the translocation channel (step 3). GDP–GTP exchange of Toc159 might then cause the relaxation (step 4) and initiation of a next round of translocation (step 5), eventually resulting in complete translocation (step 6).

Molecular chaperones or a membrane potential generated by a charge gradient do not seem to be involved in the translocation across the Toc complex. The nucleotide selectivity of translocation clearly argues against a chaperone concept (39) as a driving force but points to a more sophisticated mechanism. We envision a SecA-type mechanism (40, 41), in which parts of the cytosolic domain of Toc159 bind to the precursor in a GTP-dependent manner. This domain is retained in the 86-kDa fragment of Toc159, which is present in the proteoliposomes and which was shown to bind to preproteins (18). Precursor binding causes the activation of the endogenous Toc159 GTPase activity and a conformational change of the proteins. This activity causes movement of a preprotein binding domain, together with its cargo, toward the translocation channel and pushes the precursor into the channel. It remains to be established whether a simultaneous insertion of Toc159 domains into the Toc75 channel occurs. The precursor released from Toc159–GDP may be retained in its position by interaction with the Toc75 channel or a luminal domain of Toc159. Through multiple rounds of preprotein binding and GTP hydrolysis, Toc159 will push the polypeptide across the membrane. The diameter of the Toc75 channel of ≈25 Å (10), together with its demonstrated ability to translocate proteins with a minimal cross-section of 23 Å (42), indicates that the pore is wide enough to accommodate multiple polypeptide stretches. The proposed function of Toc159 as a GTP-dependent motor represents a previously unreported concept and will stimulate further research into the translocation process.

Abbreviations

GMP-PNP

guanosine 5′-[β,γ-imido]triphosphate

mSSU

mature form of the small subunit of Rubisco

preSSU

precursor form of the small subunit of Rubisco