Exploring ligand recognition,selectivity and dynamics of TPR domains of chloroplast Toc64 and mitochondria Om64 from Arabidopsis thaliana

The study aims to gain insight into the mode of ligand recognition by tetratricopeptide repeat (TPR) domains of chloroplast translocon at the outer envelope of chloroplast (Toc64) and mitochondrial Om64, two paralogous proteins that mediate import of proteins into chloroplast and mitochondria, respectively. Chaperone proteins associate with precursor proteins in the cytosol to maintain them in a translocation competent conformation and are recognized by Toc64 and Om64 that are located on the outer membrane of the target organelle. Heat shock proteins (Hsp70) and Hsp90 are two chaperones, which are known to play import roles in protein import. The C‐termini of these chaperones are known to interact with the TPR domain of chloroplast Toc64 and mitochondrial Om64 in Arabidopsis thaliana (At). Using a molecular dynamics approach and binding energy calculations, we identify important residues involved in the interactions. Our findings suggest that the TPR domain from AtToc64 has higher affinity towards C‐terminal residues of Hsp70. The interaction occurs as the terminal helices move towards each other enclosing the cradle on interaction of AtHsp70 with the TPR domain. In contrast, the TPR domain from AtOm64 does not discriminate between the C‐termini of Hsp70 and Hsp90. These binding affinities are discussed with respect to our knowledge of protein targeting and specificity of protein import into endosymbiotic organelles in plant cells. Copyright © 2014 John Wiley & Sons, Ltd.     

A role of Toc33 in the protochlorophyllide-dependent plastid import pathway of NADPH:protochlorophyllide oxidoreductase (POR) A

NADPH:protochlorophyllide oxidoreductase (POR) A is a key enzyme of chlorophyll biosynthesis in angiosperms. It is nucleus-encoded, synthesized as a larger precursor in the cytosol and imported into the plastids in a substrate-dependent manner. Plastid envelope membrane proteins, called protochlorophyllide-dependent translocon proteins, Ptcs, have been identified that interact with pPORA during import. Among them are a 16-kDa ortholog of the previously characterized outer envelope protein Oep16 (named Ptc16) and a 33-kDa protein (Ptc33) related to the GTP-binding proteins Toc33 and Toc34 of Arabidopsis. In the present work, we studied the interactions and roles of Ptc16 and Ptc33 during pPORA import. Radiolabeled Ptc16/Oep16 was synthesized from a corresponding cDNA and imported into isolated Arabidopsis plastids. Crosslinking experiments revealed that import of 35S-Oep16/Ptc16 is stimulated by GTP. 35S-Oep16/Ptc16 forms larger complexes with Toc33 but not Toc34. Plastids of the ppi1 mutant of Arabidopsis lacking Toc33, were unable to import pPORA in darkness but imported the small subunit precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSSU), precursor ferredoxin (pFd) as well as pPORB which is a close relative of pPORA. In white light, partial suppressions of pSSU, pFd and pPORB import were observed. Our results unveil a hitherto unrecognized role of Toc33 in pPORA import and suggest photooxidative membrane damage, induced by excess Pchlide accumulating in ppi1 chloroplasts because of the lack of pPORA import, to be the cause of the general drop of protein import.     

Toc64, a new component of the protein translocon of chloroplasts

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

Toc64/OEP64 is not essential for the efficient import of proteins into chloroplasts in Arabidopsis thaliana

Toc64/OEP64 was identified biochemically in pea as a putative component of the chloroplast protein import apparatus. In Arabidopsis, three paralogous genes (atTOC64-III, atTOC64-V and atTOC64-I) encode Toc64-related proteins, and these have been reported to localize in chloroplasts, mitochondria and the cytosol, respectively. To assess the role of the atToc64-III protein in chloroplast protein import in an in vivo context, we identified and characterized Arabidopsis knockout mutants. The absence of detectable defects in toc64-III single mutants raised the possibility of redundancy, and prompted us to also identify toc64-V and toc64-I mutants, cross them to toc64-III, and generate double- and triple-mutant combinations. The toc64 mutants were analysed carefully with respect to a variety of criteria, including chlorophyll accumulation, photosynthetic performance, organellar ultrastructure and chloroplast protein accumulation. In each case, the mutant plants were indistinguishable from wild type. Furthermore, the efficiency of chloroplast protein import was not affected by the toc64 mutations, even when a putative substrate of the atToc64-III protein (wheatgerm-translated precursor of the 33 kDa subunit of the oxygen-evolving complex, OE33) was examined. Moreover, under various stress conditions (high light, osmotic stress and cold), the toc64 triple-mutant plants were not significantly different from wild type. These results demonstrate that Toc64/OEP64 is not essential for the efficient import of proteins into chloroplasts in Arabidopsis, and draw into question the functional significance of this component.     

Toc64 is not required for import of proteins into chloroplasts in the moss Physcomitrella patens

Toc64 has been suggested to be part of the chloroplast import machinery in Pisum sativum. A role for Toc64 in protein transport has not been established, however. To address this, we generated knockout mutants in the moss Physcomitrella patens using the moss's ability to perform homologous recombination with nuclear DNA. Physcomitrella patens contains two genes that encode Toc64-like proteins. Both of those proteins appear to be localized in the chloroplast. The double-mutant plants were lacking Toc64 protein in the chloroplasts but showed no growth phenotype. In addition, these plants accumulated other plastid proteins at wild-type levels and showed no difference from wild type in in vitro protein import assays. These plants did have a slightly altered chloroplast shape in some tissues, however. The evidence therefore indicates that Toc64 proteins are not required for import of proteins in Physcomitrella, but may point to involvement in the determination of plastid shape.     

Functional analysis of the two Arabidopsis homologues of Toc34, a component of the chloroplast protein import apparatus

Two Arabidopsis Toc34 homologues, atToc34 and atToc33, components of the chloroplast protein import machinery located in the outer envelope membrane, were recently isolated. Both proteins insert into the outer envelope, are supposed to bind GTP and to interact with Toc75 as demonstrated by in vitro import assays. We studied the expression of the two genes by RNA gel blot analysis, promoter-GUS plants and in situ hybridisations as well as immunoblot analysis. The atToc34 and atToc33 genes are expressed in green as well as non-green tissues and are developmentally regulated. Despite these similarities, however, the two Arabidopsis Toc34 homologues are differentially expressed in various plant organs. To gain more insight into the in vivo function of both proteins, antisense plants were created. While antisense plants of atToc33 are characterized by a pale yellowish phenotype, antisense plants of atToc34 show a weaker phenotype. Protein interaction studies using an in vitro translated precursor protein and heterologously expressed atToc34 and atToc33 proteins showed a direct GTP-dependent interaction, but demonstrated different affinities of the two atToc proteins towards the precursor protein. Thus, our results indicate a more specialized function for both atToc34 and atToc33, suggesting specificity for certain imported precursor proteins.     

Toc159- and Toc75-independent import of a transit sequence-less precursor into the inner envelope of chloroplasts

Chloroplast envelope quinone oxidoreductase (ceQORH) is an inner plastid envelope protein that is synthesized without cleavable chloroplast transit sequence for import. In the present work, we studied the in vitro-import characteristics of Arabidopsis ceQORH. We demonstrate that ceQORH import requires ATP and is dependent on proteinaceous receptor components exposed at the outer plastid surface. Competition experiments using small subunit precursor of ribulose-bisphosphate carboxylase/oxygenase and precursor of ferredoxin, as well as antibody blocking experiments, revealed that ceQORH import does not involve the main receptor and translocation channel proteins Toc159 and Toc75, respectively, which operate in import of proteins into the chloroplast. Molecular dissection of the ceQORH amino acid sequence by site-directed mutagenesis and subsequent import experiments in planta and in vitro highlighted that ceQORH consists of different domains that act concertedly in regulating import. Collectively, our results provide unprecedented evidence for the existence of a specific import pathway for transit sequence-less inner plastid envelope membrane proteins into chloroplasts.     

Toxoplasma gondii Toc75 Functions in Import of Stromal but not Peripheral Apicoplast Proteins

Apicomplexa are unicellular parasites causing important human and animal diseases, including malaria and toxoplasmosis. Most of these pathogens possess a relict but essential plastid, the apicoplast. The apicoplast was acquired by secondary endosymbiosis between a red alga and a flagellated eukaryotic protist. As a result the apicoplast is surrounded by four membranes. This complex structure necessitates a system of transport signals and translocons allowing nuclear encoded proteins to find their way to specific apicoplast sub‐compartments. Previous studies identified translocons traversing two of the four apicoplast membranes. Here we provide functional support for the role of an apicomplexan Toc75 homolog in apicoplast protein transport. We identify two apicomplexan genes encoding Toc75 and Sam50, both members of the Omp85 protein family. We localize the respective proteins to the apicoplast and the mitochondrion of Toxoplasma and Plasmodium. We show that the Toxoplasma Toc75 is essential for parasite growth and that its depletion results in a rapid defect in the import of apicoplast stromal proteins while the import of proteins of the outer compartments is affected only as the secondary consequence of organelle loss. These observations along with the homology to Toc75 suggest a potential role in transport through the second innermost membrane.     

Import pathways of chloroplast interior proteins and the outer-membrane protein OEP14 converge at Toc75

Most chloroplast outer-membrane proteins are synthesized at their mature size without cleavable targeting signals. Their insertion into the outer membrane is insensitive to thermolysin pretreatment of chloroplasts and does not require ATP. It has therefore been assumed that insertion of outer-membrane proteins proceeds through a different pathway from import into the interior of chloroplasts, which requires a thermolysin-sensitive translocon complex and ATP. Here, we show that a model outer-membrane protein, OEP14, competed with the import of a chloroplast interior protein, indicating that the two import pathways partially overlapped. Cross-linking studies showed that, during insertion, OEP14 was associated with Toc75, a thermolysin-resistant component of the outer-membrane protein-conducting channel that mediates the import of interior-targeted precursor proteins. Whereas almost no OEP14 inserted into protein-free liposomes, OEP14 inserted into proteoliposomes containing reconstituted Toc75 with a high efficiency. Taken together, our data indicate that Toc75 mediates OEP14 insertion, and therefore plays a dual role in the targeting of proteins to the outer envelope membrane and interior of chloroplasts.     

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

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

Nucleotide binding and dimerization at the chloroplast pre‐protein import receptor,atToc33, are not essential in vivo but do increase import efficiency

The atToc33 protein is one of several pre‐protein import receptors in the outer envelope of Arabidopsis chloroplasts. It is a GTPase with motifs characteristic of such proteins, and its loss in the plastid protein import 1 (ppi1) mutant interferes with the import of photosynthesis‐related pre‐proteins, causing a chlorotic phenotype in mutant plants. To assess the significance of GTPase cycling by atToc33, we generated several atToc33 point mutants with predicted effects on GTP binding (K49R, S50N and S50N/S51N), GTP hydrolysis (G45R, G45V, Q68A and N101A), both binding and hydrolysis (G45R/K49N/S50R), and dimerization or the functional interaction between dimeric partners (R125A, R130A and R130K). First, a selection of these mutants was assessed in vitro, or in yeast, to confirm that the mutations have the desired effects: in relation to nucleotide binding and dimerization, the mutants behaved as expected. Then, activities of selected mutants were tested in vivo, by assessing for complementation of ppi1 in transgenic plants. Remarkably, all tested mutants mediated high levels of complementation: complemented plants were similar to the wild type in growth rate, chlorophyll accumulation, photosynthetic performance, and chloroplast ultrastructure. Protein import into mutant chloroplasts was also complemented to >50% of the wild‐type level. Overall, the data indicate that neither nucleotide binding nor dimerization at atToc33 is essential for chloroplast import (in plants that continue to express the other TOC receptors in native form), although both processes do increase import efficiency. Absence of atToc33 GTPase activity might somehow be compensated for by that of the Toc159 receptors. However, overexpression of atToc33 (or its close relative, atToc34) in Toc159‐deficient plants did not mediate complementation, indicating that the receptors do not share functional redundancy in the conventional sense.     

Targeting of an abundant cytosolic form of the protein import receptor at Toc159 to the outer chloroplast membrane

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.     

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

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

Pea Chloroplast DnaJ-J8 and Toc12 Are Encoded by the Same Gene and Localized in the Stroma

Toc12 is a novel J domain-containing protein identified in pea (Pisum sativum) chloroplasts. It was shown to be an integral outer membrane protein localizing in the intermembrane space of the chloroplast envelope. Furthermore, Toc12 was shown to associate with an intermembrane space Hsp70, suggesting that Toc12 is important for protein translocation across the chloroplast envelope. Toc12 shares a high degree of sequence similarity with Arabidopsis (Arabidopsis thaliana) DnaJ-J8, which has been suggested to be a soluble protein of the chloroplast stroma. Here, we isolated genes encoding DnaJ-J8 from pea and found that Toc12 is a truncated clone of one of the pea DnaJ-J8s. Protein import analyses indicate that Toc12 and DnaJ-J8s possess a cleavable transit peptide and are localized in the stroma. Arabidopsis mutants with T-DNA insertions in the DnaJ-J8 gene show no defect in chloroplast protein import. Implications of these results in the energetics and mechanisms of chloroplast protein import are discussed.Most chloroplast proteins are encoded by the nuclear genome and synthesized in the cytosol as higher molecular mass precursors with an N-terminal extension known as the transit peptide. Precursor proteins are imported into chloroplasts through a translocon complex located at the chloroplast envelope. Translocon components associated with the outer membrane are called Toc (for translocon of the outer envelope membrane of chloroplast) proteins, and those associated with the inner membrane are called Tic (for translocon of the inner envelope membrane of chloroplast) proteins. Cleavage of the transit peptide from the precursor by a specific stromal processing peptidase during translocation results in the production of the lower molecular mass mature protein. Various translocon components have been assigned functions in the basic steps of the import process (for review, see Inaba and Schnell, 2008; Jarvis, 2008; Li and Chiu, 2010). For example, Toc159 (the no. indicates the calculated molecular mass of the protein) and Toc34 are receptors for the transit peptides, and Toc75 is the protein-translocating channel across the outer membrane. Toc64, on the other hand, has a dual function: it serves as a docking site for the cytosolic Hsp90 through its cytosolic domain and as a scaffold for translocon components located in the intermembrane space through its intermembrane space domain (Qbadou et al., 2007).Protein import into chloroplasts involves at least two distinct ATP-consuming steps. The first step is called “early import intermediate” or “docking,” in which less than 100 μm ATP is required and precursors are translocated across the outer membrane and come into contact with translocon components in the inner membrane (Olsen et al., 1989; Kouranov and Schnell, 1997; Inaba et al., 2003; Inoue and Akita, 2008). It has been shown that the ATP is used in the intermembrane space (Olsen and Keegstra, 1992), most likely by a yet unidentified intermembrane space Hsp70 called imsHsp70 or Hsp70-IAP (ims for “intermembrane space” and IAP for “import intermediate-associated protein”; Marshall et al., 1990; Schnell et al., 1994; Qbadou et al., 2007). The second ATP-consuming step is the complete translocation of precursors across the two envelope membranes into the stroma. This step requires about 1 mm ATP. The ATP is most likely used by the stromal Hsp93 and chloroplast Hsc70 associated with the translocon to drive protein translocation into the stroma (Nielsen et al., 1997; Shi and Theg, 2010; Su and Li, 2010).Hsp70 family proteins are involved in many cellular processes, including protein folding, protein translocation across membranes, and regulation of protein degradation. Hsp70 proteins are often recruited to perform a certain function by specifically localized J domain-containing proteins. The J domain-containing proteins interact with Hsp70 when Hsp70 is bound to ATP and stimulate ATP hydrolysis by Hsp70. The specific J domain-containing cochaperone that recruits the stromal chloroplast Hsc70 to the inner envelope membrane to assist in protein translocation has not been identified. The specific J domain-containing cochaperone for imsHsp70 for its function in protein import into chloroplasts is proposed to be a protein named Toc12 (Becker et al., 2004).Toc12 was identified as a novel J domain-containing protein from pea (Pisum sativum) chloroplasts. It belongs to the type III J domain proteins containing only the J domain without the Gly- and Phe-rich domain (G/F domain) and the zinc-finger domain originally found in Escherichia coli DnaJ. It has been shown that the protein is synthesized at its mature size of 103 amino acids without a cleavable transit peptide. After import, the protein has been shown to anchor in the outer membrane by its N-terminal part, which has been suggested to form a β-barrel-type domain. Its C-terminal part, composed of the J domain, has been shown to localize in the intermembrane space. Toc12 has been shown to associate with imsHsp70. Toc12 and imsHsp70 interact with the intermembrane space domain of Toc64, which in turn associates with another intermembrane space translocon component, Tic22. It is proposed that the Toc12-imsHsp70-Toc64-Tic22 complex mediates protein translocation across the intermembrane space through specific precursor binding and ATP hydrolysis (Becker et al., 2004; Qbadou et al., 2007). However, the existence of imsHsp70 has only been shown on immunoblots by its reactivity to the monoclonal antibody SPA820 raised against human Hsp70. Its encoding gene has never been identified. The Arabidopsis (Arabidopsis thaliana) Hsp70 gene family has 14 members. Only two of them are localized in chloroplasts, and both have been shown to locate in the stroma (Ratnayake et al., 2008; Su and Li, 2008). A recent study has further shown that the major protein recognized by the SPA820 antibody in pea chloroplasts is located in the stroma, indicating that imsHsp70 is most likely a stromal protein (Ratnayake et al., 2008).Most translocon components were originally identified from pea chloroplasts. While all translocon components identified from pea have easily recognizable Arabidopsis homologs, Toc12 seems to be an exception. The Arabidopsis gene suggested to be the pea TOC12 homolog, At1g80920 (Inoue, 2007; Jarvis, 2008), encodes a protein that is much larger than pea Toc12 and is annotated as J8 (referred to as AtJ8 herein). The entire pea Toc12 has a high sequence similarity to the N-terminal two-thirds of AtJ8. AtJ8 contains an extra C-terminal domain of 60 amino acids that is highly conserved among J8 proteins from other higher plants. However, in contrast to pea Toc12, AtJ8 is predicted to locate in the stroma (Miernyk, 2001; www.arabidopsis.org). Indeed, a fusion protein consisting of the first 80 amino acids of AtJ8 fused at the N terminus of GFP was imported into the chloroplast stroma, and approximately 46 amino acids from the N terminus were processed after import (Lee et al., 2008), indicating that the first 46 amino acids of AtJ8 function as a cleavable stroma-targeting transit peptide. A T-DNA insertion in the AtJ8 gene that causes the truncation of the last three amino acids results in no visible phenotype. However, detailed analyses indicate that the mutant has lower CO₂ assimilation and Rubisco activity than the wild type (Chen et al., 2010).We are interested in identifying J domain-containing proteins interacting with stromal Hsp70. As part of the initial effort, we investigated the suborganellar location of J8 and examined the relationship between Toc12 and J8. We found that, in pea, there are at least two genes encoding J8, which we named PsJ8a and PsJ8b. TOC12 represents part of PsJ8b. Toc12, AtJ8, and the two PsJ8 proteins could be imported into chloroplasts and processed to stromally localized soluble mature proteins. Four alleles of AtJ8 mutants were analyzed, but none of them showed any defect in the import of various chloroplast precursor proteins.     

Toc,tic, and chloroplast protein import

The vast majority of chloroplast proteins are synthesized in precursor form on cytosolic ribosomes. Chloroplast precursor proteins have cleavable, N-terminal targeting signals called transit peptides. Transit peptides direct precursor proteins to the chloroplast in an organelle-specific way. They can be phosphorylated by a cytosolic protein kinase, and this leads to the formation of a cytosolic guidance complex. The guidance complex--comprising precursor, hsp70 and 14-3-3 proteins, as well as several unidentified components--docks at the outer envelope membrane. Translocation of precursor proteins across the envelope is achieved by the joint action of molecular machines called Toc (translocon at the outer envelope membrane of chloroplasts) and Tic (translocon at the inner envelope membrane of chloroplasts), respectively. The action of the Toc/Tic apparatus requires the hydrolysis of ATP and GTP at different levels, indicating energetic requirements and regulatory properties of the import process. The main subunits of the Toc and Tic complexes have been identified and characterized in vivo, in organello and in vitro. Phylogenetic evidence suggests that several translocon subunits are of cyanobacterial origin, indicating that today's import machinery was built around a prokaryotic core.     

Preprotein recognition by the Toc complex

The Toc core complex consists of the pore-forming Toc75 and the GTPases Toc159 and Toc34. We confirm that the receptor form of Toc159 is integrated into the membrane. The association of Toc34 to Toc75/Toc159 is GTP dependent and enhanced by preprotein interaction. The N-terminal half of the pSSU transit peptide interacts with high affinity with Toc159, whereas the C-terminal part stimulates its GTP hydrolysis. The phosphorylated C-terminal peptide of pSSU interacts strongly with Toc34 and therefore inhibits binding and translocation of pSSU into Toc proteoliposomes. In contrast, Toc159 recognises only the dephosphorylated forms. The N-terminal part of the pSSU presequence does not influence binding to the Toc complex, but is able to block import into proteoliposomes through its interaction with Toc159. We developed a model of differential presequence recognition by Toc34 and Toc159.     

Toc GTPases

Chloroplasts import more than 90% of their protein constituents from the cytosol. The import is mediated by translocon complexes located in the chloroplast envelope and the stroma. This review focuses on the two GTPases in the Toc (translocon at the outer envelope membrane of chloroplasts) complex. Hypotheses are presented about gating across the outer membrane and the possible functional states of the GTPases. This article is for the Special Issue of IMB 20th anniversary.     

Toc, Tic, and chloroplast protein import.

The vast majority of chloroplast proteins are synthesized in precursor form on cytosolic ribosomes. Chloroplast precursor proteins have cleavable, N-terminal targeting signals called transit peptides. Transit peptides direct precursor proteins to the chloroplast in an organelle-specific way. They can be phosphorylated by a cytosolic protein kinase, and this leads to the formation of a cytosolic guidance complex. The guidance complex--comprising precursor, hsp70 and 14-3-3 proteins, as well as several unidentified components--docks at the outer envelope membrane. Translocation of precursor proteins across the envelope is achieved by the joint action of molecular machines called Toc (translocon at the outer envelope membrane of chloroplasts) and Tic (translocon at the inner envelope membrane of chloroplasts), respectively. The action of the Toc/Tic apparatus requires the hydrolysis of ATP and GTP at different levels, indicating energetic requirements and regulatory properties of the import process. The main subunits of the Toc and Tic complexes have been identified and characterized in vivo, in organello and in vitro. Phylogenetic evidence suggests that several translocon subunits are of cyanobacterial origin, indicating that today's import machinery was built around a prokaryotic core.     

Neofunctionalization within the Omp85 protein superfamily during chloroplast evolution

The Toc75 and OEP80 proteins reside in the chloroplast outer envelope membrane. Both are members of the Omp85 superfamily of β-barrel proteins, and both are essential in Arabidopsis plants with important roles throughout development. Toc75 forms the translocation channel of the TOC complex, which is responsible for importing nucleus-encoded proteins into chloroplasts, while the function of OEP80 remains uncertain. Deficiency of Toc75 in plants that have artificially reduced OEP80 levels suggests that the latter may be involved in the biogenesis of β-barrel proteins, in similar fashion to Omp85-related proteins in other systems. To elucidate the evolutionary relationship between the two proteins, we conducted a phylogenetic analysis using 48 sequences from diverse species. This indicated that Toc75 and OEP80 belong to sister groups in the Omp85 superfamily, and originate from a gene duplication in an ancient eukaryotic organism > 1.2 billion years ago. Our analysis also supports the notion that the Toc75 family has undergone a phase of neofunctionalization to accommodate the organelle’s newly acquired need to import proteins.