JXB Advance Access originally published online on August 8, 2005
Journal of Experimental Botany 2005 56(419):2287-2320; doi:10.1093/jxb/eri243
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REVIEW ARTICLE |
Recognition and envelope translocation of chloroplast preproteins
Department of Biology, University of Leicester, University Road, Leicester LE1 7RH, UK
* To whom correspondence should be addressed. Fax: +44 (0)116 252 3330. E-mail: rpj3{at}le.ac.uk
Received 14 March 2005; Accepted 7 June 2005
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Abstract |
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Abstract
IntroductionPlastids are a diverse group of plant organelles that perform essential functions including important steps in many biosynthetic pathways. Chloroplasts are the best characterized type of plastid, and constitute the site of oxygenic photosynthesis in plants, a process essential to all higher life forms. It is well established that the majority (>90%) of chloroplast proteins are nucleus-encoded and must be post-translationally imported into these envelope-bound compartments. Most nucleus-encoded chloroplast proteins are translated in precursor form on cytosolic ribosomes, targeted to the chloroplast surface, and then imported across the double-membrane envelope by translocons in the outer and inner envelope membranes of the chloroplast, termed TOC and TIC, respectively. Recently, significant progress has been made in our understanding of how proteins are targeted to the chloroplast surface and translocated across the chloroplast envelope into the stroma. Evidence suggesting the existence of multiple import pathways at the outer envelope membrane for different classes of precursor proteins has been presented. These pathways appear to utilize similar TOC complexes equipped with different combinations of homologous GTPase receptors, providing preprotein recognition specificity.
Key words: Chloroplast, chloroplast protein import, membrane translocation, organelle biology, plastid, protein targeting, Tic, Toc, translocon
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Introduction |
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Plastids, their evolution and functions
Chloroplasts belong to a family of organelles known as plastids. They are ubiquitous in plants and algae, and constitute the site of photosynthesis in these photoautotrophic organisms. Oxygenic photosynthesis converts absorbed sunlight energy into a usable form of chemical energy, and is essential for plant growth and development. The redox reactions associated with photosynthesis lead to the generation of oxygen from water, and provide the energy necessary for the fixation of carbon dioxide. Photosynthesis is the most important producer of oxygen and organic matter on earth, and therefore provides the fuels essential for all higher forms of life (Nelson and Ben-Shem, 2004
).
Different types of non-photosynthetic plastids can be found in non-green tissues of plants (e.g. leucoplasts and chromoplasts), and these usually carry out specialized biosynthetic and storage functions (Whatley, 1978; Mullet, 1988). For example, leucoplasts normally accumulate lipids, starch, or proteins and can be found in petals and roots, whereas chromoplasts accumulate carotenoid pigments that provide colour in fruits, flowers, and senescing leaves. However, all plastids are separate cellular compartments containing their own genetic machinery and play essential roles in numerous biosynthetic pathways. These include steps in amino acid, lipid, nucleotide, hormone, vitamin, and secondary metabolite biosynthesis (Leister, 2003).
It is generally accepted that plastids originated from an endosymbiotic event in which a eukaryotic cell engulfed a photosynthetic prokaryote, an ancestor of today's cyanobacteria (Margulis, 1970; McFadden, 2001). These organisms thereafter developed a symbiotic relationship in which the photosynthetic prokaryote provided energy to the host, possibly in exchange for essential nutrients and protection. Through evolution, the endosymbiont lost its autonomy and came under the genetic control of the host cell. This occurred through the process of gene transfer, in which genes from the prokaryotic genome were transferred to the nuclear genome of the eukaryotic host cell (for a review, see Timmis et al., 2004). The mechanism by which gene transfer occurs is largely unknown, but transfer of plastid DNA to the nucleus has been experimentally demonstrated to occur at a relatively high frequency (Huang et al., 2003).
Gene transfer allowed the integration of plastid development into the developmental programme of plants. Chloroplasts, as well as other types of mature plastids, generally develop from undifferentiated plastids termed proplastids. Proplastids are found in undifferentiated meristematic cells and undergo division in parallel with cell division in such a way that a constant number of proplastids are found in all cells. Indeed, new plastids are not assembled de novo within plant cells but arise only from the division of pre-existing plastids (for a review, see Aldridge et al., 2005). As meristematic cells differentiate into different cell types, similarly, proplastids differentiate into different plastid types. For example, in leaves, as meristem cells develop into mesophyll cells, proplastids differentiate into chloroplasts, a process characterized by the rapid accumulation of photosynthetic proteins and the green chlorophyll pigments used to absorb light energy.
The processes of plastid division, differentiation and maturation are largely under the control of the plant cell nucleus. The cell exerts control over these events mainly by regulating the expression of plastid proteins. As a consequence of gene transfer, greater than 90% of chloroplast proteins are nucleus-encoded (Leister, 2003). Thus, plant and algal cells have had to engineer a way to ensure that plastid proteins, now translated in the cytosol, are faithfully and efficiently targeted to and imported into plastids.
General principles of chloroplast protein import
The study of plastid protein biogenesis has largely been limited to the study of chloroplast proteins. The predominance of chloroplasts over other types of plastids in plants, their ease of isolation, and the enormous importance of the photosynthetic functions that they perform are the reasons why they have received the most attention. Chloroplasts, like all plastid types, are delimited by a double membrane envelope and, in addition, contain a third membrane system, the thylakoid membranes, in which are integrated and assembled the various photosynthetic protein complexes. The chloroplast is consequently made up of three different membranes, namely the outer envelope membrane (OEM), the inner envelope membrane (IEM), and the thylakoid membrane, and three aqueous sub-compartments, namely the inter-membrane space (IMS), the stroma, and the thylakoid lumen. In order for nucleus-encoded chloroplast proteins to be correctly integrated into this complex organelle, elaborate protein sorting mechanisms are essential. Because the different membranes of the chloroplast are highly impermeable to large molecules such as proteins, selective protein translocation machineries are necessary to allow newly translated chloroplast proteins to reach internal sub-compartments.
During the last decade, a considerable amount of progress has been made in our understanding of how proteins are targeted to the chloroplast, and how these reach their final destination. Most nucleus-encoded proteins destined to the internal compartments (i.e. the IMS, IEM, stroma, thylakoid membrane, and thylakoid lumen) of the chloroplast are expressed as precursor proteins (preproteins) carrying an N-terminal extension called a transit peptide (TP). The TP plays an important role in the targeting of the preprotein to the chloroplast, as well as in directing its translocation across the envelope membranes (Bruce, 2001). It is generally believed that preproteins are post-translationally imported into the chloroplast, and that their targeting to the organelle is assisted by soluble cytosolic factors that maintain them in a loosely-folded, import-competent conformation. Once a preprotein arrives at the chloroplast surface, translocons at the outer and inner envelope membranes of the chloroplast, termed TOC and TIC, respectively, mediate its translocation (Fig. 1). These translocons are hetero-oligomeric protein complexes that actively drive the movement of the preprotein across the envelope membranes with the help of various molecular chaperones. The individual proteins making up these complexes are named using the Toc or Tic prefix, followed by a number corresponding to the calculated molecular weight of the mature protein in kilodaltons (kDa) (Schnell et al., 1997).
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Preproteins arriving at the chloroplast surface are recognized by receptor components of the TOC. The receptors specifically and selectively interact with the TP, before they mediate its insertion into the channel of the TOC. The receptor components may also act to gate the protein-conducting channel in order to maintain a barrier at the outer membrane. It is widely believed that the TOC and TIC are able to interact physically in order to allow the direct translocation of proteins simultaneously through the outer and inner envelope membranes. Components of both the TOC and the TIC protruding into the IMS, and possibly soluble IMS proteins, are likely to promote the formation of this TOC–TIC supercomplex. The TIC complex mediates the translocation of the preprotein through the IEM, and is believed to do so with the help of chaperones recruited at the stromal face of the TIC. As the preprotein emerges into the stroma, the TP is cleaved off by a stromal processing peptidase (SPP). Molecular chaperones may then assist the processed protein to reach its functional conformation, or, alternatively, the protein may be further directed to other sub-compartments of the chloroplast by additional targeting signals.
The translocation of preproteins across the chloroplast envelope is an energy-dependent process. By characterizing the import of preproteins into isolated chloroplasts in vitro, three basic steps of the translocation process have been defined based on their energy requirements. First, the initial interaction of preproteins with the TOC receptors is an energy-independent and reversible step called energy-independent binding (Perry and Keegstra, 1994). Second, under energy-limiting conditions, initial translocation leads to the formation of early import intermediates. These intermediates are formed most efficiently in the presence of GTP and low levels of ATP (
100 µM) (Olsen and Keegstra, 1992; Young et al., 1999), and correspond to precursors in which the TP has crossed the OEM and is in contact with components of the TIC complex (Wu et al., 1994; Ma et al., 1996). Similar early import intermediates can be generated by exposing preproteins to energized chloroplasts at low temperatures (
4 °C) (Leheny and Theg, 1994). In the literature, the formation of early import intermediates has also been called binding and docking; in this review, in order to be consistent and to avoid confusion with energy-independent binding, these alternative terms will be avoided. Lastly, in order for complete preprotein translocation to occur, higher levels of ATP (>100 µM) are required in the stroma (Theg et al., 1989). This higher energy requirement is attributed to stromal molecular chaperones (Pain and Blobel, 1987).
The aim of this review is to provide a detailed synthesis of the research that has improved our understanding of how preproteins bearing transit peptides are recognized at the chloroplast surface, and then imported into the stroma by the TOC and TIC complexes. Other, less well understood pathways of protein import into the chloroplast are not discussed in detail. These include protein insertion into the OEM and IEM, protein import into the IMS, and other putative, alternative pathways for protein import into the chloroplast (Jarvis, 2004; Jarvis and Robinson, 2004). Proteins destined to the chloroplast OEM generally lack a transit peptide, and have been proposed to insert directly into the outer envelope membrane, independently of the TOC apparatus (for a review, see Schleiff and Klösgen, 2001). However, recent studies on outer envelope protein 14 kDa (OEP14) from pea (Pisum sativum), a model membrane protein carrying a single transmembrane domain at its N-terminus, have revealed that insertion is assisted by Toc75, perhaps disassociated from the TOC complex (Tu and Li, 2000; Tu et al., 2004); Toc75 is the channel-forming component of the TOC complex (see section entitled, ‘The TOC channel’).
Other recent data suggest that alternative import pathways, distinct from the general TOC–TIC import pathway, do exist for certain proteins (Jarvis, 2004). For example, two nucleus-encoded chloroplast IEM proteins, the chloroplast envelope quinone oxidoreductase homologue (ceQORH) and Tic32, have each been found to lack a cleavable TP (Miras et al., 2002; Hörmann et al., 2004; Nada and Soll, 2004). Both proteins are targeted to chloroplasts by signals contained in their mature sequences, and, in addition, Tic32 was shown to be imported independently of the general TOC–TIC pathway. It is likely that these two proteins represent only the first of many, however, since a proteomics study recently identified other nucleus-encoded chloroplast proteins that appear to lack canonical TP sequences (Kleffmann et al., 2004). Interestingly, Kleffman et al. (2004) also identified nucleus-encoded chloroplast proteins that carry N-terminal sequences similar to the signal peptides that direct proteins to the endoplasmic reticulum. These proteins may therefore utilize a chloroplast targeting pathway involving the endomembrane system. Alternatively, since outer envelope proteins such as OEP14 were also found to carry signal-peptide-like N-termini (Lee et al., 2001, 2004), it is possible that these proteins follow a similar targeting pathway involving Toc75. Finally, the chlorophyll biosynthetic enzyme, NADPH:protochlorophyllide oxidoreductase (POR) A, has been proposed to utilize a substrate-dependent import pathway that is independent of the general TOC–TIC pathway (Reinbothe et al., 2000). In vivo data in support of this novel import pathway have been presented (Kim and Apel, 2004; Kim et al., 2005), and components of a putative import apparatus have been identified (Reinbothe et al., 2004a, b, 2005), but the existence of this pathway has been strongly disputed by others (Aronsson et al., 2000, 2001, 2003b; Dahlin et al., 2000; Jarvis and Soll, 2002).
Following import, or envelope translocation, four different pathways are known to be responsible for the further targeting of proteins from the stroma to the thylakoids. These pathways, which are not discussed in detail here, include the signal recognition particle (SRP)-dependent and ‘spontaneous’ insertion pathways for the insertion of proteins into the thylakoid membrane, and the Sec and Tat pathways for the translocation of proteins into the thylakoid lumen. Unlike the general protein translocation pathway of the chloroplast envelope described earlier, which appears to have evolved as a response to gene transfer and shows no clear homology to other known protein translocation systems, most of these thylakoidal pathways are highly homologous to protein translocation systems of the bacterial cell membrane. Like the protein import pathway of the envelope, these pathways are important for the proper biogenesis of the photosynthetic machinery associated with the thylakoids, and have been the subject of intensive research. For more general reviews of chloroplast protein targeting, including thylakoid protein import, readers are referred to Keegstra and Cline (1999) and Jarvis and Robinson (2004).
History of chloroplast protein import research
Chloroplast protein import research stretches back over several decades. Blair and Ellis (1973) proposed an ‘envelope carrier hypothesis’ to account for the fact that isolated pea chloroplasts are able to synthesize only a small number of their constituent proteins (including the large subunit of Rubisco, RbcL); the rest (including the small subunit of Rubisco, RbcS), it was correctly presumed, are synthesized outside of the chloroplast on cytosolic ribosomes. The hypothesis proposed the existence of specific mechanisms for the transport into chloroplasts of all those chloroplast proteins which are synthesized in the cytosol: ‘one possibility is that a membrane protein exists in the outer envelope which recognizes a site common to those proteins destined for the plastid’ (Blair and Ellis, 1973). The existence of such a ‘recognition site’ received considerable support when Dobberstein et al. (1977) demonstrated that Chlamydomonas reinhardtii RbcS is synthesized as a high molecular weight 20 kDa precursor that can be processed to a mature form of 15 kDa by a specific endoproteolytic activity present in Chlamydomonas cells. The final proof of the ‘envelope carrier hypothesis’ was provided in 1978 when two groups independently reconstituted the import of RbcS precursor (pRbcS) into isolated chloroplasts (Chua and Schmidt, 1978; Highfield and Ellis, 1978); in each case, precursor processing was observed to occur concomitantly with chloroplast import. Import occurred after precursor synthesis was complete, or post-translationally, which is consistent with the observation that the pRbcS is synthesized on free cytosolic ribosomes (Dobberstein et al., 1977), and with the fact that the chloroplast surface, unlike that of the endoplasmic reticulum (ER), is not coated with ribosomes. A year later, the amino acid sequence and N-terminal position of the Chlamydomonas RbcS TP was determined (Schmidt et al., 1979).
The nature of the envelope protein(s) mediating chloroplast protein import took longer to establish. Treatment of isolated chloroplasts with proteases such as thermolysin, which degrades only surface-exposed protein domains, provided the initial evidence for the existence of a proteinaceous import apparatus (Cline et al., 1984, 1985; Friedman and Keegstra, 1989). The demonstration that precursor binding to chloroplasts was saturable provided further support for the existence of specific protein import sites at the chloroplast surface (Friedman and Keegstra, 1989). The application of protein cross-linking techniques and the isolation of translocation complexes, led to the tentative identification of import apparatus components (Waegemann and Soll, 1991; Perry and Keegstra, 1994), and in 1994 and 1995 the identity of the first TOC and TIC components was firmly established (Hirsch et al., 1994; Kessler et al., 1994; Schnell et al., 1994; Wu et al., 1994; Seedorf et al., 1995; Tranel et al., 1995). Since then, several more components of the import apparatus have been identified, and the exact sequence of events that occurs during import has been established (Keegstra and Cline, 1999; Chen et al., 2000b; Hiltbrunner et al., 2001a; Jarvis and Soll, 2002). More recently, attention has turned to the determination of the specific functions performed by individual TOC and TIC components, as discussed in detail in this review. In addition, the model organism, Arabidopsis thaliana, has been utilized so that genomics, genetics, and other in vivo approaches can be used to complement the biochemical approaches that have been used traditionally, with pea chloroplasts, to study protein import (Jarvis et al., 1998; Bauer et al., 2000; Chen et al., 2002; Chou et al., 2003).
Overview of protein import into mitochondria
Mitochondria are organelles found in almost all eukaryotic cells. They are mainly responsible for the generation of usable energy by oxidative respiration, but also perform other essential metabolic functions. In many ways, mitochondria are strikingly similar to chloroplasts. They also arose from an endosymbiotic event, and their evolution similarly involved gene-transfer from the endosymbiont to the nucleus of the eukaryotic progenitor cell. Like chloroplasts, mitochondria are semi-autonomous organelles: they retain a functional genetic system and a few protein-coding genes, but most mitochondrial proteins are nucleus-encoded and translated on cytosolic ribosomes. Thus, in order to carry out their metabolic functions, mitochondria must import most of their proteome post-translationally from the cytosol. Mitochondria are composed of two membranes, namely the outer membrane and the inner membrane, and two aqueous compartments, namely the inter-membrane space and the matrix. Since the majority of mitochondrial proteins are found either in the matrix or the inner membrane, each membrane is equipped with hetero-oligomeric protein complexes which mediate protein translocation across the hydrophobic membrane barriers. These are the translocases of the outer and inner membranes of mitochondria, or the TOM and TIM complexes, respectively.
Most components of the chloroplast envelope and mitochondrial translocases are not homologous, but the processes of chloroplast protein import into the stroma and mitochondrial protein import into the matrix appear to be generally analogous. Due to the importance of mitochondrial biology for human health, and the ease of working with yeast as a model organism, the processes of mitochondrial protein targeting and import are understood in much greater detail than the analogous processes in chloroplasts. Because of this, mitochondrial import is often used as a model to predict how chloroplast protein targeting and import may function. A short summary of mitochondrial preprotein targeting and import is therefore included below, in order to provide background for the comparisons that will be drawn throughout this review. For a more detailed description of protein import into mitochondria, readers are referred to the following reviews: Neupert and Brunner (2002), Endo et al. (2003), Truscott et al. (2003), Rehling et al. (2004), and Wiedemann et al. (2004).
Three general mitochondrial protein import pathways have been defined in yeast and animal cells. (i) The presequence import pathway utilized by preproteins carrying an N-terminal targeting sequence (presequence), which includes most proteins targeted to the mitochondrial matrix and some proteins of the inner membrane and inter-membrane space. (ii) The carrier protein import pathway utilized by inner membrane proteins that carry multiple internal targeting signals. (iii) The outer membrane protein insertion pathway utilized by ß-barrel proteins which also carry internal targeting signals. All three pathways share a common path through the outer membrane at the TOM complex. The TOM complex is equipped with different receptors that can recognize preproteins carrying presequences and internal targeting signals. Once translocation across the outer membrane has been achieved, the different pathways diverge. Outer membrane ß-barrel proteins are targeted to the sorting and assembly machinery (SAM), which assists their insertion into the membrane, whereas preproteins following the other two pathways are targeted to distinct TIM complexes. Presequence preproteins are targeted to the presequence translocase, or TIM23 complex, and carrier proteins are targeted to the protein insertion machinery of the inner membrane, or TIM22 complex.
The import pathway followed by chloroplast stromal proteins carrying an N-terminal TP is generally believed to be most analogous to the presequence protein import pathway of mitochondria. After initial binding at the TOM receptors, the positively-charged presequence of mitochondrial precursors is thought to be translocated through the outer membrane in step-wise fashion, by progression along a chain of binding sites on the TOM translocase. Forward movement of the preprotein is driven by the increasing affinity of the binding sites for the presequence along the translocase. Like the TOC and TIC of the chloroplast envelope, the TOM and TIM are known to form supercomplexes, allowing for simultaneous translocation through both membranes. Translocation of the presequence through the TIM translocase is driven by a pulling force exerted by the electric potential across the inner membrane. Once the presequence emerges at the inner face of the TIM, translocation of the preprotein is completed by the action of a molecular chaperone in the matrix, called matrix Hsp70 (mtHsp70). Together with soluble and membrane-bound co-chaperones, mtHsp70 forms a presequence translocase-associated protein import motor (PAM) that drives protein import in an ATP-dependent fashion. Translocation is completed by the removal of the presequence by the mitochondrial processing peptidase (MPP).
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Targeting of preproteins from the cytosol to the chloroplast |
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N-terminal targeting signals are commonly used in eukaryotic cells to direct nucleus-encoded proteins to various cellular sub-compartments, including the ER, mitochondria, and chloroplasts (Blobel and Sabatini, 1971
; Schnell and Hebert, 2003). In order to prevent mis-targeting of proteins, these signals must be specifically recognized by the cellular protein sorting machinery. Like the presequence of mitochondrial preproteins, the TP of chloroplast precursors is an essential targeting signal and plays several key roles to ensure that the protein reaches its destination (Bruce, 2001). First, as preproteins are synthesized on cytosolic ribosomes, TPs are probably recognized by soluble cytosolic factors that assist the targeting of the preprotein to the chloroplast. Later, as they reach the chloroplast, they are known to interact directly and specifically with receptors of the TOC complex, leading to translocation initiation. TPs also interact with other downstream components of the translocation machinery, and are therefore likely to play important roles to ensure the progression of the protein through the TOC and TIC complexes. Finally, they must be correctly recognized by the stromal processing machinery so that preproteins can attain their mature and functional form, or so that downstream N-terminal targeting signals are recognized to allow further targeting (Keegstra and Cline, 1999; Jarvis and Robinson, 2004).
Surprisingly, although TPs play such important roles, they are highly variable in length, amino acid composition, and sequence. Typically, higher plant TPs are between 20 and 100 residues in length, are rich in hydrophobic and hydroxylated residues, and almost completely lack acidic residues such that they show an overall positive charge (Zhang and Glaser, 2002). Since TPs lack detectable blocks of conserved amino acid sequence, they are thought to share similar secondary or tertiary structural properties that would allow their specific recognition and targeting. The detailed analysis of a few TPs from a small number of algal and higher plant proteins suggests that they are largely unstructured in aqueous environments and form a random coil (von Heijne and Nishikawa, 1991; Bruce, 2001). However, they have been found to form amphipathic helix domains when exposed to membrane-like environments. These properties are very similar to those of mitochondrial presequences, and it is therefore unclear how specificity of targeting is achieved in plants, which possess both types of organelle (Macasev et al., 2000; Chew and Whelan, 2004). The targeting sequences of plant mitochondrial and chloroplast proteins are proposed to be made of several domains, which may mediate their different functions and may also display different properties that could account for the specificity of targeting (Bruce, 2000).
The proposed, largely unstructured nature of presequences and TPs in the cytosol has led to the suggestion that they may serve as ideal substrates for the binding of Hsp70 molecular chaperones (von Heijne and Nishikawa, 1991). Indeed, the majority of known presequences and TPs have been found to display at least one putative Hsp70 binding site, and several have been shown to interact with Hsp70s experimentally (Ivey and Bruce, 2000; Ivey et al., 2000; Rial et al., 2000; Zhang and Glaser, 2002). Cytosolic Hsp70s, therefore, probably interact with the TP in the cytosol, as it emerges from the ribosome, in order to prevent preprotein aggregation and to maintain a translocation-competent conformation. In addition, Hsp70s have been suggested to participate in protein translocation across the chloroplast envelope membranes at several stages, at both the outer and inner faces of the outer envelope, as well as in the stroma (Marshall et al., 1990; Tsugeki and Nishimura, 1993; Schnell et al., 1994; Kourtz and Ko, 1997). It is possible that the TP is important in promoting binding by these Hsp70s during translocation.
Interestingly, TPs of chloroplast preproteins are proposed to be phosphorylated at a serine or threonine residue within a loosely conserved motif by a soluble, cytosolic, ATP-dependent kinase (Waegemann and Soll, 1996). Furthermore, the phosphopeptide motif of TPs appears to be specifically recognized by 14-3-3 proteins (May and Soll, 2000). Because plant mitochondrial preproteins are neither phosphorylated nor recognized by 14-3-3 proteins, it was proposed these TP-specific events may confer specificity for targeting to the chloroplast. It was further shown that chloroplast preproteins translated and phosphorylated in a wheat-germ lysate associate with 14-3-3 and Hsp70 proteins in a complex termed the guidance complex. Preproteins assembled into such hetero-oligomeric complexes were imported into isolated chloroplasts at a rate three- to four-times higher than free preproteins, suggesting that these complexes constitute true targeting intermediates. Recently, however, the physiological relevance of TP phosphorylation and 14-3-3 protein interaction was disputed, since disruption of the TP phosphorylation site did not affect targeting or import in vivo (Nakrieko et al., 2004).
The change in structure of parts of the TP from a random coil to an amphipathic helix in membrane-mimetic conditions led to the proposal that chloroplast preproteins may interact specifically with the unique lipids of the OEM prior to association with TOC receptors (Bruce, 2000). Although this remains a possibility, the TP has now been shown to interact directly and specifically with recombinant TOC receptors in aqueous solution (Sveshnikova et al., 2000b; Becker et al., 2004b; Ivanova et al., 2004; Smith et al., 2004). It is possible that a similar change in structure may be induced directly via an interaction with the receptor proteins at the TOC, since mitochondrial presequences have been shown to assume an amphipathic helical structure upon interaction with the Tom20 receptor (Abe et al., 2000; Muto et al., 2001). The amphipathic nature of the helices formed by chloroplast preproteins appears to be provided by the clustering of hydroxylated residues on one side of the helix, rather than by the clustering of basic residues as observed in mitochondrial presequence helices (Bruce, 2000). The different nature of these secondary structures may therefore also contribute to the specificity of targeting. However, a larger number of TP structures and the nature of the interactions between TPs and the TOC receptors must be established before this can be confirmed.
Finally, the TOC component, Toc159, has recently been proposed to play a role in preprotein targeting from the cytosol to the chloroplast. Toc159 has traditionally been thought of as a receptor for chloroplast precursor proteins acting exclusively at the chloroplast OEM (see below). However, re-investigation of its localization profile suggested that a large proportion (50%) of the protein behaves as a soluble cytosolic protein (Hiltbrunner et al., 2001b). This prompted the suggestion that Toc159 may shuttle between the cytosol and the chloroplast OEM to mediate the transport of nucleus-encoded preproteins between their site of synthesis and the TOC complex. This particular role of Toc159 needs to be investigated further, however, since the existence of a soluble form of the protein has been disputed (Becker et al., 2004b).
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Preprotein binding and recognition at the TOC complex |
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Receptor components of the TOC complex
Preprotein recognition at the OEM is mediated by at least two receptor components of the TOC complex, Toc34 and Toc159 (Fig. 1). These were originally identified as proteins in close proximity to preproteins bound to the chloroplast OEM translocon under energy-limiting conditions at the initial stage of translocation (Perry and Keegstra, 1994
; Schnell et al., 1994). Both proteins display homology to GTPases (Bourne et al., 1991), and show substantial levels of similarity to each other across their GTPase (G) domains. In Toc34, the G-domain spans almost the entire length of the protein except for short amino acid extensions at the N- and C-termini that lack homology with Toc159. The C-terminal extension contains a single, short stretch of hydrophobic amino acids, predicted to form a transmembrane 
-helix, which anchors Toc34 in the OEM (Kessler et al., 1994; Seedorf et al., 1995). By contrast, in Toc159, the central G-domain is flanked by a large acidic (A) domain at the N-terminus, and an unusual membrane-anchoring (M) domain at the C-terminus (Bölter et al., 1998a; Chen et al., 2000a). Initially, Toc159 was identified as an 86 kDa proteolytic fragment (Toc86) lacking the large A-domain. The full-length pea protein was only recently discovered (Bölter et al., 1998a) and characterized (Chen et al., 2000a) as a result of the Arabidopsis genome project (The Arabidopsis Genome Initiative, 2000), which led to the identification of a homologous gene encoding a protein much larger than 86 kDa. The A-domain of Toc159 was found to be rich in acidic residues, and to be essentially made up of two different tandemly-repeated motifs (Chen et al., 2000a).
Isolated chloroplasts in which the Toc159 protein remained intact displayed a significantly greater import rate than those in which Toc159 had been proteolysed (Bölter et al., 1998a), demonstrating that this non-essential domain does perform some role in import. Due to its acidic nature, the A-domain was proposed to interact directly with the basic TPs of precursor proteins and to help mediate the preprotein receptor function of Toc159. The M-domain of Toc159 lacks a clear stretch of hydrophobic amino acids that could span the membrane as an -helix, and it predominantly consists of hydrophilic residues (Hirsch et al., 1994; Kessler et al., 1994). However, the M-domain was shown to be necessary for the correct insertion of Toc159 into the OEM (Hirsch et al., 1994; Kessler et al., 1994). In addition, it was shown to become highly resistant to proteases as well as to salt and alkaline extraction upon membrane insertion, indicating that it behaves as a true integral membrane anchor (Hirsch et al., 1994; Kessler et al., 1994). The bulk of Toc34 and a large proportion of Toc159, consisting of its A- and G-domains, protrude into the cytosol where they can interact specifically with the TPs of incoming preproteins (Hirsch et al., 1994; Kessler et al., 1994; Ma et al., 1996; Chen et al., 2000a; Sveshnikova et al., 2000b).
A third putative preprotein receptor, Toc64, has been described (Sohrt and Soll, 2000). Toc64 was initially identified as a pea OEM protein co-fractionating with the solubilized TOC complex in sucrose density gradients. It was further shown, by cross-linking in isolated chloroplasts, to be in close physical proximity with known components of the TOC and TIC complexes, as well as with an arrested translocation intermediate. Interestingly, Toc64 carries three tetratricopeptide repeat (TPR) motifs that are exposed in the cytosol. Such TPR domains have previously been shown to mediate protein–protein interactions (Lamb et al., 1995), and commonly mediate the functional interaction of proteins with molecular chaperones (e.g. Hsp70, Hsp90) (Frydman and Höhfeld, 1997). Thus, Toc64 has been proposed to participate in import as a docking receptor for phosphorylated preproteins delivered to the chloroplast by cytosolic Hsp70s (Sohrt and Soll, 2000).
Receptor proteins equipped with TPR motifs are also known to play roles in the recognition of preproteins targeted to other protein translocation systems. For example, Pex5 is a soluble protein involved in the translocation of proteins to the peroxisome, and Tom70 is a receptor for TIM22 substrates (e.g. carrier proteins) at the outer membrane of mitochondria. The Pex5 TPR motifs are known to interact directly with preproteins (Terlecky et al., 1995; Gatto et al., 2000), whereas the TPR motifs of Tom70 serve as a docking site for the cytosolic chaperones, Hsp70 and Hsp90, and also interact directly with internal targeting domains of carrier proteins (Young et al., 2003). It is therefore possible that Toc64 could bind directly to chloroplast preproteins or, as suggested previously, interact with a soluble targeting factor associated with chloroplast preproteins.
In Arabidopsis, three proteins similar to pea Toc64 (psToc64) were identified by homology searches (Jackson-Constan and Keegstra, 2001). Recently, the localization of these Toc64-like proteins has been investigated (Chew et al., 2004; Lee et al., 2004). The most similar protein to psToc64 (67% identity), Arabidopsis Toc64-III (atToc64-III), was shown to be associated with the chloroplast OEM, indicating that it is likely the functional orthologue of psToc64. By contrast, atToc64-I, a truncated Toc64-like protein, was not found to localize to organelles. Interestingly, mtOM64 (atToc64-V), the third Toc64-like protein, which displays 52% identity to psToc64, was found to associate with the mitochondrial outer membrane. This suggests that plant mitochondria, which appear to have different preprotein recognition properties from the mitochondria of other organisms (Macasev et al., 2000), may also utilize a Toc64-like receptor. This led to the hypothesis that Toc64 may be specifically involved in the recognition of so-called dual-targeted proteins (Chew et al., 2004): proteins that are normally targeted to both organelle types (Peeters and Small, 2001).
Core components and structure of the TOC complex
The receptor proteins, Toc34 and Toc159, have been shown by co-immunoprecipitation studies to form a stable trimeric complex with the Toc75 channel protein (Ma et al., 1996; Hiltbrunner et al., 2001b) (see section entitled, ‘The TOC channel’). Recently, the core TOC complex, found to be composed solely of these three proteins, was successfully purified from pea chloroplast OEM vesicles (OEVs) by sucrose gradient centrifugation (Schleiff et al., 2003c). The absence of Toc64 from this complex suggests that it may only transiently associate with the complex. Analysis of the stoichiometry of the three core components within the complex revealed that Toc86 (the proteolytic fragment of Toc159), Toc75, and Toc34 are assembled in a ratio of 1:4:4. The predicted size of 522 kDa for such a complex was found to be consistent with the apparent molecular mass of 500 kDa observed by size exclusion chromatography.
The isolated translocon was shown to interact specifically with the TP of precursor proteins in a GTP-dependent manner, and was therefore proposed to represent an intact and functional TOC complex. Electron microscopy analysis of core translocon particles incubated with gold-labelled TPs indicated that each particle could interact with up to four peptides, suggesting that each translocon may contain four independent channels. This idea was further supported by two-dimensional structural analysis of the translocon, again using electron microscopy. The translocon was shown to form a spherical complex, made up of four curved channels separated by a finger-like central structure. It is possible, as suggested by Schleiff et al. (2003c), that individual Toc75 proteins, each associated with a Toc34 receptor, form the four observed channels, and that these are assembled around a single Toc159 unit that forms part of the central finger-like domain.
GTP-regulated receptors of the TOC
GTP is necessary during the early steps of protein translocation (Olsen and Keegstra, 1992; Kessler et al., 1994; Young et al., 1999). GTP has been shown to promote the formation of early import intermediates (Ma et al., 1996; Kouranov and Schnell, 1997; Young et al., 1999), whereas, by contrast, non-hydrolysable GTP analogues inhibit the formation of such intermediates (Kessler et al., 1994; Young et al., 1999). These observations established a role for GTP-hydrolysis in mediating translocation initiation. Because of their homology to GTP-binding proteins and their capacity to bind GTP in vitro, Toc34 and Toc159 were originally proposed to regulate the initial steps of protein import through a GTP binding and hydrolysis cycle (Kessler et al., 1994). Shortly after this, Toc34 was shown to display intrinsic GTPase activity, further supporting a function in GTP-dependent regulation of protein import (Seedorf et al., 1995). More recently, it was also revealed that Toc34 interacts directly and specifically with the TP of RbcS in vitro, and that this interaction is stimulated by GTP (Sveshnikova et al., 2000a). Since their discovery, a considerable amount of effort has been invested in experiments to elucidate how GTP influences the TOC GTPases in mediating preprotein recognition and translocation initiation.
Based on sequence analysis of the Toc34 and Toc159 G-domains, the receptors were proposed to form a novel class of GTPase proteins (Kessler et al., 1994; Seedorf et al., 1995). This was recently confirmed by the resolution of the crystal structure of the pea Toc34 (psToc34) G-domain (Sun et al., 2002), which, due to its similarity to the Toc159 G-domain, serves as general model for the TOC GTPases. Analysis of the crystal structure revealed that the motifs and residues most likely involved in GTP binding and hydrolysis (Bourne et al., 1991) are uniquely arranged in Toc34, and differ significantly in sequence compared with those of the model GTPase, Ras. This was taken to suggest that the TOC GTPases probably utilize a novel GTP binding and hydrolysis mechanism, an idea that has taken further support from the finding that atToc33, the Arabidopsis orthologue of psToc34, appears to display unusual nucleotide binding properties (Aronsson et al., 2003a).
Interestingly, GDP-bound Toc34 G-domains were found to crystallize as dimers in which residues of each Toc34 monomer contributed to the GDP/GTP-binding pocket of its partner. Moreover, it was suggested that each monomer provides a catalytic ‘arginine finger’ (Arg133) necessary to promote GTP hydrolysis by the other monomer. Because of this, dimerization was proposed to lead to reciprocal stimulation of GTPase activity in each monomer. In most common GTPase systems, such as those of the small GTPases family including Ras, a non-homologous GTPase-activating protein (GAP) associates with the GTP-bound GTPase to provide such an arginine finger (Scheffzek et al., 1998). In the case of Toc34, the interaction properties of the two monomers within a dimer led to the suggestion that Toc34 might serve as its own GAP. However, the discovery that preprotein binding to the TOC GTPases strongly stimulates their GTPase activity (Jelic et al., 2002; Becker et al., 2004b) argues against this possibility, and suggests that TPs may actually serve as GAPs. Furthermore, the idea that dimerization stimulates the GTPase activity of Toc34 is inconsistent with the finding that substitution of Arg130 for an alanine residue (R130A) in atToc33 does not affect its GTPase activity, but instead greatly inhibits dimerization (Weibel et al., 2003); Arg130 in atToc33 corresponds to Arg133 in psToc34, the arginine residue proposed to act as an arginine finger (Sun et al., 2002).
Because the G-domains of Toc34 and Toc159 are very similar, and particularly because the residues determined to be involved in Toc34 dimerization are also conserved in Toc159, it was proposed that Toc34 and Toc159 may heterodimerize in vivo (Kessler and Schnell, 2002). This idea was supported by the finding that the G-domains of Toc34 and Toc159 interact in vitro (Hiltbrunner et al., 2001b). Furthermore, it can be proposed that such heterodimerization might be favoured in vivo, since, when Sun et al. (2002) assessed whether Toc34 G-domains could homodimerize in solution, only 20% of polypeptides were found as dimers. Another interesting feature of the dimer is that there appears to be no exit or entrance for nucleotide exchange at the GTP-binding pocket, suggesting that either a large conformational change or dimer dissociation must occur to allow GDP/GTP exchange. Many GTPases also associate with a second partner protein, a guanine nucleotide exchange factor (GEF), that promotes the exchange of GDP for GTP at the binding pocket of the GTPase (Bourne et al., 1991). As yet, however, GEFs for Toc34 and Toc159 have not been identified.
Overall, the study by Sun et al. (2002) provided significant insight into how the TOC GTPases may function together as GTP-regulated receptors (Kessler and Schnell, 2002). In addition, the identification of the actual motifs that form the GDP-binding pocket and the residues that are likely to mediate GTP binding and hydrolysis provided the information necessary to design useful mutants to analyse the function of the TOC GTPases further. Interestingly, biochemical assessment of the relationship between GTP- and preprotein- binding by Toc34 led to the suggestion that the GTPase activity of the TOC GTPases may be regulated by phosphorylation (Sveshnikova et al., 2000a). It was found that recombinant Toc34 could be phosphorylated in vitro, and that phosphorylation inhibits GTP binding and, as a consequence, preprotein binding. The possibility that protein import is regulated in this way is further supported by the fact that Toc34 and Toc159 can each be phosphorylated by a different protein kinase of the chloroplast OEM (Fulgosi and Soll, 2002). However, it has yet to be demonstrated that phosphorylation of the TOC GTPases truly occurs in vivo.
Recognition of preproteins by the TOC GTPases
Recent efforts to define the roles of Toc34 and Toc159 receptors in preprotein recognition have led to the emergence of two distinct models (Fig. 2): the targeting model, and the motor model (Jarvis and Robinson, 2004; Kessler and Schnell, 2004). The targeting model proposes that Toc159 is the primary receptor, and that it also plays a role in preprotein targeting from the cytosol to the chloroplast surface. By contrast, the motor model suggests that Toc34 carries out the role of primary receptor, whereas Toc159 acts as a molecular motor to drive preprotein translocation across the outer envelope.
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The targeting model:
In the targeting model, after preprotein recognition, Toc159 associates with the secondary receptor, Toc34, to promote the transfer of the precursor to the Toc75 channel. The idea that Toc159 acts as the primary receptor is based on the early finding that Toc159 is the predominant protein cross-linking with preproteins arrested at the initial stages of translocation (Perry and Keegstra, 1994
; Ma et al., 1996; Kouranov and Schnell, 1997). This notion is further supported by the fact that Toc159 antibodies were found to block formation of early import intermediates in vitro (Hirsch et al., 1994). However, the model also incorporates the more recent finding that an abundant cytosolic form of Toc159 exists in addition to its integral OEM form (Hiltbrunner et al., 2001b). In this study, analysis of Arabidopsis Toc159 (atToc159) in Arabidopsis protoplasts by immunofluorescence microscopy suggested that a certain proportion of the protein is cytosolic. This observation was supported by subcellular fractionation experiments in Arabidopsis and pea, which revealed that Toc159, but neither Toc34 nor Toc75, could be found in a soluble, cytosolic form. The discovery of a soluble form of Toc159 led to the suggestion that the protein may shuttle between the cytosol and the chloroplast OEM in order to target newly translated, nucleus-encoded chloroplast proteins. Such a model implies that it is necessary for Toc159 to be able to both insert and retract from the membrane, and therefore provides an attractive explanation for its unusual, hydrophilic membrane-anchoring domain.
The discovery of a cytosolic soluble form of Toc159 prompted new initiatives to gain a better understanding of the factors that control and direct its targeting and insertion into the chloroplast OEM. In vitro targeting studies revealed that Toc159 binding and insertion are regulated in part by the protein's intrinsic GTPase activity. GTP binding by Toc159 promotes binding of the receptor to the chloroplast OEM, and GTP hydrolysis is required for efficient insertion of the M-domain into the membrane (Bauer et al., 2002; Smith et al., 2002). A role for the G-domain in Toc159 targeting has been supported by two independent in vivo studies (Bauer et al., 2002; Lee et al., 2003). These both showed that a functional GTPase domain is required for Toc159 transgenes to fully complement an Arabidopsis atToc159 null mutant called ppi2 (see section entitled, ‘Arabidopsis Toc159 homologues’). In addition, both studies revealed that Toc159 GTPase mutants with reduced GTP-binding and/or -hydrolysis activity failed to localize at the OEM in Arabidopsis chloroplasts and, instead, appeared to accumulate in the cytosol.
Surprisingly, however, the G-domain of Toc159 is not essential for targeting in vivo, since a truncated form of Toc159 composed solely of the M-domain was found to accumulate at the OEM, and to mediate partial complementation of ppi2 (Lee et al., 2003). These findings led Lee et al. (2003) to suggest that a non-functional Toc159 GTPase domain has a dominant negative effect on the targeting of the M-domain. In agreement with this idea, GTP hydrolysis by Toc159 does not appear to directly drive insertion of the M-domain into the membrane in vitro, but rather generates the GDP-bound form of the receptor required for efficient insertion (Smith et al., 2002). This was suggested by the fact that Toc159 was found to insert equally well in the presence of either GTP or GDP. It is therefore likely that GTPase mutants that cannot bind and/or hydrolyse GTP efficiently maintain an insertion-incompetent conformation, perhaps because the M-domain is masked by the G-domain. By contrast, the Toc159 M-domain (lacking the G-domain) appears to be in an insertion-competent form, although both targeting and insertion are most likely rather inefficient.
Studies by both Hiltbrunner et al. (2001a) and Smith et al. (2002) suggested that Toc159 targeting to the chloroplast is mediated by an interaction with Toc34. Smith et al. (2002) further showed that this specific interaction corresponds to a homotypic interaction between the G-domains of both proteins. While association did occur in the presence of GTP (Hiltbrunner et al., 2001a; Smith et al., 2002), this interaction was stimulated in the presence of GDP (Smith et al., 2002). In a recent study taking advantage of a newly-developed protocol allowing reconstitution of the TOC components into liposomes (Hinnah et al., 1997; Schleiff et al., 2003b), it was revealed that binding and insertion of Toc159 into the OEM is also dependent upon Toc34 GTPase activity (Wallas et al., 2003), confirming the importance of a GTP-regulated interaction between the homologous G-domains of Toc34 and Toc159. It is possible that initial binding occurs between Toc159-GTP and Toc34-GTP, and that this induces GTP hydrolysis by both GTPases. Hydrolysis would induce stable dimerization and insertion of the M-domain of Toc159 into the membrane. In addition, Wallas et al. (2003) revealed that the presence of Toc75 is essential for the membrane insertion of the M-domain of Toc159. It is possible that, due to its overall hydrophilic nature, the insertion of the Toc159 M-domain involves the formation of ß-strands (Schleiff et al., 2003a), and that these are stabilized through interactions with Toc75; alternatively, Toc75 may actively participate in the insertion of the Toc159 M-domain.
As described earlier, the regulated targeting and insertion of Toc159 into the OEM is proposed to be coupled with the targeting of precursor proteins to the TOC complex, leading to translocation initiation. Such a scenario is reminiscent of co-translational protein targeting to the ER and bacterial inner membrane by the SRP and the SRP-receptor (SR/FtsY) GTPases. In these systems, the nascent signal peptide of a preprotein associates with the SRP, and then makes its way as a complex along with the ribosome to the target membrane. At the membrane, a GTP-regulated interaction between the SRP GTPase and the homologous, membrane-associated SR/FtsY GTPase leads to the transfer of the precursor protein to the Sec translocase and the initiation of co-translational translocation (Keenan et al., 2001; Schnell and Hebert, 2003). The targeting hypothesis proposes similar roles for the Toc159 and Toc34 GTPases, as shown in Fig. 2A. Soluble Toc159 interacts with chloroplast preproteins in the cytosol and mediates their targeting to the chloroplast surface. Upon arrival at the chloroplast membrane, a similar homotypic interaction between Toc159 and membrane-bound Toc34, coupled with GTP hydrolysis, leads to transfer of the preprotein to the TOC channel. Further support for this hypothesis was recently provided by the finding that soluble Toc159, either from cell extracts or in vitro translation mixtures, can interact specifically with the TP of chloroplast preproteins (Smith et al., 2004).
The motor model:
In the recently proposed motor model, Toc159 functions as a GTP-dependent motor that pushes preproteins through the TOC channel via multiple rounds of GTP hydrolysis. This hypothesis stems from the results of recent studies in which the structure of the purified TOC complex, and the functions of individual TOC components of the reconstituted complexes (see section entitled, ‘Translocation through the TOC complex’), were examined. The proposed central function of Toc159 as a motor and the near 1:1 stoichiometric association of Toc34 with the Toc75 channel in the core complex (Schleiff et al., 2003c), led to the suggestion that Toc34 is more likely to function as the primary receptor (Becker et al., 2004b). This view can be supported by the fact that Toc34 was found to cross-link to precursors only at the initial energy-independent binding stage of import; by contrast, Toc159 appears to remain in close proximity with precursors throughout their translocation through the OEM (Kouranov and Schnell, 1997). In addition, the direct and specific interaction of Toc34 with the TP of various precursor proteins has been clearly demonstrated (Sveshnikova et al., 2000b; Schleiff et al., 2002; Becker et al., 2004b), further supporting its role as a receptor.
Recently, Becker et al. (2004b) presented strong evidence in support of the motor model. First, detailed subcellular fractionation studies suggested that the previously-observed soluble form of Toc159 (Hiltbrunner et al., 2001b) is an artefact produced by the fractionation method used. They argued that the Toc159 detected in the cytosolic fraction was associated with low-density membrane shreds released from the chloroplast during tissue homogenization. In light of these results, they concluded that Toc159 is only present as an integral protein of the chloroplast OEM. Having concluded that productive interactions between preproteins and the TOC GTPases occur only at the OEM, Becker et al. (2004b) demonstrated that, like Toc34, Toc159 interacts specifically with the TP of RbcS, and that GTP stimulates this preprotein binding. However, in contrast with Toc34, which was previously shown to bind phosphorylated TPs with high affinity (Sveshnikova et al., 2000a), Toc159 only recognizes the non-phosphorylated form of the RbcS TP. Furthermore, by using peptides spanning either the C- or N-proximal regions of the RbcS TP, they revealed that a Toc159 fragment spanning the G- and M-domains (Toc86) displays high affinity for the N-terminal part of the TP. This, again, is in contrast with Toc34, which binds the phosphorylated C-terminal peptide with high affinity (Sveshnikova et al., 2000b; Becker et al., 2004b).
The different peptide-binding properties of Toc34 and Toc159 were used to show that Toc34 functions before Toc159 in the binding and translocation of precursor proteins. This was demonstrated by using the different peptides to inhibit binding and import into proteoliposomes containing the reconstituted core TOC complex (TOC proteoliposomes). It was found that only the phosphorylated C-proximal peptide was able to significantly inhibit binding of pRbcS to TOC proteoliposomes, whereas both the phosphorylated C-proximal peptide and the N-proximal peptide inhibited import of the precursor. This indicates that in order to bind to the chloroplasts, precursors must first interact with Toc34. Subsequently, these precursors need to interact with Toc86 to be imported. These experiments were designed under the assumption that the TP is phosphorylated in the cytosol and requires dephosphorylation in order to become import-competent. However, as described previously, the importance and even the occurrence of TP phosphorylation in vivo is still a case open to debate. Regardless of this caveat, the results of these experiments may nevertheless be valid, since, even though a phosphorylated peptide was used as a specific competitor for Toc34 binding, the pRbcS used in the import assays was not phosphorylated. Inhibition of precursor binding to TOC proteoliposomes was only caused by the high-affinity binding of a peptide to Toc34, implying that Toc34 plays the initial receptor role.
In addition to assessing the hierarchy of the TOC receptors, Becker et al. (2004b) also investigated how GTP and GDP affect the stability of the TOC complex. By performing immunoprecipitation experiments from OEVs with antibodies against both receptors, they established that the association of Toc34 with the TOC is destabilized by GDP. By contrast, in the presence of a non-hydrolysable form of GTP (GMP-PNP) the association of Toc34 with the TOC was favoured. Interestingly, the association of Toc159 with Toc75 was nucleotide-insensitive. The effect of both nucleotides on the stability of the TOC complex was further confirmed by sucrose gradient centrifugation of solubilized OEVs. When OEVs were solubilized in the presence of GDP, the core TOC components were enriched in low-density fractions as compared with in the absence of nucleotides. By contrast, in the presence of GMP-PNP, TOC components, especially Toc34, were enriched in high-density fractions. To determine further how the TOC receptors might affect the stability of the TOC core complex, Becker et al. (2004b) assessed interactions between Toc34 and Toc86, individually preloaded with either GMP-PNP or GDP, by affinity chromatography. Their results revealed that interaction between the two GMP-PNP-bound receptors is greatly stimulated in the presence of TPs, whereas the GDP-bound forms interact poorly, regardless of the presence or absence of TPs. Thus, Becker et al. (2004b) proposed that the TOC complex is stabilized by GTP, and that Toc159-GDP (or Toc86-GDP) promotes the dissociation of Toc34 from the complex.
The motor model is drawn from the results described above, and from additional results suggesting that Toc159 is a GTP-powered import motor (see section entitled, ‘Translocation through the TOC complex’) (Schleiff et al., 2003b). The model proposes that Toc34-GTP initially recognizes and binds to the phosphorylated C-proximal region of an incoming TP. Close association between Toc34-GTP and Toc159-GTP would then be induced by the binding of the N-proximal part of the phosphorylated TP to Toc159. Hydrolysis of GTP by Toc34 subsequently causes the release of the C-proximal region of the TP, allowing an unidentified phosphatase to dephosphorylate the Toc159-bound TP. After dephosphorylation, the C-proximal part of the TP also binds to Toc159, promoting its GTPase activity. GTP hydrolysis by Toc159 induces a change in conformation of the protein, which, in turn, promotes dissociation of Toc34-GDP from the TOC complex and the insertion of the TP into the Toc75 channel. Toc159 is then proposed to undergo multiple rounds of GTP binding and hydrolysis in order to mediate the full translocation of the preprotein through the OEM. All of these events are summarized in Fig. 2B.
Comparison between the models:
The models presented above differ significantly in their mechanistic details, but nonetheless are in agreement on larger issues. In both cases, an interaction between the GTPase receptors is believed to co-ordinate, in step-wise fashion, GTP-regulated preprotein recognition at the TOC complex. In addition, Toc159 is recognized as a key player mediating steps in preprotein recognition and/or insertion into the Toc75 channel. In order to gain a better understanding of the initial processes controlling protein import, however, important functional details will need to be resolved. Evidently, further investigation will be required to confirm the localization of the Toc159 receptor protein. The effects of GTP and GDP on receptor interaction at the TOC, in the presence and absence of preproteins, will also need to be clarified; Smith et al. (2002) proposed that the Toc34 and Toc159 G-domains interact with greater affinity in the presence of GDP, whereas Becker et al. (2004b) suggested that GDP destabilizes the TOC complex and leads to the dissociation of Toc34. Most likely, the context in which these processes are assessed (i.e. in the membrane versus in the solution, or in the presence versus in the absence of certain Toc159 domains) has a bearing on the results obtained. In addition, further details on the effects that GTP binding and hydrolysis have on the conformation of Toc159 will be required in order to determine how preprotein translocation is initiated.
Preprotein recognition specificity by the Toc GTPases
In Arabidopsis, small gene families encode homologues of pea Toc34 and Toc159 (termed psToc34 and psToc159, respectively). Two Arabidopsis genes, atTOC33 and atTOC34, encode homologues of psToc34, whereas four genes, atTOC159, atTOC132, atTOC120, and atTOC90, encode homologues of psToc159 (Jarvis et al., 1998; Bauer et al., 2000; Hiltbrunner et al., 2001a; Jackson-Constan and Keegstra, 2001). The atToc33 and atToc34 proteins are both highly similar to psToc34 (59% and 64% identity, respectively) as well as to each other (61% identity) (Jarvis et al., 1998). The Toc159 homologues show more variation, particularly across the A-domain, which varies greatly in length and sequence between the different isoforms. However, the G- and M-domains of each homologue display significant levels of homology with psToc159. The atToc159 isoform shows the greatest level of homology to psToc159 (48% identity overall, 74% across the G-and M-domains), indicating that the two proteins are functional orthologues (Bauer et al., 2000). The G- and M-domains of atToc132 and atToc120 are very similar. These two receptors show greater homology to each other than to atToc159, and thus appear to form a subgroup within the Toc159 family (Ivanova et al., 2004; Kubis et al., 2004). Lastly, atToc90 is the only Toc159 homologue that lacks a true A-domain; instead, it carries a short, non-acidic N-terminal extension. Based on its G- and M-domains, atToc90 may be slightly more closely related to atToc159 than to atToc132 and atToc120 (Ivanova et al., 2004). However, a phylogenetic analysis conducted by Kubis et al. (2004) suggested that atToc90 represents a unique subtype of Toc159-related proteins, distinct from the atToc159- and atToc132/atToc120-related subgroups.
The discovery of these small gene families in Arabidopsis, together with the identification and characterization of mutants for these receptors, led to the proposal that the TOC receptors may assemble together in various combinations to form TOC complexes with some level of preprotein recognition specificity (Jarvis et al., 1998; Bauer et al., 2000; Hiltbrunner et al., 2001a). The idea that different import pathways may exist for different classes of preproteins was proposed by Wan et al. (1996) after they compared the import efficiencies of different preproteins into leucoplasts and chloroplasts. The study revealed that some precursor proteins are imported with similar efficiency into both types of plastids, whereas others are preferentially imported into one type or the other. It was also shown that the TPs of these precursors were the principal determinants of this differential import behaviour, suggesting that the different plastid types may have different TP recognition preferences. The properties of TPs that could define their differential behaviour are unknown at present. However, the research reviewed below has provided some insight into how the different TOC GTPase isoforms can associate together in various combinations and differentially recognize certain classes of precursor proteins.
Arabidopsis Toc34 homologues
The first identified Arabidopsis plastid protein import mutant, ppi1, was originally identified in a screen for mutants displaying reduced expression of nucleus-encoded photosynthetic genes (Jarvis et al., 1998). The ppi1 mutant carries a T-DNA insertion that abolishes the expression of the atTOC33 gene. Young plants homozygous for the insertion display a uniformly pale phenotype caused by a defect in chloroplast biogenesis. As the plant becomes older, mature leaves develop a greener appearance similar to wild-type leaves, whereas young expanding leaves still display a chlorotic phenotype. This late greening of the leaves is associated with late maturation of leaf chloroplasts, indicating that chloroplast development is impeded in ppi1. The small chloroplasts with poorly developed thylakoid membranes observed in young leaves of ppi1 were found to display a reduced capacity to import proteins, whereas ppi1 chloroplasts isolated from older leaves imported proteins as efficiently as wild-type chloroplasts of the same age. The capacity of chloroplasts to import proteins is developmentally regulated: high in young leaf tissue where immature chloroplasts divide and develop, and low in older leaves where chloroplasts have reached maturity (Dahlin and Cline, 1991). The observations described above therefore suggested that atToc33 is necessary for efficient import of chloroplast proteins in young, expanding tissues.
Analysis of the expression levels of atTOC33 and atTOC34 in developing seedlings revealed that both genes were developmentally regulated, showing higher levels of expression in young seedlings than in older plants. By comparing the levels of steady-state mRNA found in young and old leaves of older plants, both genes were found to be up-regulated in young developing leaves. In addition, atTOC33 was expressed at 5-fold higher levels than atTOC34, in both young and old leaves. This led to the conclusion that atToc33 is important to cope with the high import requirements associated with rapid chloroplast biogenesis in young, expanding green tissues (Jarvis et al., 1998). Furthermore, because atToc34 was found to be able to complement ppi1 when expressed using a strong, constitutive promoter, it was proposed that atToc33 and atToc34 share significant functional similarity. Although the presence of two isoforms of Toc34 could be explained simply by the fact that having two genes might enable expression to be more easily tailored to suit developmental and tissue-specific requirements than would be the case with a single gene, the possibility that functional differences exist between the two proteins was not ruled out.
More detailed expression studies of atTOC33 and atTOC34 further established that the two genes display different patterns of expression (Gutensohn et al., 2000; Kubis et al., 2003). Promoter-GUS fusions and in situ hybridization studies revealed that the two genes are expressed differentially in tissues of various organs including roots, stems, and flowers (Gutensohn et al., 2000). In addition, quantitative and comparative analyses of the steady-state expression levels of both genes in different tissues and at different developmental stages confirmed that atTOC33 is strongly up-regulated in young, developing green tissues (Kubis et al., 2003). By contrast, atTOC34 expression was considerably lower than that of atTOC33, and was relatively more uniform. Interestingly, in roots, the two genes were found to be expressed at similar levels, indicating that atTOC34 may play a relatively more important role in root plastid development than in chloroplast biogenesis. Consistent with this idea was the finding that root plastid development does not seem to be affected in the ppi1 mutant (Yu and Li, 2001).
Experimental evidence suggesting that the two proteins display a certain level of functional specificity has also been presented. Gutensohn et al. (2000) initially showed that soluble atToc33, but not atToc34, can compete with chloroplasts for the binding of RbcS preproteins in the presence of GTP. It was further revealed, by testing the affinity of atToc33 and atToc34 for various preproteins in vitro, that the two Toc34 receptor homologues display some level of recognition specificity towards the TPs of different precursors (Jelic et al., 2003). In addition, preproteins that displayed greater binding affinity for one of the two Toc34 homologues also had a greater stimulatory effect on the GTPase activity of the same receptor. Interestingly, the binding-affinity and GTPase-stimulatory preferences of the different preproteins varied considerably, such that some displayed strong preference for one of the two receptors (e.g. ferredoxin NADP oxidoreductase for atToc33, and ATP synthase 
subunit for atToc34), whereas others seemed to show relatively lower, but equal affinity for both receptors (e.g. chlorophyll a oxygenase).
The question of functional specificity between the two Arabidopsis Toc34 receptor homologues has also recently been addressed in vivo by taking advantage of the ppi1 mutant, other similar T-DNA insertion mutants, and transgenic antisense lines (Kubis et al., 2003; Constan et al., 2004b; Gutensohn et al., 2004). Because the atTOC33 gene is strongly up-regulated in tissues where chloroplast biogenesis occurs, it was suggested that the atToc33 receptor may be involved specifically in the import of highly-expressed photosynthetic proteins (Kubis et al., 2003). Characterization of the ppi1 chloroplast protein content by immunoblotting to determine the levels of representative nucleus-encoded photosynthetic (e.g. RbcS, and light-harvesting chlorophyll a/b binding protein, or LhcII) and non-photosynthetic (e.g. stromal Hsp70 and triose phosphate/phosphate translocator) proteins suggested that ppi1 may be specifically deficient in photosynthetic proteins. This was further confirmed by taking a proteomics approach in which the ppi1 chloroplast proteome was compared with that of wild-type plants by difference gel electrophoresis (DIGE). Identification of proteins that were of altered abundance in ppi1 chloroplasts, by mass spectrophotometry, revealed that all deficient proteins were components of the photosynthetic apparatus, whereas enriched proteins were non-photosynthetic (Kubis et al., 2003).
Interestingly, transcriptomics analysis of ppi1 revealed that nuclear genes for photosynthetic chloroplast genes were moderately but specifically down-regulated in the mutant (Kubis et al., 2003). This observation is consistent with the criteria by which the ppi1 mutant was identified, but raised the possibility that reduced photosynthetic gene expression, rather than a specific import defect, might be responsible for the photosynthetic protein accumulation defect of ppi1. By determining the import rates of one non-photosynthetic and two photosynthetic preproteins, Kubis et al. (2003) established that the reduced level of photosynthetic proteins in ppi1 chloroplasts is at least partly due to a specific import defect: it was found that the import rate of both photosynthetic preproteins was significantly reduced in ppi1 chloroplasts whereas the non-photosynthetic preprotein was imported as efficiently as in wild-type chloroplasts. This led to the conclusion that the ppi1 mutant is specifically defective in the import of photosynthetic chloroplast proteins (those linked directly or indirectly to photosynthesis, as opposed to housekeeping proteins or proteins involved in unrelated, metabolic pathways which are more-or-less constitutively expressed) and that the reduced expression of photosynthetic genes in the mutant is most likely an adaptive response to this import defect.
A role of atToc33 as a specific receptor for photosynthetic preproteins and, by extension, that of atToc34 as a non-photosynthetic preprotein receptor, has recently been addressed by Gutensohn et al. (2004), who identified and characterized a new knockdown allele of atToc33. This allele carries a T-DNA insertion within the first exon of the gene, in the upstream untranslated region, resulting in near complete loss of atToc33 expression and in a phenotype similar to that of ppi1. By taking similar immunoblotting and proteomics approaches to those taken by Kubis et al. (2003), it was shown that photosynthetic proteins are, by and large, less abundant in the mutant than in the wild type, although some did appear to be unaffected. When the expression of atToc34 was also repressed, by the introduction of an antisense transgene into the atToc33 knockdown mutant, it was revealed that the photosynthetic proteins unaffected in the Toc33 mutant were now significantly reduced. This result suggests that the proposed specificities of atToc33 and atToc34 for photosynthetic and non-photosynthetic preproteins, respectively, are not absolute.
From the results summarized above, it appears clear that the atToc33 and atToc34 receptor proteins display at least some level of preprotein recognition specificity. However, the functional complementation of ppi1 by the overexpression of atToc34, as described previously, strongly suggests that the receptors are unlikely to be totally exclusive for particular preprotein types. The recent identification and characterization of ppi3, a null T-DNA insertion mutant of atToc34 further supports these general conclusions (Constan et al., 2004b). The only phenotype reported for ppi3 consists of a slight reduction in root growth, further supporting the prediction that atToc34 may be involved in root plastid development. However, a ppi1 ppi3 double homozygous mutant was revealed to be embryo lethal. This indicates that the Toc34 homologues are essential for plastid biogenesis, and that atToc34 is probably responsible for the residual level of chloroplast development observed in ppi1. The latter conclusion was supported by the observation that ppi1 homozygotes bearing just a single copy of the atTOC34 gene (genotype: ppi1/ppi1; +/ppi3) exhibit more severe chloroplast biogenesis defects than ppi1 single mutants (Constan et al., 2004b). This further demonstrates that these proteins are not exclusive receptors for particular preprotein types. It is possible that the specificity of the TOC for certain precursors is not determined by individual receptors, but by a combinatorial effect involving both Toc34 and Toc159 homologues (see below).
Arabidopsis Toc159 homologues
The second Arabidopsis plastid protein import mutant to be identified, termed ppi2, is a null T-DNA insertion mutant of atTOC159 (Bauer et al., 2000). Initial RNA blot analysis of steady-state mRNA levels indicated that atTOC159 is expressed at a level approximately 7-fold higher than atTOC132 and atTOC120, in both green and etiolated young, wild-type seedlings. In addition, the expression of all three receptors appeared to be stimulated roughly 2-fold by light. These results suggested that atToc159 is probably the predominant Toc159 receptor homologue during seedling development. This is in line with the finding that ppi2 is seedling-lethal, since it develops albino cotyledons but fails to produce first true leaves when grown on soil. Remarkably, however, growth on medium supplemented with sucrose can partially ‘rescue’ the mutant, allowing it to develop into a small bleached plant. This indicates that it can still carry out essential plastid functions other than photosynthesis. Indeed, transmission electron micrographs of the plastids in ppi2 cotyledons indicated that these remained largely undifferentiated, lacking thylakoid membranes which are a specific characteristic of chloroplasts associated directly with photosynthesis.
Further characterization of the mutant revealed that the major photosynthetic proteins, namely RbcL, RbcS, and LhcII, were present in ppi2 plastids, but only at much lower levels than in wild-type chloroplasts. This indicated that, although accumulation of photosynthetic proteins was severely reduced in plastids lacking atToc159, these organelles were still capable of importing these proteins. By contrast, non-photosynthetic proteins such as Toc75 and Tic110 (two nucleus-encoded chloroplast proteins known to be inserted into the envelope after import through the TOC) appeared to accumulate normally. This led to the conclusion that atToc159 is an essential receptor for the efficient import of photosynthetic chloroplast preproteins, and for chloroplast biogenesis. In addition, it was proposed that other Toc159 homologues are responsible for the normal accumulation of non-photosynthetic plastid proteins, as well as for the limited accumulation of photosynthetic proteins observed.
Recently, more direct evidence has been presented supporting a specific role for atToc159 in the import of photosynthetic preproteins (Smith et al., 2004). Since it is probably impossible to isolate sufficient plastids from ppi2 seedlings to assess protein import in vitro, Smith et al. (2004) used an alternative in vivo approach based on transgenic expression of TP-GFP fusion proteins. The TPs of pRbcS and the pyruvate dehydrogenase E1 subunit precursor (pE1) were used to generate photosynthetic and non-photosynthetic TP-GFP fusions, respectively. The endogenous forms of these two proteins were initially shown to display reduced and normal accumulation, respectively, in ppi2 seedlings compared with the wild type. The fusions were cloned downstream of the strong, constitutive 35S cauliflower mosaic virus promoter and introduced into ppi2 plants. Analysis of the import efficiency of each preprotein by immunoblotting and confocal microscopy revealed that pE1-GFP was efficiently imported into ppi2 plastids, whereas pRbcS-GFP was not, instead accumulating as an unprocessed preprotein. Because the 35S promoter displays constitutive activity and is not affected by adaptive responses of the plant to import defects, the differential accumulation of the two preproteins was most likely due to the specificity of the import defect in ppi2 plastids. Therefore, in combination with the previous data (Bauer et al., 2000), these results provided strong evidence that the atToc159 is a selective receptor for photosynthetic chloroplast proteins.
The molecular basis of the specific defect in the import of photosynthetic preproteins observed in ppi2 was shown to lie, at least in part, in the preferential binding of atToc159 to the TP of photosynthetic preproteins (Smith et al., 2004). In vitro, specific binding of atToc159 to immobilized fusion proteins carrying the TP of either RbcS or ferredoxin (Fd), two photosynthetic proteins, could be efficiently competed by adding soluble photosynthetic preproteins. By contrast, addition of soluble non-photosynthetic preproteins, including pE1 and the L11 ribosomal protein precursor (pL11), had no inhibitory effect. Recently, a similar strategy was used to demonstrate that atToc132 displays significantly greater binding affinity for the TP of a non-photosynthetic preprotein (pE1) than that of a photosynthetic preprotein (pRbcS) (Ivanova et al., 2004). This was the first biochemical evidence suggesting that Toc159 receptor homologues, other than atToc159, are likely to function as preprotein receptors. Furthermore, this finding provided strong support for the proposal that the other Toc159 homologues are responsible for the efficient import of non-photosynthetic preproteins observed in ppi2.
More evidence in support of the presence of multiple import pathways at the OEM has recently been provided by the identification and characterization of knockout mutants for the other Toc159 homologues in Arabidopsis (Hiltbrunner et al., 2004; Ivanova et al., 2004; Kubis et al., 2004). Homozygous knockout mutants for atToc120 (toc120) and atToc90 (toc90 or ppi4) have been reported to display no phenotypic differences from wild-type plants. However, atToc132 knockout mutants (toc132) were reported to have a subtle chlorotic phenotype in young seedlings, which develops into a clear yellow-green and reticulate phenotype in older, soil-grown plants (Kubis et al., 2004). These observations indicate that none of the three corresponding Toc159 isoforms are essential for normal development, and suggest that their absence can be compensated for (only partially in the case of toc132) by the remaining Toc159 isoforms.
Double mutants for the different Toc159 homologues have also been generated, in all possible combinations, to determine the functional relationships between them. Interestingly, a strong relationship was observed between toc132 and toc120, producing double homozygotes with a severely chlorotic phenotype (Ivanova et al., 2004; Kubis et al., 2004; D Schnell, personal communication). Such an effect suggests that the corresponding proteins are redundant in function. This was further confirmed by the finding that overexpression of either atToc132 or atToc120 in a toc120 toc132 double mutant fully complements the pale phenotype of these plants (Kubis et al., 2004). Functional redundancy between atToc132 and atToc120 is in line with the observation that these proteins are the two most closely-related Arabidopsis Toc159 homologues, and with the fact that they are both expressed at relatively low and uniform levels in different tissues and across different developmental stages (Ivanova et al., 2004; Kubis et al., 2004). In addition, the fact that overexpression of atToc159 does not complement the toc120 toc132 double mutant further indicates that atToc159 and atToc132/atToc120 participate in different import pathways (Kubis et al., 2004). This is consistent with the originally proposed function of atToc120 and atToc132 as import receptors for non-photosynthetic proteins (Bauer et al., 2000), as well as with the biochemical data of Smith et al. (2004) and Ivanova et al. (2004).
Curiously, toc90 was not found to generate any distinct, new phenotypes when combined with the other Toc159 homologue mutants (Kubis et al., 2004). In addition, its overexpression did not result in any significant complementation of either ppi2 or the toc132 toc120 double mutant. This observation indicates that atToc90 does not share substantial functional redundancy with any of the other homologues. Moreover, the expression pattern of atTOC90 differs from those of atTOC159 and the atTOC132/atTOC120 subgroup in that it is uniformly expressed at a relatively high level in all tissues and during all developmental stages (Kubis et al., 2004). However, it was also shown that atTOC90 expression is up-regulated approximately 2-fold by light (Hiltbrunner et al., 2004), much like the expression of atTOC159 (Bauer et al., 2000; Ivanova et al., 2004). A possible explanation for these results is that atToc90 only plays a non-essential, accessory or supportive role in import that is not specific to any particular pathway.
Association of Toc159 and Toc34 isoforms into different TOC complexes
Additional biochemical evidence supporting the idea that atToc159 and the atToc120/atToc132 subgroup of Toc159 homologues function in different import pathways has been provided by assessing their association with Toc34 homologues. Because the insertion of Toc159 into the OEM was previously shown to be assisted by Toc34, Ivanova et al. (2004) investigated the possibility that the different Arabidopsis Toc159 homologues might associate preferentially with different Toc34 homologues. These experiments were particularly relevant since atToc132 and atToc120 were both found to partition between soluble, cytosolic, and envelope-associated pools, like Toc159 (Ivanova et al., 2004). Remarkably, co-immunoprecipitation experiments using specific antibodies for atToc159, atToc132, and atToc120 established that atToc159 assembles preferentially with atToc33 and Toc75 to form one subclass of TOC complexes, whereas atToc132, and atToc120 associate preferentially with atToc34 to form another. Interestingly, this preferential association of the TOC receptors does not appear to be exclusive since small amounts of Toc34 were co-immunoprecipitated with atToc159, and vice versa. To determine how this preferential association of the receptors was determined, Ivanova et al. (2004) assessed the interactions between the atToc159 homologues (excluding atToc90) and the Toc34 homologues in a solid-phase interaction assay. The atToc159 receptor was found to bind atToc33 with very high affinity, and atToc34 with very low affinity; by contrast, atToc132 and atToc120 displayed an equal, intermediate affinity for both atToc33 and atToc34. This revealed that the pairing of the receptors was most likely determined by preferential binding of atToc159 to atToc33, which, as a consequence, partially excludes binding of atToc132 and atToc120 to atToc33, favouring their binding to atToc34.
The preferential association of atToc159 with atToc33, in combination with the fact that these two components are both strongly expressed in young green tissues, and with the various genetic data described above, strongly suggests that they associate together to form TOC complexes specialized for the import of highly-expressed, photosynthetic preproteins during chloroplast biogenesis. Interestingly, this also raises the possibility that, in ppi1, the specific defect in the import of photosynthetic preproteins may be due, at least in part, to an atToc159 accumulation/targeting defect at the chloroplast OEM in these plants. Since atToc132, atToc120, and atToc34 all share largely similar expression patterns (Kubis et al., 2003, 2004; Ivanova et al., 2004), they are likely to associate together to form TOC complexes specialized for the import of non-photosynthetic, general plastid proteins. This fits very well with the hypothesis that preprotein recognition specificity amongst the TOC receptors evolved to prevent the import of essential but low-level plastid proteins from being out-competed by the import of highly-abundant photosynthetic proteins during chloroplast biogenesis (Bauer et al., 2000). These ideas are summarized in Fig. 3.
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The co-immunoprecipitation experiments performed by Ivanova et al. (2004)
revealed that neither atToc120 nor atToc132 was associated with atToc159. Therefore, TOC complexes carrying the atToc159 receptor are envisaged to be pure atToc159 complexes. By contrast, significant amounts of atToc120 were found to co-immunoprecipitate with atToc132, and vice versa, indicating that these two receptor components can associate together to form mixed atToc132/atToc120 TOC complexes, possibly in addition to ‘pure’ atToc120 and atToc132 complexes. This can be taken as further evidence that atToc120 and atToc132 perform very similar functions by recognizing preproteins with similar TPs. These results are also noteworthy regarding the structure of the TOC complex, since they indicate that at least two Toc159 homologues can associate together in a stable fashion within the same complex. This suggests that individual TOC complexes, contrary to what was proposed by Schleiff et al. (2003c), are not composed of Toc75, Toc34, and Toc159 arranged in a simple 4:4:1 stoichiometry. Either the proposed stoichiometry is correct and individual complexes are made up of at least twice the number of components (8:8:2) or, alternatively, the predicted stoichiometry is incorrect. Another possibility is that TOC complexes with specificity for non-photosynthetic preproteins have a different structural organization. Overall, the new findings reviewed in this section strongly suggest that different protein import pathways across the chloroplast OEM exist. Preproteins imported via these different pathways appear to be translocated through similar TOC channels that are simply associated with different combinations of TOC receptor isoforms. Recognition specificity at the TOC is mediated by the preferential binding of receptors to the TPs of different preproteins. Assembly of the receptor isoforms at the TOC channel appears to occur in a preferential, but non-restrictive manner; association preference seems to be governed by atToc159, which displays high affinity for atToc33 and low affinity for atToc34. Two distinct pathways have been defined so far: one with preference for photosynthetic preproteins, defined by atToc33 and atToc159, and another with preference for non-photosynthetic preproteins, defined by atToc34 and atToc132/atToc120. However, it is possible that other, more specific pathways exist, particularly for different subsets of non-photosynthetic proteins, given the possibility of various arrangements between atToc33/atToc34 and atToc132/atToc120.
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Translocation through the outer envelope membrane |
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After binding to the TOC receptors, a chloroplast preprotein destined to the internal sub-compartments is transferred in a GTP-dependent manner to Toc75 and translocation is initiated. It is generally accepted that Toc75 plays a major role in the formation of a channel that allows unfolded preproteins to cross the hydrophobic lipid bilayer of the OEM (Fig. 1).
The TOC channel
Toc75 is the predominant protein cross-linked to early import intermediates, indicating that it is in close proximity to the preprotein as it progresses through the OEM (Perry and Keegstra, 1994; Ma et al., 1996). It was also found to co-purify with arrested translocation intermediates isolated from outer envelopes by solubilization, further indicating that it associates closely with translocating preproteins (Schnell et al., 1994). In addition, antibodies raised against Toc75 were shown to inhibit import of preproteins (Tranel et al., 1995). Together, these results provided strong evidence for a role of Toc75 in chloroplast protein import. Initial characterization of Toc75 demonstrated that it behaves as an integral OEM protein, resistant to extraction by salt or high pH treatment (Schnell et al., 1994; Tranel et al., 1995). It was further found to be protected from thermolysin treatment, and to produce a large 52 kDa proteolysis-protected fragment after trypsin treatment (Schnell et al., 1994; Tranel et al., 1995); these two proteases are frequently used to analyse the topology of chloroplast envelope proteins, since thermolysin cannot penetrate the OEM whereas trypsin damages the OEM and gains access to proteins exposed in the IMS but not the stroma (Cline et al., 1984; Jackson et al., 1998). The proteolysis data indicated that Toc75 is largely embedded in the OEM, and led to the early suggestion that it might form the protein translocation channel of the OEM (Schnell et al., 1994).
Initial analysis of the amino acid sequence of Toc75 revealed that it probably contains multiple membrane-spanning ß-strands allowing it to form a ß-barrel (Schnell et al., 1994). Topology studies using various proteases and computational tools further suggested that Toc75 is likely to form either 16 (Sveshnikova et al., 2000a) or 18 (Schleiff et al., 2003c) amphiphilic ß-strands. Reconstitution of recombinant Toc75 protein and electrophysiological experiments confirmed that Toc75 forms a membrane channel. Initially, patch-clamp studies in liposomes suggested that the constriction site of Toc75 was 8–9 Å in diameter (Hinnah et al., 1997), a size sufficient to allow the translocation of only completely-unfolded proteins. More recently, however, improvement of the reconstitution technique allowed electrophysiological studies to be carried out in planar lipid bilayers (Hinnah et al., 2002). These studies suggested that the constriction site of the pore was 14 Å in diameter, and would therefore allow the translocation of partially-folded preproteins. These data are broadly consistent with the previous observation that the translocation channels of the envelope are sufficiently flexible to allow the passage of a precursor carrying a permanently folded domain 23 Å in diameter (Clark and Theg, 1997). The corrected pore size measurement is also more comparable with those of other protein translocation channels. For example, Tom40 of the mitochondrial outer membrane, and the flexible Sec61 complex of the ER, were estimated to form channels of 22 Å and 6–60 Å diameter, respectively (Hill et al., 1998; Wirth et al., 2003). Hinnah et al. (2002) also showed that the Toc75 channel is cation-selective, and can differentiate between chloroplast TPs and mitochondrial presequences. This indicates that Toc75 does not bind to preproteins based solely on electrostatic interactions with the positively-charged TP, and suggests that it also selects for other characteristics such as conformation (Hinnah et al., 2002).
Remarkably, Toc75 is the only TOC component identified so far that displays significant homology (22% identity) with a cyanobacterial protein (Bölter et al., 1998b; Reumann et al., 1999). While this homology suggests that the plastid OEM is of bacterial origin, the lack of other, similar homologous relationships indicates that the Toc machinery has evolved from a combination of endosymbiont and host cell proteins. Although Toc75 and its Synechocystis homologue, SynToc75, are quite dissimilar in sequence, SynToc75 has been shown to form a channel with in vitro functional properties very similar to those of psToc75 (Bölter et al., 1998a). Interestingly, SynToc75 was recently proposed to carry three repetitive polypeptide-transport-associated (POTRA) domains at its N-terminus (Sánchez-Pulido et al., 2003). POTRA domains are composed of three ß-strands and two -helices, and are normally associated with ß-barrel membrane proteins. They are present in bacterial channel proteins that are evolutionarily-related to SynToc75, in which they are found to associate with unstable or unfolded proteins. This led to the suggestion that POTRA domains carry out a chaperone function. At present, it remains unclear if such domains are also present in plastidic Toc75. If such a chaperone domain was at the IMS face of the channel, however, it could help mediate translocation and transfer of the unfolded preproteins to the TIC.
Three closely-related homologues of pea Toc75 (psToc75) have been identified in Arabidopsis (Jackson-Constan and Keegstra, 2001). One of these, atToc75-III, is highly similar (73% identity) to psToc75 throughout its mature sequence, whereas the other two, atToc75-I and atToc75-IV, appeared to have significant N-terminal truncations (Inoue and Potter, 2004). Recently, atTOC75-III has been shown to display a developmental expression pattern similar to that originally observed for psToc75 (Tranel et al., 1995), with high steady-state mRNA levels in young, developing green tissues and significantly reduced levels in older tissues (Baldwin et al., 2005). In addition, of the two other homologues, only atTOC75-IV was found to be expressed; atTOC75-I was shown to be a pseudogene due to the presence of a retrotransposon insertion. The atTOC75-IV gene is expressed at a very low and uniform level throughout development and in different tissues (Baldwin et al., 2005). Together, these observations suggest that atToc75-III may be the unique functional orthologue of psToc75 in Arabidopsis. This is further supported by the finding that a null allele of atTOC75-III is embryo-lethal in the homozygous state, indicating that atToc75-III is essential for plastid and embryo development (Baldwin et al., 2005). By contrast, homozygous atTOC75-IV knockouts appear to be completely normal during growth in the light. Nevertheless, these toc75-IV mutants do display significant defects following growth in the dark, especially during de-etiolation, suggesting an important role for the protein in etioplasts (Baldwin et al., 2005). However, the precise role of the atToc75-IV protein remains to be determined.
Translocation through the TOC complex
Three putative models can be envisaged for the translocation of preproteins through the TOC. The first, the binding chain hypothesis, is drawn by comparison with protein translocation through the mitochondrial outer membrane. In this system, it has been proposed that translocation is driven by the passage of the presequence along a sequence of different binding sites of increasing affinity on the TOM translocase (Komiya et al., 1998; Meisinger et al., 2001). At the moment, no direct evidence has been provided for the existence of such a binding chain along the TOC. However, since chloroplast protein precursors carry a similar N-terminal signal sequence, it seems possible that such a translocation mechanism could occur. The fact that the TOC and TIC have been shown to interact physically during the translocation of stromal preproteins to form a continuous channel through both envelope membranes (Akita et al., 1997; Nielsen et al., 1997), makes this hypothesis an attractive possibility. As in mitochondria, this would allow the TP to reach the TIC components and initiate translocation through the IEM.
Recently, experimental evidence for a second model, the GTP motor hypothesis, has been presented (Schleiff et al., 2003b). In this model, as previously mentioned, Toc159 is proposed to act as a GTP-driven motor that pushes the preprotein through the TOC channel. The study by Schleiff et al. (2003b) used purified and reconstituted core TOC complexes and individual TOC components to determine the minimal requirements for translocation. They showed that proteoliposomes loaded with both Toc86 (the proteolytic fragment of Toc159) and Toc75 were able to import preproteins in a GTP-dependent manner. Since proteoliposomes loaded with Toc34 and Toc75 in combination, or Toc75 alone, were not able to support translocation, it was concluded that Toc159 and Toc75 make up the minimal translocase. To explain the GTP dependence of this minimal translocase, it was proposed that GTP hydrolysis by Toc159 (or Toc86) induces a change in conformation that would lead to the insertion of the preprotein into the TOC channel. By completing several rounds of GTP-hydrolysis and GDP/GTP exchange, Toc159 would drive the translocation of the preprotein across the TOC channel into the proteoliposome (Fig. 2B).
The GTP motor model is reminiscent of protein translocation across the bacterial cell membrane, where the ATPase, SecA, drives preprotein translocation by going through cycles of membrane insertion and de-insertion that push the preprotein through the Sec channel (Vrontou and Economou, 2004). However, the central role of the GTPase domain implied by this model is in disagreement with previously presented data indicating that the Toc159 GTPase domain is not essential for protein import in vitro (Chen et al., 2000a). In addition, the fact that non-hydrolysable GTP analogues still allow protein translocation in vitro further demonstrates that GTP hydrolysis is not essential for protein transport to occur (Kessler et al., 1994; Young et al., 1999). Moreover, the recent demonstration that the M-domain of Toc159 is sufficient to partially complement the ppi2 mutant argues strongly against an essential role for the G-domain of Toc159 in driving translocation (Lee et al., 2003). Together, these results seem to indicate that only the M-domain of Toc159 is essential for protein translocation to occur and that, in the reconstituted proteoliposomes used by Schleiff et al. (2003b), GTP-hydrolysis was required for the M-domain to achieve the correct conformation to allow translocation. It has recently been suggested that the M-domain of Toc159 inserts into the membrane as a ß-barrel structure (Schleiff et al., 2003a), and so could potentially participate in the structure of the TOC channel. In addition, it is possible that the chaperone function of putative Toc75 POTRA domains may be sufficient to drive the low levels of translocation observed in proteoliposomes (Sánchez-Pulido et al., 2003).
Neither of the two previously described models account for the fact that a low concentration of ATP (<100 µM) is required in the IMS to promote maximal formation of early translocation intermediates (Olsen et al., 1989; Olsen and Keegstra, 1992; Young et al., 1999). This ATP requirement, together with the finding by several groups that an Hsp70 protein associates with the TOC complex (Marshall et al., 1990; Waegemann and Soll, 1991; Schnell et al., 1994), forms the basis for the third model: the IMS chaperone motor hypothesis. In this model, an OEM-associated Hsp70 protein linked with the TOC is proposed to utilize ATP to bind to incoming preproteins, promote their forward movement through the channel, and assist in their transfer to TIC components. Strong evidence in support of this model has recently been presented by the identification of Toc12, a new component of the TOC complex (Becker et al., 2004a). Toc12 is a small DnaJ-like protein anchored to the OEM by its N-terminus, exposing its C-terminal J-domain in the IMS. DnaJ is a co-chaperone known to associate with Hsp70, and to stimulate its ATPase activity via its J-domain (Walsh et al., 2004). Interestingly, Toc12 was shown to be associated with the TOC complex and to promote the ATP-dependent binding of the Hsp70 to incoming preproteins in the IMS. This observation suggests a role for the OEM Hsp70 in the formation of early import intermediates, and offers an explanation for the ATP requirements of this step.
Hsp70s have previously been demonstrated to play a motor role in other protein translocation systems. For example, post-translational protein translocation across the ER membrane and the mitochondrial inner membrane are two processes known to require Hsp70 function at the trans side of the membrane, in the lumen and matrix, respectively. In both systems, the Hsp70 is known to function as an ATP-dependent protein import motor that drives the import of preproteins. In the ER, the Hsp70 protein, BiP, has been shown to function as a ratchet (Matlack et al., 1999). The preprotein slides back and forth through the Sec channel, as a result of Brownian movement, and Hsp70s bind to segments of the preprotein as they emerge from the translocon, thereby blocking retrograde movement and favouring forward movement into the ER lumen. In the mitochondria, the exact means by which mtHsp70 drives import is still under debate. Both a pulling model, in which the mtHsp70 actively pulls on incoming preproteins, and a ratchet model, like that described above for the ER, have been proposed to explain the motor function of mtHsp70 (Neupert and Brunner, 2002). By contrast with this mitochondrial system, the chloroplast OEM Hsp70 described above would, in most cases, only promote the transfer of preproteins to components of the TIC, and would not drive the complete translocation of preproteins. Complete translocation of preproteins across both envelope membranes is believed to be driven by an analogous chaperone system at the stromal face of the TIC, where greater levels of energy are known to be required for import (Theg et al., 1989).
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Formation of TOC–TIC supercomplexes |
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Translocation of preproteins can occur independently across each membrane, indicating that each membrane carries a fully functional translocase that can recognize and translocate preproteins (Scott and Theg, 1996
). However, early studies established that translocation of preproteins normally occurs simultaneously through the OEM and IEM of the chloroplast (Schnell and Blobel, 1993; Perry and Keegstra, 1994; Schnell et al., 1994; Wu et al., 1994). This was initially shown when late translocation intermediates spanning both membranes were identified (Schnell and Blobel, 1993). The N-termini of these intermediates had reached the stroma where the TP was processed, but their C-terminal ends were still accessible to thermolysin proteolysis at the cytosolic face of the chloroplast. In addition, translocation was shown to occur in distinct patches at the chloroplast surface, called contact sites, where the outer and inner membranes of the envelope are in close physical proximity (Schnell and Blobel, 1993; Perry and Keegstra, 1994)
Contact sites are generally believed to represent areas of the chloroplast surface where the TOC complexes interact with TIC complexes to facilitate the direct translocation of preproteins from the cytosol to the stroma. It has been shown that the TOC and TIC components can be cross-linked together in the presence of translocating preproteins (Akita et al., 1997). After formation of early import intermediates at low ATP concentration, and cross-linking, large supercomplexes containing both TOC and TIC components were isolated from solubilized chloroplast envelope membranes. Immunoprecipitation with antibodies against Toc75 and Tic110 (a component of the TIC; see below) indicated that both TOC and TIC complexes could be cross-linked to early import intermediates. Interestingly, only a proportion of TOC complexes appeared to be associated with TIC complexes, and this was found to be the case both in the presence and absence of added preproteins. Furthermore, TOC–TIC supercomplexes could also be isolated under native conditions (in the absence of cross-linkers) indicating that they represent stable associations, even in the absence of added preproteins (Nielsen et al., 1997; Kouranov et al., 1998).
Contact between the two complexes is probably mediated by the interaction of TOC and TIC components that protrude into the IMS, and by soluble IMS proteins that participate in protein translocation. Two TIC components, Tic20 and Tic22, were identified as proteins that associate with the TP of precursors under conditions that promote the formation of early import intermediates (Ma et al., 1996; Kouranov and Schnell, 1997; Kouranov et al., 1998). Tic22 appeared to interact with preproteins before Tic20, as they emerged from the TOC channel, and both proteins were found to be predominant interactors under conditions promoting full translocation (Kouranov and Schnell, 1997). Tic22 is a soluble IMS protein that associates peripherally with the IEM and is proposed to mediate the passage of preproteins to the TIC channel (Kouranov et al., 1998). By contrast, Tic20 is a highly hydrophobic integral protein believed to be largely embedded in the IEM, and is thus proposed to form a part of the TIC channel.
In immunoprecipitation studies, it was found that proportions of the Tic22 and Tic20 pools were in TOC–TIC supercomplexes, being associated with both the TOC complex and Tic110, again either in the presence or absence of added preproteins (Kouranov et al., 1998). Surprisingly, however, neither Tic22 nor Tic20 were found to immunoprecipitate with Tic110 outside of this supercomplex assembly. This led Kouranov et al. (1998) to propose that the assembly of functional TIC complexes and TOC–TIC supercomplexes may be mediated by the direct interaction of TIC components with the TOC complex. These complexes, and, consequently, contact sites of the chloroplast envelope, would be dynamic in nature, associating and disassociating in response to unknown factors. In mitochondria, the TOM and TIM complexes have also been found to interact together at contact sites between the outer and inner membranes, but these interactions only occur in the presence of precursors (Horst et al., 1995).
Recently, the identification of Toc12 and its interaction partners has provided some insight into how the formation of supercomplexes and contact sites might be induced (Becker et al., 2004a). An IMS translocase complex composed of, at least, Toc64, Toc12, Tic22, and the IMS-facing OEM Hsp70, was identified by sucrose density centrifugation of solubilized OEVs in the presence of ATP. The ATP requirement for the assembly of this IMS translocase can be attributed to Hsp70, which is only recruited to the TOC translocon by Toc12 in its ATP-bound state. Because Toc12 was shown to promote the ATP-dependent binding of Hsp70 to preproteins, and Toc64 is predicted to play a role as a guidance complex receptor, it is possible that the assembly of the IMS translocase is promoted by the arrival of a precursor protein at the TOC. In addition, because Tic22, a component associated with the TIC, is recruited in the assembly of the IMS translocase, it can be proposed that this further induces assembly of the TOC–TIC supercomplex (Fig. 1). If this is true, this would indicate that the formation of TOC–TIC supercomplexes is linked to the presence of preproteins and ATP. It is possible that, in the previously described studies, the observation of TOC–TIC supercomplexes in the absence of added precursors was due to the presence of endogenous translocating preproteins trapped during the chloroplast isolation process.
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Translocation through the inner envelope membrane |
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Several proteins have been identified as putative components of the TIC complex, but the functions of most of these components are still largely unclear. Two previously introduced components, Tic20 and Tic110, are proposed to form at least part of the TIC channel.
The TIC channel
Tic20 is distantly related to bacterial branched amino acid transporters and to the TIM channel proteins, Tim23, Tim22, and Tim17 (Reumann and Keegstra, 1999). Tim23 forms the channel of the TIM23 translocase, which mediates the translocation of proteins carrying an N-terminal presequence (Truscott et al., 2001). Tim22 forms the channel of the TIM22 translocase, which mediates the inner membrane integration of proteins carrying several membrane-spanning hydrophobic helices and internal signal sequences, such as the metabolite carriers of the mitochondrial inner membrane (Kovermann et al., 2002). All three TIM proteins are predicted to carry four transmembrane -helices (Rassow et al., 1999). The TIM23 translocase, to which the TIC translocase appears to be functionally analogous, possesses a channel of 13 Å, as deduced by reconstitution of Tim23 (Truscott et al., 2001). Tic20 is also predicted to span the membrane with four transmembrane -helices (Kouranov et al., 1998; Chen et al., 2002), and it is therefore possible that it forms a channel similar to that formed by Tim23. The ability of Tic20 to form such a channel, however, has not yet been demonstrated experimentally.
In Arabidopsis, four genes appear to encode homologues of pea Tic20 (psTic20). The atTic20-I homologue is highly similar to psTic20 (62% identity in regions aligning), whereas the other three putative homologues, atTic20-IV, atTic20-II and atTic20-V, are considerably less conserved (35%, 25%, and 25% identical, respectively) (Jackson-Constan and Keegstra, 2001; Chen et al., 2002). As a consequence, atTic20-I is believed to be the functional orthologue of psTic20. The atTIC20-I gene is expressed in most tissues, including roots, stems, leaves, flowers, and siliques (Chen et al., 2002). Its expression was found to be highest in young, rapidly-expanding tissues, which is consistent with the high protein import requirements of these tissues. Interestingly, atTIC20-I was shown to be expressed at similarly high levels in young, etiolated plants and light-grown seedlings, suggesting that Tic20 is required for general protein import into all types of plastids.
Functional analysis of Tic20 in vivo, in transgenic antisense Arabidopsis plants showing reduced atTIC20-I expression levels, revealed that Tic20 is probably essential for plant growth and development (Chen et al., 2002). A proportion (10%) of the primary transformants was found to develop albino cotyledons and, as observed in ppi2, did not survive beyond this stage. In contrast to ppi2, however, these albino seedlings could not be rescued on medium supplemented with sucrose, providing further evidence that Tic20 plays a role in a general import pathway. Other, less sick transformants displayed a strong chlorotic phenotype due to poor chloroplast development. Chloroplasts in these plants were smaller and contained poorly developed thylakoid membrane systems. It was further demonstrated that these defects were associated with a reduced rate of general chloroplast protein import. In support of a role of Tic20 as a component of the TIC channel, chloroplasts were shown to be specifically defective in translocation of proteins across the IEM.
Tic110, the other known TIC component proposed to form at least a part of the channel, was initially identified as an IEM protein that associates closely with late translocation intermediates (Schnell et al., 1994; Wu et al., 1994). Primary structure analysis suggested that Tic110 is composed of two hydrophobic transmembrane -helices at the N-terminus, and a large (97 kDa) hydrophilic C-terminal domain displaying no homology to any known protein (Kessler and Blobel, 1996; Lübeck et al., 1996). Tic110 is generally accepted as a core component of the TIC machinery, but its topology and precise function have been strongly debated. The large hydrophilic domain of Tic110 was initially proposed to protrude into the IMS, and to play a role in the assembly of TOC–TIC supercomplexes (Lübeck et al., 1996). A second model, supported by more thorough topology studies, proposes that the C-terminal domain protrudes into the stroma where it may recruit stromal factors involved in import (Kessler and Blobel, 1996; Nielsen et al., 1997; Jackson et al., 1998; Inaba et al., 2003). Finally, it has recently been proposed that the large hydrophilic domain of Tic110 forms a ß-barrel that serves as the protein import channel of the IEM (Heins et al., 2002).
The proposal that Tic110 forms a channel is derived from the observation that reconstitution of urea-denatured Tic110 into liposomes leads to the formation of a cation-selective channel (Heins et al., 2002). Structural analysis of both the full-length Tic110 protein and the large C-terminal domain by circular dichroism (CD) spectroscopy, after dilution in detergent, suggested that it is predominantly composed of ß-sheets, indicating that it may form a ß-barrel structure in the IEM. Electrophysiological analysis of the reconstituted C-terminal domain after its transfer into a planar lipid bilayer indicated the presence of a channel with similar properties to those of the channel formed by the full-length protein, as well as to those of a cation-selective channel present in IEM vesicles. The channel formed by reconstituted Tic110 was proposed to be 15 Å in diameter, suitable for the translocation of partially folded proteins, and was further shown to interact specifically with a positively charged TP, leading to blockage of the channel.
Curiously, Heins et al. (2002) were unable to reconstitute native Tic110 obtained from IEM vesicles into a functional channel, which raises questions about the physiological relevance of the electrophysiological data described above. The idea that Tic110 forms a ß-barrel channel was placed in further doubt, recently, by the characterization of Tic110 C-terminal domain expressed in bacteria and transgenic Arabidopsis plants (Inaba et al., 2003). In this study, the C-terminal end of the unique Arabidopsis homologue of Tic110, atTic110, was found to accumulate mainly as a soluble protein when expressed heterologously in Escherichia coli. Also, in contrast to the findings of Heins et al. (2002), CD spectroscopic analysis of this recombinant protein, in buffer lacking detergent, indicated that it was predominantly constituted of -helices. Moreover, the C-terminal end of Tic110 was found to accumulate as a soluble, stromal protein when a construct lacking the two putative, N-terminal transmembrane -helices was expressed in transgenic Arabidopsis plants. Together, these results strongly indicate that the C-terminus of Tic110 folds into a large, soluble domain.
Interestingly, Inaba et al. (2003) further demonstrated that the bulk of the endogenous atTic110 protein protrudes into the stroma, and is protected from trypsin proteolysis in isolated chloroplasts. In addition, they demonstrated that the stromal C-terminus carries a domain that can specifically interact with the TP of preproteins, a finding which seems to be consistent with IEM vesicle binding data reported by Heins et al. (2002), although one must assume that the IEM vesicles were of inside-out orientation, rather than right-side-out, as was reported. The data presented by Inaba et al. (2003) argue against (but do not completely disprove) the possibility that the Tic110 C-terminus may, under certain conditions, undergo a conformational change leading to its integration into the membrane as a ß-barrel. It appears more likely that, if Tic110 does indeed form a part of the TIC channel, it would do so by contributing its two transmembrane -helices, and that the C-terminal domain protrudes into the stroma where it might co-ordinate late translocation events.
Previously, an anion channel was identified as a putative component of the IEM translocase by inside-out, patch-clamp analysis of the chloroplast envelope (van den Wijngaard and Vredenberg, 1997). This protein import related anion channel (PIRAC) was specifically blocked by the addition of chloroplast precursor proteins or TPs (van den Wijngaard and Vredenberg, 1997; van den Wijngaard et al., 2000). In addition, this blocking effect was only obtained in conditions that allow the formation of early import intermediates and the interaction of precursors with the TIC machinery. These observations strongly suggested that PIRAC is associated with the TIC complex, a notion that was further supported by the finding that PIRAC is completely inactivated by antibodies against Tic110, but remains unaffected by Toc75 antibodies (van den Wijngaard and Vredenberg, 1999). Finally, a direct involvement of PIRAC in protein import was proposed when 4,4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS), a known anion channel blocker, was shown to inhibit protein import efficiency (van den Wijngaard et al., 2000).
The PIRAC pore was roughly estimated to be 6.5 Å in diameter, which would be sufficient to allow the translocation of fully unfolded preproteins (van den Wijngaard and Vredenberg, 1999). However, due to the prevalence of positive charges in TPs of chloroplast preproteins, it seems unlikely that an anion channel would function as the TIC channel. To account for this inconsistency, it has been proposed that, upon interaction of a preprotein with the TIC complex, PIRAC may undergo a conformational change resulting in its transition from an anion channel to a cation channel that is active for protein import (van den Wijngaard et al., 2000). The fact that Tic110 antibodies block PIRAC activity suggests that the channel is in close association with Tic110 or, alternatively, that it is formed, at least in part, by Tic110. Given the proposed dynamic assembly of the TIC complex, it appears possible that Tic110 might form a small anion channel that, in response to incoming preproteins, associates with other integral IEM proteins, such as Tic20, to form the TIC channel.
Translocation through the TIC complex
The mechanism by which the TP of a preprotein crosses the IEM through the TIC is unknown. In mitochondria, translocation of the presequence through the TIM23 complex is driven by the electrical potential across the inner membrane (negative charge inside), which exerts an electrophoretic effect on the positively-charged presequence (Martin et al., 1991; Geissler et al., 2000). In chloroplasts, however, translocation of the TP across the IEM must be mechanistically different since: (i) unlike the inner membrane of mitochondria, the IEM of chloroplasts is not believed to maintain a strong electric potential; and (ii) a membrane potential in chloroplasts has previously been shown not to be necessary for protein translocation across the envelope (Theg et al., 1989).
The recent identification of a TP binding site within the C-terminal stromal domain of Tic110 suggests that a binding chain mechanism, much like that proposed to mediate translocation through the TOM in mitochondria, may operate. The TP binding site of Tic110 is at the N-proximal part of the large soluble domain, which is probably positioned at the exit of the TIC channel (Inaba et al., 2003). This binding site could constitute the end of a putative binding chain, such that the TP would protrude into the stroma as it completes translocation through the TIC. If this were the case, translocation of the TP across the TIC would not require any additional energy input. Thus, it is possible that the TPs of early import intermediates are bound to this site, since abundant evidence suggests that the N-termini of such intermediates are associated with the TIC complex (Akita et al., 1997; Kouranov and Schnell, 1997; Nielsen et al., 1997; Kouranov et al., 1998). More support for this idea was provided by the recent demonstration, by label-transfer cross-linking, that the TPs of early import intermediates are in close proximity to Tic110 (Inaba et al., 2003).
A binding chain pathway that would expose the TP at the exit of the translocation channel would allow stromal factors involved in protein translocation to gain access to the translocating preprotein. This fits well with the fact that a high level of energy (
100 µM ATP) is required in the stroma to complete preprotein translocation across the envelope (Pain and Blobel, 1987; Theg et al., 1989). The high energy requirement has been attributed to an unidentified, stromal ATPase (Pain and Blobel, 1987). By analogy with the import of presequence-carrying preproteins into the mitochondrial matrix, it has been proposed that this stromal ATPase is a molecular chaperone necessary to drive import (Akita et al., 1997; Nielsen et al., 1997). Such a model is consistent with the fact that molecular chaperones, involved in the late stages of protein transport, associate with the stromal face of the TIC complex (Kessler and Blobel, 1996; Akita et al., 1997; Nielsen et al., 1997). Two stromal molecular chaperones have been reported to associate specifically with the TIC complex: Cpn60 (Kessler and Blobel, 1996), and Hsp93 (Akita et al., 1997; Nielsen et al., 1997): plastid homologues of bacterial GroEL (a member of the Hsp60 family) and ClpC (a member of the Hsp100 family), respectively.
Cpn60 was initially identified as the predominant protein co-immunoprecipitating with Tic110 from solubilized chloroplasts (Kessler and Blobel, 1996), and was later shown to associate with TOC–TIC supercomplexes (Kouranov et al., 1998). Cpn60, like the bacterial chaperonin, GroEL, is an oligomeric ATPase that assists protein folding in its central cavity (Bukau and Horwich, 1998). The absence of other significantly co-immunoprecipitated proteins under the experimental conditions used by Kessler et al. suggested that Cpn60 may interact directly with the stromal domain of Tic110. The interaction of Cpn60 with the TIC was further shown to be specific and to be destabilized by ATP, suggesting that it is a functional interaction. The Tic110–Cpn60 complex was shown to be associated with both precursor and mature forms of preprotein applied exogenously to the chloroplasts, prior to solubilization and immunoprecipitation, indicating a role for these proteins in the late stages of translocation. Finally, it was shown that the interaction with the processed form of the preprotein was abolished by the addition of ATP, implying that Cpn60 probably mediates the direct interaction of processed preproteins with the TIC. These results led to the suggestion that Cpn60 is recruited by the stromal domain of Tic110 and interacts with the incoming unfolded protein as it emerges from the translocase to promote its folding before or as it is released in the stroma.
Hsp93, the second stromal molecular chaperone found to interact with the TIC complex, was identified in cross-linking experiments with early import intermediates (Akita et al., 1997). Since Hsp93 was found to be a functional homologue of ClpA, the ATPase subunit of the bacterial caseinolytic protease (Clp), it was proposed to play a role in plastid protein degradation (Shanklin et al., 1995). In E. coli, ClpA (which is an Hsp100 protein like Hsp93), associates with the protease subunit, ClpP (Schirmer et al., 1996). Together, the ClpA and ClpP subunits of the ATP-dependent protease assemble into an oligomeric structure composed of two face-to-face ClpP heptameric rings associated with hexameric rings of ClpA at one or both extremities. The ClpA protein acts as a regulatory subunit of the protease by specifically binding and unfolding proteins targeted for degradation in an ATP-dependent fashion. It is further known to translocate the substrate protein into the central proteolytic chamber of the ClpP oligomer. In addition, ClpA can function independently of the protease subunit, mediating the disaggregation and unfolding of protein aggregates and complexes. In chloroplasts, Hsp93 has been reported to associate with ClpP proteases, providing strong evidence for a role in degradation (Sokolenko et al., 1998; Halperin et al., 2001). Therefore, it appears likely that, in plastids, Hsp93 participates in protein translocation through an association with the TIC complex at the IEM, and in protein degradation via an interaction with ClpP in the stroma.
The interaction of Hsp93 with the import machinery was shown to be stable and to occur even in the absence of added preproteins (Nielsen et al., 1997; Kouranov et al., 1998). As for Cpn60, the interaction of Hsp93 with the TIC was also found to be destabilized by ATP, suggesting that the interaction is physiologically relevant. Furthermore, Hsp93 was shown to associate with the TIC complex independently of ClpP, strongly suggesting that its presence is unrelated to protein degradation. While studying the association of Hsp93 with the TIC complex, Nielsen et al. (1997) also investigated the possibility that stromal Hsp70 is associated with the import machinery. Interestingly, stromal Hsp70 was only detected in non-soluble translocation complexes together with other proteins not involved in protein translocation. This observation suggested that the association of stromal Hsp70 with these complexes was not functionally relevant. In support of this conclusion, translocating preproteins were only successfully co-immunoprecipitated from solubilized chloroplasts with Hsp93, and not with Hsp70. Since only Hsp93 was found to associate functionally with the translocating preproteins, it was proposed that this Hsp100 homologue, rather than an Hsp70 protein as is the case in mitochondria, functions as an ATP-dependent protein import motor to drive preprotein translocation.
In mitochondria, the core component of the protein import motor, mtHsp70, interacts with inner membrane co-chaperones as well as with a soluble matrix co-chaperone, which altogether form the PAM motor complex (Rehling et al., 2004). These proteins co-operate with mtHsp70 in order to assist and regulate its function in driving the complete translocation of preproteins as they exit the TIM channel. As mentioned previously, the PAM may drive translocation using either a Brownian ratchet mechanism, or an active pulling mechanism (Neupert and Brunner, 2002). Tim44 is a peripheral inner membrane protein associated with the TIM23 complex at the matrix side, and serves to anchor mtHsp70 to the channel exit. Two other, recently discovered inner membrane proteins, Pam18 and Pam16, are DnaJ-like proteins which project their J-domains into the matrix and regulate mtHsp70 by modulating its ATPase activity. ATP hydrolysis by mtHsp70 leads to its conversion from a low-substrate-affinity, ATP-bound state to a high-substrate-affinity, ADP-bound state (Bukau and Horwich, 1998). Therefore, the Pam18 and Pam16 co-chaperones regulate the binding of the Hsp70 to unfolded preproteins as they emerge through the TIM23 channel. Finally, the soluble matrix co-chaperone, Mge1, functions as a nucleotide-exchange factor, inducing ADP release by mtHsp70 and allowing it to bind a new ATP molecule. Nucleotide exchange leads to the release of the preprotein substrate by Hsp70, and the initiation of a new substrate binding and release cycle (Neupert and Brunner, 2002; Rehling et al., 2004).
By analogy with this system, Tic110 is proposed to play a role similar to that performed by Tim44, recruiting Hsp93 at the stromal face to the TIC complex. Although Hsp93 has not been demonstrated to interact directly with Tic110, several studies have suggested that it is likely to do so (Caliebe et al., 1997; Nielsen et al., 1997; Kouranov et al., 1998). Such a role would be consistent with the proposal that the large stromal domain of Tic110 acts as a scaffold to co-ordinate the stromal steps of protein import (Inaba et al., 2003). Interestingly, Tic40, another component of the TIC complex, shares homology with eukaryotic Hsp70-interacting protein (Hip) and Hsp70/Hsp90-organising protein (Hop) co-chaperones, and may also be involved in a stromal import motor complex. The Hip and Hop co-chaperones possess one and two TPR domains, respectively, and share a small (60 residue) but highly conserved C-terminal domain (Frydman and Höhfeld, 1997). Both proteins interact with Hsp70 via their TPR domains, and Hop also interacts with Hsp90. The co-chaperones are believed to modulate the activity of the chaperones, and to facilitate the transfer of substrates from Hsp70 to Hsp90. In addition, Sti1, a yeast homologue of Hop, has been shown to interact with Hsp104, another Hsp100 protein (Abbas-Terki et al., 2001).
Tic40 was originally identified as a member of a putative family of immunologically-related envelope proteins named Com/Cim44 (Wu et al., 1994; Ko et al., 1995). Two forms of Tic40 were detected by immunoblotting in pea (estimated sizes were 44 kDa and 42 kDa), and both forms were found to be in close proximity with translocating preproteins, predominantly at the IEM but also at the OEM. In a later study, however, it was argued that only a single form of Tic40 exists in pea, and that this form is localized exclusively in the IEM (Stahl et al., 1999). It was suggested that Tic40 was prone to proteolysis and that the previously observed 42 kDa form was an artefact resulting from proteolysis of the intact 44 kDa protein. Tic40 was further shown to behave as an integral membrane protein, and was proposed to be anchored in the IEM by a single, transmembrane -helix at the N-terminus. The C-terminal part of Tic40 is largely hydrophilic, and was recently shown to be protected from trypsin proteolysis in isolated chloroplasts, suggesting that it protrudes into the stroma (Chou et al., 2003). Tic110 and Tic40 are therefore likely to have a similar topology. This, in addition to the finding that the two proteins were found to be cross-linked together by a disulphide bond under oxidative conditions (Stahl et al., 1999), leads to the suggestion that they work together as a complex facilitating protein translocation.
Interestingly, the C-terminus of Tic40 is particularly similar to the corresponding regions of Hip and Hop (Stahl et al., 1999; Chou et al., 2003). In addition, recent analysis of the predicted secondary structure of Tic40 revealed that the polypeptide sequence adjacent to the C-terminal domain carries seven -helices predicted to fold into a structure similar to that of known TPR domains (Chou et al., 2003). In order to provide experimental support for this prediction, Chou et al. (2003) demonstrated that an antibody against TPR1, a protein containing a single TPR domain, could cross-react with Tic40. Together, these observations suggest that the soluble, C-terminal part of Tic40 is structurally similar to the C-terminal regions of Hip and Hop, and that the protein may, therefore, function as a co-chaperone. This raises the possibility that Tic40 associates with unfolded preproteins as they emerge from the TIC channel, and/or recruits or modulates the activity of a chaperone protein that associates with the stromal face of the TIC complex, potentially Hsp93. The former possibility is supported by the fact that the C-terminal part of Tic40 appears to interact specifically with preproteins (Ko et al., 2004), whereas the latter possibility is consistent with the finding that Tic40 has been shown to associate into supercomplexes along with Hsp93 (Chou et al., 2003). These observations point to the tantalizing possibility that Tic110, Tic40, and Hsp93 form a chloroplast protein import motor complex, and co-operate together at the stromal face of the TIC to drive the import of the preproteins in an ATP-dependent manner (Fig. 1). This model is supported by the fact that all three proteins were shown to associate with precursor proteins during the late stages of protein translocation (Chou et al., 2003).
In vivo studies of Tic110, Tic40, and Hsp93
Null Arabidopsis mutants for Tic110, Tic40, and Hsp93 have recently been identified and characterized (Chou et al., 2003; Constan et al., 2004a; Sjögren et al., 2004; Kovacheva et al., 2005). In Arabidopsis, two genes encoding Hsp93 homologues are present, whereas single genes encode orthologues of Tic110 and Tic40 (Jackson-Constan and Keegstra, 2001). The two Hsp93 genes, atHSP93-III and atHSP93-V, encode proteins with very high levels of homology throughout their mature regions (91% identity), but have significantly different expression patterns (Kovacheva et al., 2005). Steady-state mRNA abundance of atHSP93-V is approximately 15-fold higher than that of atHSP93-III in young light-grown seedlings. atHSP93-III appears to be expressed at similarly low levels in different tissue types and developmental stages, except in expanding and mature rosette leaves where the expression is significantly reduced. By contrast, the expression of atHSP93-V is strongly up-regulated in rosettes. This suggests that the two Hsp93 isoforms are expressed in a complementary fashion. In comparison, atTIC110 and atTIC40 expression patterns are not strongly regulated, as the two genes show comparable levels of expression across different tissues and developmental stages (Kovacheva et al., 2005). The fact that all four genes are expressed in all tissues analysed, including roots and etiolated seedlings, suggests that the corresponding proteins are likely to be involved in protein import in all types of plastids.
Knockout mutants for the most highly expressed HSP93 homologue, atHSP93-V, as well as for atTIC110 and atTIC40, were all found to display a distinct chlorotic phenotype (Chou et al., 2003; Constan et al., 2004a; Sjögren et al., 2004; Kovacheva et al., 2005), indicative of a defect in chloroplast function. By contrast, knockout mutants for atHSP93-III are not phenotypically different from wild-type plants (Constan et al., 2004; Kovacheva et al., 2005; S Kovacheva, J Bédard, P Jarvis, unpublished data). The absence of a visible phenotype in hsp93-III homozygotes is believed to be due to a compensatory effect of atHsp93-V, since the isoforms appear to be largely redundant in function (Kovacheva et al., 2005; S Kovacheva, J Bédard, P Jarvis, unpublished data). Remarkably, heterozygous tic110 seedlings were found to display a subtle chlorotic phenotype during periods of rapid growth and expansion (Kovacheva et al., 2005) when leaf chloroplasts divide and develop rapidly and, consequently, need to import large quantities of proteins. Furthermore, in the homozygous state, the tic110 mutation is embryo lethal, indicating that atTic110 is essential for plastid protein import and embryo development. These observations are consistent with the biochemical evidence that Tic110 plays an important function in plastid protein import and constitutes a core component of the TIC complex (Heins et al., 2002; Inaba et al., 2003). By contrast, although Arabidopsis plants possess only a single atTIC40 gene, homozygous tic40 plants are viable, albeit with a strong chlorotic phenotype, suggesting that Tic40 plays only an accessory role in protein import (Chou et al., 2003; Kovacheva et al., 2005).
The tic110, tic40, and hsp93-V knockout mutants were all found to display reductions in chloroplast development and chloroplast protein import efficiency, in each case to a degree proportional with the severity of the chlorotic phenotype. These observations established a role in vivo for Tic110, Tic40, and Hsp93 in chloroplast protein import (Chou et al., 2003; Constan et al., 2004a; Kovacheva et al., 2005). In addition, the fact that each mutant showed comparable import defects for both photosynthetic and non-photosynthetic precursors (pRbcS and pL11, respectively) suggests that the three proteins play a role in a general import pathway (Kovacheva et al., 2005). The possibility that all three proteins function in a common process, such as in the driving of translocation, is supported by the fact that double mutants involving tic110, tic40, and hsp93-V, in all possible combinations, did not display phenotypic additivity (Kovacheva et al., 2005). Detailed analysis of the chloroplast protein import defect in tic40 chloroplasts revealed that translocation through the IEM was specifically defective (Chou et al., 2003). Interestingly, it was further proposed that the absence of atTic40 in tic40 chloroplasts led to the release of processed precursors back into the import assay medium, suggesting that Tic40 may be required to prevent retrograde movement of translocating proteins as they reach the stromal face. An analogous role is played by the PAM complex in mitochondria.
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Redox regulation of chloroplast protein import |
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The action of the recently identified Toc12 protein has been proposed to be subject to redox regulation, since the structure of its J-domain is thought to be stabilized by an intramolecular disulphide bond (Becker et al., 2004a
). Under reductive conditions, this bond could be disrupted, leading to the inability of Toc12 to stimulate the ATPase activity of the OEM Hsp70 chaperone. Such a scenario would probably hinder protein import, and is reminiscent of the mechanism by which the insertion of polytopic proteins into the mitochondrial inner membrane is redox-regulated (Curran et al., 2004; Lu et al., 2004). In the latter import pathway, small Tim proteins (Tim9 and Tim10) form a chaperone complex (TIM10) in the IMS, which mediates the delivery of polytopic proteins to the TIM22 complex. The assembly of TIM10 has been shown to be dependent on the formation of intramolecular disulphide bonds within the small Tim proteins, following their import into the IMS (Lu et al., 2004). A novel pathway for the formation of disulphide bonds, involving an IMS-localized sulphydryl oxidase, is proposed to regulate the folding and assembly of the TIM10 chaperone complex (Curran et al., 2004). It therefore seems possible that the folding of Toc12 is controlled in a similar fashion, and that this regulates import through the TOC and/or assembly of the IMS translocase and the transfer of preproteins to the TIC.
Interestingly, the identification of three putative TIC components that may be capable of sensing the redox state of the chloroplast also led to the suggestion that chloroplast protein import may be regulated by redox signals (Caliebe et al., 1997; Küchler et al., 2002; Hörmann et al., 2004). Two of these proteins, Tic55 and Tic62, were identified as components of a pea chloroplast IEM complex detected by blue native polyacrylamide gel electrophoresis (BN-PAGE) (Caliebe et al., 1997; Küchler et al., 2002). This complex was proposed to correspond to the TIC since it also contained Tic110, a previously identified marker of the protein import machinery of the IEM. The third protein, Tic32, was more recently identified as an IEM protein that can tightly interact with the N-terminal transmembrane domain of Tic110 (Hörmann et al., 2004).
Tic55 carries a Rieske-type iron–sulphur centre and a mononuclear iron-binding site, two domains typically found in a specific class of bacterial oxygenases (Caliebe et al., 1997). The iron–sulphur cluster associated with the Rieske centre of proteins normally functions in electron transfer reactions as it can act as an electron acceptor and donor. Diethylpyrocarbonate (DEPC), a molecule known to disrupt Rieske centres through the modification (ethoxyformylation) of their histidine residues, was found to inhibit the progression of translocating preproteins across the IEM in isolated chloroplasts, indicating that Tic55 may be involved in import. Furthermore, Tic55 was found to be associated with translocating preproteins, along with known components of both the TOC and TIC machineries. Based on these results, it was proposed that Tic55 might act as a biosensor of the redox state of the chloroplast, acting to regulate chloroplast protein import in response to putative redox signals. However, a role for Tic55 in protein import remains a matter of debate, since two independent research groups have been unable to detect Tic55 in import complexes (Kouranov et al., 1998; Reumann and Keegstra, 1999).
Additional support for a role of Tic55 as a redox sensor was provided when the protein was later shown to form a stable IEM complex along with Tic110 and a previously unidentified putative TIC component, Tic62 (Küchler et al., 2002). Tic62 carries a functional NAD(P) binding site at its N-terminus, which could also serve as an electron transfer site. The C-terminal end of the protein was shown to protrude into the stroma where it interacts with ferredoxin-NAD(P) reductase (FNR). Because FNR normally mediates the transfer of electrons from ferredoxin to NAD(P) at the thylakoid membrane, it is possible that FNR may reduce the NAD(P) molecule associated with Tic62. An electron transfer reaction from Tic62 to Tic55 might ensue, possibly leading to further signalling or to an allosteric conformational change in Tic55 that directly affects protein import. However, compositional discrepancies exist between the complex identified by Caliebe et al. (1997), and that described by Küchler et al. (2002). In the initial study, the TIC complex was reported to be composed of at least six components; these were 110 (Tic110), 100 (Hsp93), 60 (presumably Tic62), 52 (Tic55), 45, and 36 kDa in size. In the second study, however, only three components were present in the complex, namely, Tic55, Tic62, and Tic110. Strangely, both complexes were found to have roughly the same size (280 kDa and 230 kDa, respectively). This raises the possibility that Tic110 may associate with different proteins of the IEM to form two distinct complexes of roughly the same size, which may have co-fractionated under the conditions of the first study.
The recently identified Tic32 protein might act as a third link in a putative regulatory electron transport chain (Hörmann et al., 2004). Tic32 is a conserved short-chain dehydrogenase/reductase (SDR), similar to the hydrophobic human retinol dehydrogenases that are bound to the ER membrane (Simon et al., 1999). Consistent with this, Tic32 was found to behave as an integral IEM protein. It was further proposed to associate with other known components of the TIC complex, including Tic40 and Tic22, as well as with translocating precursors. Two mutant lines each carrying a T-DNA insertion within the putative Arabidopsis orthologue of pea TIC32 were identified, and their analysis suggested that Tic32 may be essential for embryo development. Because Tic32 was found to carry the necessary domains for SDR activity, including an NAD(P) binding site, it was proposed to perform an essential regulatory function. Due to its tight association with Tic110, it was further proposed that it might act as a redox-regulated gate of the TIC channel in similar fashion to the ß-subunit of some potassium channels, which couple oxidoreductase activity to channel inactivation (Bähring et al., 2001).
Some experimental evidence for the redox regulation of protein translocation across the IEM was recently presented. The import of a constitutively expressed maize, non-photosynthetic precursor protein, ferredoxin-III (pFd-III), was found to be differentially regulated in dark and light conditions (Hirohashi et al., 2001). In the presence of light, pFd-III was mis-targeted to the chloroplast IMS as an intact precursor, and was not imported and processed in the stroma, its normal site of accumulation. By contrast, the precursor of the light-regulated, photosynthetic ferredoxin-I (pFd-I) was imported normally into the stroma, both in dark and light conditions. Because Fd-III has a significantly higher redox potential than Fd-I, it was proposed that accumulation of this isoform in the stroma under light conditions could interfere with electron transport at the thylakoid membrane. Therefore, preventing the import of this isoform may serve to ensure maximal photosynthetic efficiency. The differential import regulation of these two isoforms was shown to be determined by their different TPs. This suggests that two or more differentially regulated TIC complexes may exist, with different specificities for the two different ferredoxin precursors, or, alternatively, that a single TIC channel exists which becomes more restrictive under light conditions.
Interestingly, Hirohashi et al. (2001) also showed that two FNR isoforms display a similar differential import pattern, indicating that the ferredoxins are not a unique case and that this mode of regulation may be utilized to inhibit the import of a subclass of precursor proteins in the presence of light. It has been proposed that differences in redox state within the chloroplast under light and dark conditions may control this differential import regulation (Küchler et al., 2002). In the presence of light and active photosynthetic electron transport, a greater pool of reduced NAD(P) over the oxidized form may trigger the closure or increased selectivity of the import machinery. Such a regulatory mechanism is supported by the fact that deamino-NAD, a non-reducible NAD analogue, and ruthenium hexamine trichloride, an oxidant of NAD, were found to affect the in vitro import of two FNR isoforms differently (Küchler et al., 2002). It is possible that redox changes in the NAD(P) pool affected either Tic32 or Tic62 directly or, alternatively, that induced changes in redox state were somehow perceived by FNR and further signalled to the TIC complex via an interaction between FNR and Tic62. However, further work is required to determine exactly how light inhibits the import of certain precursors, and whether redox signalling truly plays a role in these processes.
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Processing and folding of preproteins in the stroma |
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Processing of preproteins to their mature form is mediated by the SPP. The N-terminus of SPP carries an HXXEH zinc-binding motif characteristic of the pitrilysin metalloendopeptidase family, which also includes MPP of the mitochondrial matrix (VanderVere et al., 1995
). The SPP and MPP likely evolved from a common ancestor since the domains containing the zinc-binding motif are homologous. However, the two processing enzymes differ considerably in structure. SPP is a large monomeric enzyme whereas MPP is a heterodimer formed by the association of two homologous subunits (the - and ß-subunits), of which only one (the ß-subunit) contains the HXXEX motif (Oblong and Lamppa, 1992; Taylor et al., 2001). Both peptidases are capable of recognizing and processing a large number of targeting sequence substrates, which vary greatly in sequence. In a recent study of the MPP structure by X-ray crystallography, it was demonstrated that mitochondrial presequences bind to the peptidase in an extended conformation within the acidic cavity formed between the - and ß-subunits (Taylor et al., 2001). The HXXEH motif was shown to be important for catalysis of the single endopeptidic reaction that leads to presequence removal. Similarly, the zinc-binding motif was recently found to be essential for preprotein processing by SPP (Richter and Lamppa, 2003). Therefore, despite their structural differences, the two processing enzymes may remove signal sequences through a similar catalytic reaction.
Processing of chloroplast precursors is believed to occur almost immediately after the N-terminus of the translocating preprotein emerges from the TIC complex into the stroma, since processed precursor proteins spanning the envelope have been identified (Schnell and Blobel, 1993). This is consistent with the fact that SPP displays a high level of affinity for TPs (Richter and Lamppa, 1999). The SPP interacts directly with a 10–15 amino acid sequence at the C-terminal end of TPs, adjacent to the processing site, where the basic residues are generally concentrated (Richter and Lamppa, 2002). It was previously believed that processing occurs at a loosely conserved motif, but the results of a recent study suggest that processing involves recognition of specific physicochemical properties rather than a particular amino acid sequence (Rudhe et al., 2004). Rhudhe et al. (2004) showed that, in contrast to MPP, SPP is tolerant of single amino acid substitutions in a signal sequence substrate, and is only affected by double and triple mutations introduced near the processing site. Cleavage of the TP by SPP leads to the immediate release of the mature protein but the TP remains bound to the enzyme until the peptidase catalyses at least one second proteolytic event (Richter and Lamppa, 1999). This corresponds to the trimming of the C-terminal part of the TP such that the peptide is no longer recognized by the protease and is released into the stroma. Trimming and release of the TP leads to recycling of the SPP, and prevents further binding of the protease to free TPs. The released TP sub-fragments are then further proteolysed by a second unidentified metalloprotease of the stroma in an ATP-dependent fashion (Richter and Lamppa, 1999).
Investigation of the role of SPP in vivo by antisense suppression of SPP expression established that the processing enzyme plays an important role in chloroplast biogenesis (Wan et al., 1998; Zhong et al., 2003). In a recent antisense study in Arabidopsis, a large proportion of the transformants displayed a seedling-lethal phenotype suggesting that SPP is essential for chloroplast development (Zhong et al., 2003). Such a finding is not surprising as it can be assumed that removal of the TP is necessary for proper folding and functionality of the majority of chloroplast proteins. Arabidopsis seedlings with reduced expression of SPP displayed albino sectors, reduced numbers of chloroplasts per cell, and strong defects in chloroplast ultrastructure. Interestingly, plants with reduced levels of SPP were also shown to have a strong defect in chloroplast protein import, as determined by in vitro import assays (Wan et al., 1998) and an in vivo assay using a TP-GFP fusion (Zhong et al., 2003). This defect is most likely due to the fact that Toc75, as well as most components of the TIC, are synthesized as precursor proteins and most likely require processing by SPP before they can assemble into functional TOC and TIC complexes.
Once precursor proteins destined for the stroma have been processed by SPP, the mature proteins are believed to require assistance from stromal molecular chaperones to fold properly into their functional conformation. Stromal Hsp70, Cpn60, and Cpn10 homologues are believed to mediate the folding of newly imported polypeptide chains (Tsugeki and Nishimura, 1993). These chaperone proteins bind to exposed hydrophobic surfaces to prevent aggregation and promote folding (Bukau and Horwich, 1998). Because Cpn60 was previously found to associate with the stromal surface of the TIC complex in an ATP-dependent fashion (Kessler and Blobel, 1996), it is likely that folding of the incoming polypeptide chain may occur simultaneously with translocation. Alternatively, the chaperone may bind to the incoming polypeptide simply to prevent mis-folding or aggregation and, once translocation is complete, the chaperone-substrate could dissociate from the TIC to initiate folding in the stroma.
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Outlook and conclusions |
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Presumably, the majority of the core components making up the TOC and TIC machinery have now been identified. However, the recent, unexpected identifications of Toc12, which is likely to play an important role in translocation across the IMS, and Tic32, a putative regulatory component, suggest that some peripheral or regulatory components may not yet have been identified. Cross-linking studies and interaction studies with preproteins arrested at different stages of import have provided insight into the putative roles of different components. Research efforts in chloroplast protein import are now mostly aimed at developing a better understanding of the functions of individual TOC and TIC components, as well as of the structural organization of the TOC and TIC complexes. The recent integration of Arabidopsis molecular-genetic studies into the field of chloroplast protein import research, and the development of reconstitution systems, should help to address these issues.
At present, the events leading to the complete translocation of a preprotein from the cytosol to the chloroplast stroma, via the TOC and TIC, can be described in general terms. However, the mechanistic details underlying these events are still largely unknown. Our understanding of how preproteins are initially recognized at the TOC by the GTPase receptors is considerably more advanced than that of other import steps. Even at this recognition step, however, the exact mechanism by which the GTP-regulated interplay between the Toc159 and Toc34 receptors leads to the initiation of preprotein translocation is debated. The context in which these receptor components can interact with each other, and the exact outcome of these interactions, both still need to be resolved. The recent discovery that multiple isoforms of these receptors exist in Arabidopsis, and that these assemble preferentially together to form different import pathways, adds to the complexity of preprotein targeting to the plastids. Determining how Toc34 and Toc159 co-operate to recognize TP signals is essential to our understanding of how targeting specificity is achieved.
The targeting sequences of plastidic and mitochondrial preproteins are very similar, and little is known regarding how specificity of targeting between these organelles is achieved. Since the major determinants of specificity are found within targeting signals, the detailed characterization of a greater number of targeting sequences must be completed in order to identify and define putative differentiating signals or properties. In parallel, analyses of TP interactions with different TOC and TIC components should provide us with a better understanding of the features of these peptides that are important for recognition and translocation. Phosphorylation of TPs by a cytosolic protein kinase has been suggested as a possible means to achieve specificity of targeting to plastids. A guidance complex composed of 14-3-3 and Hsp70 has been proposed to target phosphorylated preproteins to the chloroplast surface via a possible interaction with the TPR-protein, Toc64. Further investigation is required, however, to determine if preprotein phosphorylation has any relevance in vivo.
Finally, studies investigating the structure and possible dynamics of the TIC complex, aimed at providing a better view of how this complex assists transport across the IEM, will be of particular importance. For example, the putative role of Tic20 as a component of the import channel requires further investigation. The presence of multiple isoforms of this protein in Arabidopsis leads to the suggestion that chloroplasts, like mitochondria, may possess different translocon complexes in the inner membrane which serve to mediate the translocation of different subclasses of preproteins. In addition, further characterization of the putative molecular motors of the IMS and stroma will be necessary in order to understand fully how transport across the envelope via the TOC and TIC complexes is driven.
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Acknowledgements |
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We thank Sabina Kovacheva and Sybille Kubis for their helpful comments on the manuscript. Financial support was provided by a Universities UK Overseas Research Students (ORS) Award (to JB), the Royal Society Rosenheim Research Fellowship (to PJ), and Biotechnology and Biological Sciences Research Council (BBSRC) grants 91/C12976, 91/P12928, and 91/C18638 (to PJ).
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T. Kleffmann, A. von Zychlinski, D. Russenberger, M. Hirsch-Hoffmann, P. Gehrig, W. Gruissem, and S. Baginsky Proteome Dynamics during Plastid Differentiation in Rice Plant Physiology, February 1, 2007; 143(2): 912 - 923. [Abstract] [Full Text] [PDF]
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E. Lopez-Juez Plastid biogenesis, between light and shadows J. Exp. Bot., January 1, 2007; 58(1): 11 - 26. [Abstract] [Full Text] [PDF]
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M. W. Murcha, D. Elhafez, R. Lister, J. Tonti-Filippini, M. Baumgartner, K. Philippar, C. Carrie, D. Mokranjac, J. Soll, and J. Whelan Characterization of the Preprotein and Amino Acid Transporter Gene Family in Arabidopsis Plant Physiology, January 1, 2007; 143(1): 199 - 212. [Abstract] [Full Text] [PDF]
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M. Li and D. J. Schnell Reconstitution of protein targeting to the inner envelope membrane of chloroplasts J. Cell Biol., October 23, 2006; 175(2): 249 - 259. [Abstract] [Full Text] [PDF]
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 Y. Xiang, K. Kakani, R. Reade, E. Hui, and D. Rochon A 38-amino-Acid sequence encompassing the arm domain of the cucumber necrosis virus coat protein functions as a chloroplast transit Peptide in infected plants. J. Virol., August 1, 2006; 80(16): 7952 - 7964. [Abstract] [Full Text] [PDF]
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