Analysis of the Interactions of Preproteins with the Import Machinery over the Course of Protein Import into Chloroplasts

We have investigated the interactions of two nuclear-encoded preproteins with the chloroplast protein import machinery at three stages in import using a label-transfer crosslinking approach. During energy-independent binding at the outer envelope membrane, preproteins interact with three known components of the outer membrane translocon complex, Toc34, Toc75, and Toc86. Although Toc75 and Toc86 are known to associate with preproteins during import, a role for Toc34 in preprotein binding previously had not been observed. The interaction of Toc34 with preproteins is regulated by the binding, but not hydrolysis of GTP. These data provide the first evidence for a direct role for Toc34 in import, and provide insights into the function of GTP as a regulator of preprotein recognition. Toc75 and Toc86 are the major targets of cross-linking upon insertion of preproteins across the outer envelope membrane, supporting the proposal that both proteins function in translocation at the outer membrane as well as preprotein recognition. The inner membrane proteins, Tic(21) and Tic22, and a previously unidentified protein of 14 kD are the major targets of crosslinking during the late stages in import. These data provide additional support for the roles of these components during protein translocation across the inner membrane. Our results suggest a defined sequence of molecular interactions that result in the transport of nuclear-encoded preproteins from the cytoplasm into the stroma of chloroplasts.     

Characterization of the preprotein translocon at the outer envelope membrane of chloroplasts by blue native PAGE

The preprotein translocon at the outer envelope membrane of chloroplasts (Toc) mediates the recognition and import of nuclear-encoded preproteins into chloroplasts. Two receptor components, Toc159 and Toc34, and the channel Toc75 form the Toc complex. In this study, we have analyzed the molecular architecture and organization of the Toc complex by blue native PAGE (BN-PAGE), which is a high-resolution method for separating membrane protein complexes under non-denaturing conditions. Pea chloroplasts isolated in the presence of a protease inhibitor cocktail were directly solubilized in detergent solution and analyzed by BN-PAGE and size exclusion chromatography. Subsequent immunoblot analyses indicated that the complex composed of Toc75, Toc159 and Toc34 has a molecular mass of 800-1,000 kDa. Limited proteolysis revealed a core of the Toc complex, which was resistant to proteases and detergent treatments. The stoichiometry of the three Toc proteins was calculated as approximately 1 : 3 : 3 between Toc159 : Toc75 : Toc34. We have also analyzed the Toc complex of etioplasts and root plastids. These plastids were found to have essentially the same sized Toc complex as that of the chloroplast.     

Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts

The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.     

Toc Receptor Dimerization Participates in the Initiation of Membrane Translocation during Protein Import into Chloroplasts

The post-translational import of nucleus-encoded preproteins into chloroplasts occurs through multimeric translocons in the outer (Toc) and inner (Tic) membranes. The high fidelity of the protein import process is maintained by specific recognition of the transit peptide of preproteins by the coordinate activities of two homologous GTPase Toc receptors, Toc34 and Toc159. Structural and biochemical studies suggest that dimerization of the Toc receptors functions as a component of the mechanism to control access of preproteins to the membrane translocation channel of the translocon. We show that specific mutations that disrupted receptor dimerization in vitro reduced the rate of protein import in transgenic Arabidopsis compared with the wild type receptor. The mutations did not affect the GTPase activities of the receptors. Interestingly, these mutations did not decrease the initial preprotein binding at the receptors, but they reduced the efficiency of the transition from preprotein binding to membrane translocation. These data indicate that dimerization of receptors has a direct role in protein import and support a hypothesis in which receptor-receptor interactions participate in the initiation of membrane translocation of chloroplast preproteins as part of the molecular mechanism of GTP-regulated protein import.     

Toc64, a new component of the protein translocon of chloroplasts

A subunit of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) of 64 kD is described, Toc64. Toc64 copurifies on sucrose density gradients with the isolated Toc complex. Furthermore, it can be cross-linked in intact chloroplasts to a high molecular weight complex containing both Toc and Tic subunits and a precursor protein. The 0 A cross-linker CuCl(2) yields the reversible formation of disulfide bridge(s) between Toc64 and the established Toc complex subunits in purified outer envelope membranes. Toc64 contains three tetratricopeptide repeat motifs that are exposed at the chloroplast cytosol interface. We propose that Toc64 functions early in preprotein translocation, maybe as a docking protein for cytosolic cofactors of the protein import into chloroplasts.     

The targeting of the atToc159 preprotein receptor to the chloroplast outer membrane is mediated by its GTPase domain and is regulated by GTP

The multimeric translocon at the outer envelope membrane of chloroplasts (Toc) initiates the recognition and import of nuclear-encoded preproteins into chloroplasts. Two Toc GTPases, Toc159 and Toc33/34, mediate preprotein recognition and regulate preprotein translocation. Although these two proteins account for the requirement of GTP hydrolysis for import, the functional significance of GTP binding and hydrolysis by either GTPase has not been defined. A recent study indicates that Toc159 is equally distributed between a soluble cytoplasmic form and a membrane-inserted form, raising the possibility that it might cycle between the cytoplasm and chloroplast as a soluble preprotein receptor. In the present study, we examined the mechanism of targeting and insertion of the Arabidopsis thaliana orthologue of Toc159, atToc159, to chloroplasts. Targeting of atToc159 to the outer envelope membrane is strictly dependent only on guanine nucleotides. Although GTP is not required for initial binding, the productive insertion and assembly of atToc159 into the Toc complex requires its intrinsic GTPase activity. Targeting is mediated by direct binding between the GTPase domain of atToc159 and the homologous GTPase domain of atToc33, the Arabidopsis Toc33/34 orthologue. Our findings demonstrate a role for the coordinate action of the Toc GTPases in assembly of the functional Toc complex at the chloroplast outer envelope membrane.     

Modifications at the A-domain of the chloroplast import receptor Toc159

Two families of GTPases, the Toc34 and Toc159 GTPase families, take on the task of preprotein recognition at the translocon at the outer membrane of chloroplasts (TOC translocon). The major Toc159 family members have highly acidic N-terminal domains (A-domains) that are non-essential and so far have escaped functional characterization. But recently, interest in the role of the A-domain has strongly increased. The new data of three independent studies provide evidence that the Toc159 A-domain (I) participates in preprotein selectivity, (II) has typical features of intrinsically unfolded proteins and (III) is highly phosphorylated and possibly released from the rest of the protein by a proteolytic event. This hints at a complex regulation of A-domain function that is important for the maintenance of the preprotein selectivity at the TOC translocons.Key words: chloroplast, import, Toc159, acidic domain, kinase, protease     

Is chloroplast import of photosynthesis proteins facilitated by an actin-TOC-TIC-VIPP1 complex?

Actin filaments are major components of the cytoskeleton that interact with chloroplast envelope membranes to allow chloroplast positioning and movement, stromule mobility and gravitropism perception. We recently reported that Toc159, a component of the TOC complex of the chloroplast protein import apparatus, interacts directly with actin. The interaction of Toc159 and actin was identified by co-immunoprecipitation and co-sedimentation experiments with detergent-solubilised pea chloroplast envelope membranes. In addition, many of the components of the TOC-TIC protein import apparatus and VIPP1 (vesicle-inducing protein in plastids 1) were identified by mass spectroscopy in the material co-immunoprecipitated with antibodies to actin. Toc159 is the receptor for the import of photosynthesis proteins and VIPP1 is involved in thylakoid membrane formation by inducing vesicle formation from the chloroplast inner envelope membrane, suggesting we may have identified an actin-TOCTIC-VIPP1 complex that may provide a means of channeling cytosolic preproteins to the thylakoid membrane. The interaction of Toc159 with actin may facilitate exchange between the putative soluble and membrane forms of Toc159 and promote the interaction of cytosolic preproteins with the TOC complex.Key words: actin, chloroplast, protein import, TOC complex, TIC complex, VIPP1Actin is a ubiquitous protein of eukaryotic cells and a major component of the cytoskeleton as microfilaments. In plant cells, plastids are closely associated with actin microfilaments.^1,2 A direct interaction of plastids with the actin cytoskeleton has been postulated to anchor chloroplasts at appropriate intracellular positions,³ to support chloroplast light-intensitydependent movement,⁴ to facilitate plastid stromule (stroma-filled tubule) mobility^5,6 and to participate in gravity perception.⁷ The known proteins implicated in plastid-actin interaction are CHUP1 (chloroplast unusual positioning 1), a protein exclusively targeted to the chloroplast outer envelope membrane that is essential for chloroplast anchorage to the plasma membrane,⁸ and myosin XI proteins that play a role in stromule movement9 and in gravitropism.^10,11 Recently, we found that Toc159 also interacts with actin.¹²Toc159 is a component of the TOC complex, which is part of the chloroplast protein translocation apparatus. 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 (reviewed in ref. ¹³). The translocon at the outer envelope membrane of chloroplasts (TOC complex) mediates the initial recognition of preproteins and their translocation across the outer membrane.¹⁴ 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 (ClpC and Hsp70), which assist the transfer of imported proteins^15–17 (Fig. 1).Open in a separate windowFigure 1Schematic diagram of Toc159-actin interactions and the import of photosynthesis proteins. Toc159, linked to actin by its A-domain, recruits a newly synthesized photosynthesis preprotein by its G-domain. Actin filaments facilitate Toc159 movement to the chloroplast outer envelope membrane for integration into the TOC complex. The core TOC complex is formed by Toc159, Toc34 and Toc75. Tic22 acts to facilitate the passage of preproteins across the intermembrane space and interacts with the TIC complex. The core TIC complex is composed of Tic110, Tic20 and/or Tic21, and Tic40. The Tic110 protein recruits stromal molecular chaperone ClpC. On arrival in the stroma, the transit peptide is cleaved by SPP, and other chaperones (Hsp90 or Hsp70) may assist in the folding. VIPP1 interacts with the chaperones and polymerises, inducing chloroplast inner envelope membrane budding, leading to thylakoid formation.The interaction between actin and Toc159 was identified by co-immunoprecipitation and co-sedimentation experiments with detergent-solubilised pea chloroplast envelope membranes, and confirmed with Toc159 expressed in Escherichia coli. In addition, many other components of the TOC-TIC protein import apparatus were co-immunoprecipitated by antibodies to actin and co-sedimented with added F-actin filaments.¹² Using mass spectrometry, we identified the principal components of the TOC complex (Toc159, Toc75 and Toc34) and three accepted components of the TIC core complex (Tic110, Tic40 and ClpC). The presence of Tic20/21 and Tic22 could not be examined because they migrate in the same position on SDS-PAGE as the light chains of antibody molecules but, since they are involved in linking the TOC and TIC complexes,⁶ they may also be part of the complex with actin.The identification of the region of Toc159 that interacts with actin is an important feature to help establish whether any of the other Toc159 isoforms (such as Toc132 and Toc120) are likely to interact with actin. Toc159 family proteins are composed of three different domains: the A (acidic) domain, the G (GTPase) domain and the M (membrane) domain.¹⁸ The interaction of Toc159 with actin appears most likely to be through the A-domain; the G-domain did not co-sediment with actin filaments¹² and the M-domain is embedded in the chloroplast envelope outer membrane and therefore is unlikely to be accessible to 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.¹⁸ For import of photosynthesis proteins, two models have been proposed for preprotein recognition by the TOC complex: 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 in the outer envelope membrane.¹³ In support of the first model, Toc159 has been reported to exist in both cytosolic and membrane-bound forms^19,20 and the soluble form of Toc159 is able to bind preproteins.^20,21 Toc159 is proposed to be the major point of contact for preproteins during the early stages of protein import through its A-domain.²² The interaction of Toc159 with actin might provide a means to favor exchange between the putative soluble and membrane forms of Toc159 and potentially facilitate chloroplast photosynthesis protein import (Fig. 1).Several features of this model require additional experimental evidence. The involvement of a soluble form of Toc159 is highly controversial,¹³ and evidence for a physiological role in vivo is required. Experimental evidence for a facilitating role of the actin cytoskeleton in chloroplast protein import is also required. Does the presence of a basket of actin filaments surrounding the chloroplasts² provide a means of concentrating cytosolic Toc159 in the vicinity of the chloroplasts? Or do actin filaments provide a trackway for movement of Toc159 to or from chloroplasts? Myosin, the motor protein for movement along actin filaments, was not detected in the co-immunoprecipitated complex, but this does not necessarily rule out its involvement.VIPP1 was also identified in the complex with actin. VIPP1 is involved in thylakoid membrane formation by vesicle formation from the chloroplast inner envelope membrane²³ and the quantity of thylakoid membrane proteins is closely correlated to the amount of VIPP1 in chloroplasts.²⁴ VIPP1 is also known to interact with Hsp70 and Hsp90 chaperones^25–27 and these chaperones may associate with the stromal face of the TIC complex to support protein folding.¹⁵ This raises the possibility that an actin-TOC-TIC-VIPP1 complex may facilitate thylakoid formation by channeling the import of thylakoid-located photosynthesis proteins through the chloroplast envelope membrane into vesicles directed to the thylakoid membrane (Fig. 1).Our study of actin-binding proteins in the chloroplast envelope membrane may have provided an initial glimpse at previously unrecognized mechanisms facilitating the import of photosynthesis proteins by chloroplasts. The formation of an actin-TOC-TIC-VIPP1 complex may provide a means of channeling cytosolic preproteins to the thylakoid membrane.     

A 1-Megadalton Translocation Complex Containing Tic20 and Tic21 Mediates Chloroplast Protein Import at the Inner Envelope Membrane

Chloroplast protein import is mediated by two hetero-oligomeric protein complexes, the Tic and Toc translocons, which are located in the inner and outer envelope membranes. At the inner membrane, many Tic components have been identified and characterized, but it remains unclear how these Tic proteins are organized to form a protein-conducting channel or whether a stable Tic core complex that binds translocating preproteins exists. Here, we report the identification of a 1-megadalton (MD) translocation complex as an intermediate during protein translocation across the inner membrane in Arabidopsis thaliana and pea (Pisum sativum). This complex can be detected by blue native PAGE using the mild detergent digitonin without any chemical cross-linkers. The preprotein arrested in the 1-MD complex can be chased into its fully translocated form after a subsequent incubation. While Tic20 and Tic21 appear to be involved in the 1-MD complex, Tic110, a well-characterized Tic component, exists as a distinct entity from the complex. Several lines of evidence suggest that the 1-MD complex functions in between the Toc and Tic110-containing complexes, most likely as a protein-conducting channel at the inner envelope.     

The roles of toc34 and toc75 in targeting the toc159 preprotein receptor to chloroplasts

The Toc complex at the outer envelope of chloroplasts initiates the import of nuclear-encoded preproteins from the cytosol into the organelle. The core of the Toc complex is composed of two receptor GTPases, Toc159 and Toc34, as well as Toc75, a beta-barrel membrane channel. Toc159 is equally distributed between a soluble cytoplasmic form and a membrane-inserted form, suggesting that assembly of the Toc complex is dynamic. In the present study, we used the Arabidopsis thaliana orthologs of Toc159 and Toc34, atToc159 and atToc33, respectively, to investigate the requirements for assembly of the trimeric Toc complex. In addition to its intrinsic GTPase activity, we demonstrate that integration of atToc159 into the Toc complex requires atToc33 GTPase activity. Additionally, we show that the interaction of the two GTPase domains stimulates association of the membrane anchor of atToc159 with the translocon. Finally, we employ reconstituted proteoliposomes to demonstrate that proper insertion of the receptor requires both Toc75 and Toc34. Collectively these data suggest that Toc34 and Toc75 act sequentially to mediate docking and insertion of Toc159 resulting in assembly of the functional translocon.     

The role of GTP binding and hydrolysis at the atToc159 preprotein receptor during protein import into chloroplasts

The majority of nucleus-encoded chloroplast proteins are targeted to the organelle by direct binding to two membrane-bound GTPase receptors, Toc34 and Toc159. The GTPase activities of the receptors are implicated in two key import activities, preprotein binding and driving membrane translocation, but their precise functions have not been defined. We use a combination of in vivo and in vitro approaches to study the role of the Toc159 receptor in the import reaction. We show that atToc159-A864R, a receptor with reduced GTPase activity, can fully complement a lethal insertion mutation in the ATTOC159 gene. Surprisingly, the atToc159-A864R receptor increases the rate of protein import relative to wild-type receptor in isolated chloroplasts by stabilizing the formation of a GTP-dependent preprotein binding intermediate. These data favor a model in which the atToc159 receptor acts as part of a GTP-regulated switch for preprotein recognition at the TOC translocon.     

Chloroplast Hsp93 Directly Binds to Transit Peptides at an Early Stage of the Preprotein Import Process

Three stromal chaperone ATPases, cpHsc70, Hsp90C, and Hsp93, are present in the chloroplast translocon, but none has been shown to directly bind preproteins in vivo during import, so it remains unclear whether any function as a preprotein-translocating motor and whether they have different functions during the import process. Here, using protein crosslinking followed by ionic detergent solubilization, we show that Hsp93 directly binds to the transit peptides of various preproteins undergoing active import into chloroplasts. Hsp93 also binds to the mature region of a preprotein. A time course study of import, followed by coimmunoprecipitation experiments, confirmed that Hsp93 is present in the same complexes as preproteins at an early stage when preproteins are being processed to the mature size. In contrast, cpHsc70 is present in the same complexes as preproteins at both the early stage and a later stage after the transit peptide has been removed, suggesting that cpHsc70, but not Hsp93, is important in translocating processed mature proteins across the envelope.Most chloroplast proteins are encoded by the nuclear genome as higher M_r preproteins that are fully synthesized in the cytosol before being imported into the chloroplast. The import process is initiated by binding of the N-terminal transit peptide of the preprotein to the translocon at the outer envelope membrane of chloroplasts (TOC) complex, in which Toc159 and Toc34 function as receptors and Toc75 is the outer membrane channel. This step is followed by binding of the transit peptide to the translocon at the inner envelope membrane of chloroplasts (TIC) machinery, the central components of which include the Tic20/Tic56/Tic100/Tic214 channel complex and Tic110. Tic110 functions as the stromal receptor for transit peptides and also as a scaffold for tethering other translocon components (for reviews, see Li and Chiu, 2010; Shi and Theg, 2013; Paila et al., 2015). The actual translocation of the bound preproteins across the envelope is powered by hydrolysis of ATP in the stroma (Pain and Blobel, 1987; Theg et al., 1989), and it is therefore assumed that some stromal ATPase motor proteins bind the preproteins as they emerge from the inner membrane and use the energy of ATP hydrolysis to translocate the preproteins across the envelope into the stroma.Three stromal ATPases have been identified in the translocon complex: cpHsc70 (chloroplast heat shock cognate protein 70 kD), Hsp90C (chloroplast heat shock protein 90), and Hsp93/ClpC (93-kD heat shock protein). Hsp93, the first to be identified, belongs to the Hsp100 subfamily of AAA+ proteins (ATPases associated with various cellular activities) and was detected in coimmunoprecipitation experiments in complexes containing other translocon components and preproteins undergoing import (Akita et al., 1997; Nielsen et al., 1997; Chou et al., 2003; Rosano et al., 2011). In Arabidopsis (Arabidopsis thaliana), Hsp93 exists as two isoforms encoded by the genes HSP93III and HSP93V. Removal of the more abundant Hsp93V results in protein import defects, while double knockout of the two genes causes lethality (Constan et al., 2004; Kovacheva et al., 2007; Chu and Li, 2012; Lee et al., 2015). Purified recombinant Hsp93III can bind to the transit peptide of pea (Pisum sativum) ferredoxin-NADP+ reductase in vitro (Rosano et al., 2011). In addition, the N-terminal domain of Hsp93 is critical both for its in vivo functions and its association with chloroplast membranes and Tic110, suggesting that one of the major functions of Hsp93 requires it to be localized at the envelope with Tic110 (Chu and Li, 2012). However, because many prokaryotic Hsp100 family proteins function as the regulatory components of the Clp proteases (Kress et al., 2009; Nishimura and van Wijk, 2015), and, in Arabidopsis, some Clp proteolytic core components have also been found at the envelope fraction, it has been proposed that Hsp93 is involved in degradation of misfolded or damaged proteins at the envelope (Sjögren et al., 2014). However, whether the Clp proteolytic core can form a stable complex with Hsp93 in higher plant chloroplasts remains to be shown.In mitochondria and the endoplasmic reticulum, protein import is driven by the Hsp70 family of proteins. In chloroplasts, accumulating evidence also supports that Hsp70 is important for chloroplast protein import. Purified recombinant Hsp70 can bind in vitro to the transit peptide of the small subunit of RuBP carboxylase preprotein (prRBCS; Ivey et al., 2000). Stromal Hsp70 can be coimmunoprecipitated with preproteins undergoing import and with other translocon components, and mutations resulting in reduced or altered stromal Hsp70 activity cause protein import defects (Shi and Theg, 2010; Su and Li, 2010). Recently, it has been shown, in moss, that increasing the K_m for Hsp70 ATP hydrolysis results in an increased K_m for ATP usage in chloroplast protein import, indicating that stromal Hsp70 is indeed one of the proteins supplying ATP-derived energy to power import (Liu et al., 2014). Finally, stromal Hsp90C has been shown to be part of active translocon complexes in coimmunoprecipitation experiments (Inoue et al., 2013). As further evidence that Hsp90 is important for protein import into chloroplasts, the Hsp90 ATPase activity inhibitor radicicol reversibly inhibits the import of preproteins into chloroplasts (Inoue et al., 2013).Presence of the three ATPases in the translocon was demonstrated by coimmunoprecipitation after solubilization of chloroplast membranes under conditions that preserve the large membrane protein complexes, either by solubilization with nonionic detergents or by treating chloroplasts with crosslinkers that link all proteins in a complex together (Akita et al., 1997; Nielsen et al., 1997; Shi and Theg, 2010; Su and Li, 2010; Inoue et al., 2013). These complexes contain translocon components that directly bind to preproteins, and also other proteins that are associated with these translocon components but have no direct contacts with the preproteins. For example, Nielsen et al. (1997) demonstrated the presence of Hsp93 in the translocon by binding of prRBCS to isolated pea chloroplasts and then solubilization of chloroplast membranes with the nonionic detergent decylmaltoside. Under these conditions, an anti-Hsp93 antibody specifically immunoprecipitated Hsp93 together with Toc159, Toc75, Toc34, Tic110, and prRBCS (Nielsen et al., 1997). The result showed that Hsp93 is in the same complexes with these proteins but did not provide information whether Hsp93 directly binds to them. It is possible that Hsp93 only has direct contacts with, for example, Tic110, which then binds to prRBCS. Direct binding, in particular to the transit peptide region, would provide strong evidence that an ATPase functions as a protein translocating motor, rather than in assisting the assembly of other translocon components or in the folding or degradation of imported proteins. Furthermore, if all three ATPases were found to be involved in preprotein translocation, it would be important to understand how they work together; for example, whether they preferentially bind different preproteins, bind to different regions of a preprotein, or act at different stages of the import process.Here, we examined whether Hsp93 can directly bind to preproteins undergoing import into chloroplasts, and compared the timing of the binding of Hsp93 and cpHsc70 to the preproteins. We used isolated pea chloroplasts, rather than isolated Arabidopsis chloroplasts, because pea chloroplasts exhibit more robust import ability (Fitzpatrick and Keegstra, 2001). Various crosslinkers that react with cysteines were then used to achieve more specific crosslinkings, followed by solubilization with the ionic detergent lithium dodecyl sulfate (LDS) to thoroughly solubilize chloroplast membranes and to disrupt noncovalent protein-protein interactions. Our results show that Hsp93 directly binds to preproteins undergoing import. Import time course experiments further revealed that Hsp93 functions primarily during the early stage of import, whereas cpHsc70 associates with substrates being imported at both the early stage and a later stage after transit peptide removal.     

Mechanism of protein import across the chloroplast envelope

The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes.     

Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor, Toc34

Toc34 is a transmembrane protein located in the outer envelope membrane of chloroplasts and involved in transit peptide recognition. The cytosolic region of Toc34 reveals 34% alpha-helical and 26% beta-strand structure and is stabilized by intramolecular electrostatic interaction. Toc34 binds both chloroplast preproteins and isolated transit peptides in a guanosine triphosphate- (GTP-) dependent mechanism. In this study we demonstrate that the soluble, cytosolic domain of Toc34 (Toc34deltaTM) functions as receptor in vitro and is capable to compete with the import of the preprotein of the small subunit (preSSU) of ribulose-1,5-bisphosphate carboxylase-oxygenase into chloroplasts in a GTP-dependent manner. We have developed a biosensor assay to study the interaction of Toc34deltaTM with purified preproteins and transit peptides. The results are compared with the interactions of both a full-size preprotein and the transit peptide of preSSU with the translocon of the outer envelope of chloroplasts (Toc complex) in situ. Several mutants of the transit peptide of preSSU were evaluated to identify amino acid segments that are specifically recognized by Toc34. We present a model of how Toc34 may recognize the transit peptide and discuss how this interaction may facilitate interaction and translocation of preproteins via the Toc complex in vivo.     

Redox-regulation of protein import into chloroplasts and mitochondria: Similarities and differences

Redox signals play important roles in many developmental and metabolic processes, in particular in chloroplasts and mitochondria. Furthermore, redox reactions are crucial for protein folding via the formation of inter- or intramolecular disulfide bridges. Recently, redox signals were described to be additionally involved in regulation of protein import: in mitochondria, a disulfide relay system mediates retention of cystein-rich proteins in the intermembrane space by oxidizing them. Two essential proteins, the redox-activated receptor Mia40 and the sulfhydryl oxidase Erv1 participate in this pathway. In chloroplasts, it becomes apparent that protein import is affected by redox signals on both the outer and inner envelope: at the level of the Toc complex (translocon at the outer envelope of chloroplasts), the formation/reduction of disulfide bridges between the Toc components has a strong influence on import yield. Moreover, the stromal metabolic redox state seems to be sensed by the Tic complex (translocon at the inner envelope of chloroplasts) that is able to adjust translocation efficiency of a subgroup of redox-related preproteins accordingly. This review summarizes the current knowledge of these redox-regulatory pathways and focuses on similarities and differences between chloroplasts and mitochondria.Key words: protein import, chloroplasts, mitochondria, redox-regulation, disulfide bridges, NADP(H), Toc, Tic, Tom     

Initial binding of preproteins involving the Toc159 receptor can be bypassed during protein import into chloroplasts

Two integral outer envelope GTPases, Toc34 and Toc86, are proposed to regulate the recognition and translocation of nuclear-encoded preproteins during the early stages of protein import into chloroplasts. Defining the precise roles of Toc86 and Toc34 has been complicated by the inability to distinguish their GTPase activities. Furthermore, the assignment of Toc86 function is rendered equivocal by recent reports suggesting that the standard protocol for the isolation of chloroplasts results in significant proteolysis of Toc86 (B. Bolter, T. May, J. Soll [1998] FEBS Lett 441: 59-62; G. Schatz [1998] Nature 395: 439-440). We demonstrate that Toc86 corresponds to a native protein of 159 kD in pea (Pisum sativum), designated Toc159. We take advantage of the proteolytic sensitivity of Toc159 to selectively remove its 100-kD cytoplasmic GTPase domain and thereby distinguish its activities from other import components. Proteolysis eliminates detectable binding of preproteins at the chloroplast surface, which is consistent with the proposed role of Toc159 as a receptor component. Remarkably, preprotein translocation across the outer membrane can occur in the absence of the Toc159 cytoplasmic domain, suggesting that binding can be bypassed. Translocation remains sensitive to GTP analogs in the absence of the Toc159 GTP-binding domain, providing evidence that Toc34 plays a key role in the regulation of translocation by GTP.     

Arabidopsis tic110 is essential for the assembly and function of the protein import machinery of plastids

The translocon at the inner envelope membrane of chloroplasts (Tic) plays a central role in plastid biogenesis by coordinating the sorting of nucleus-encoded preproteins across the inner membrane and coordinating the interactions of preproteins with the processing and folding machineries of the stroma. Despite these activities, the precise roles of known Tic proteins in translocation, sorting, and preprotein maturation have not been defined. In this report, we examine the in vivo function of a major Tic component, Tic110. We demonstrate that Arabidopsis thaliana Tic110 (atTic110) is essential for plastid biogenesis and plant viability. The downregulation of atTic110 expression results in the reduced accumulation of a wide variety of plastid proteins. The expression of dominant negative mutants of atTic110 disrupts assembly of Tic complexes and the translocation of preproteins across the inner envelope membrane. Together, these data suggest that Tic110 plays a general role in the import of nuclear-encoded preproteins as a common component of Tic complexes.