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.     

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.     

The chloroplastic protein import machinery contains a Rieske-type iron-sulfur cluster and a mononuclear iron-binding protein.

Transport of precursor proteins across the chloroplastic envelope membranes requires the interaction of protein translocons localized in both the outer and inner envelope membranes. Analysis by blue native gel electrophoresis revealed that the translocon of the inner envelope membranes consisted of at least six proteins with molecular weights of 36, 45, 52, 60, 100 and 110 kDa, respectively. Tic110 and ClpC, identified as components of the protein import apparatus of the inner envelope membrane, were prominent constituents of this complex. The amino acid sequence of the 52 kDa protein, deduced from the cDNA, contains a predicted Rieske-type iron-sulfur cluster and a mononuclear iron-binding site. Diethylpyrocarbonate, a Rieske-type protein-modifying reagent, inhibits the translocation of precursor protein across the inner envelope membrane, whereas binding of the precursor to the outer envelope membrane is still possible. In another independent experimental approach, the 52 kDa protein could be co-purified with a trapped precursor protein in association with the chloroplast protein translocon subunits Toc86, Toc75, Toc34 and Tic110. Together, these results strongly suggest that the 52 kDa protein, named Tic55 due to its calculated molecular weight, is a member of the chloroplastic inner envelope protein translocon.     

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

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

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

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

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

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

Tic40, a new "old" subunit of the chloroplast protein import translocon

The protein import translocon at the inner envelope of chloroplasts (Tic complex) is a heteroligomeric multisubunit complex. Here, we describe Tic40 from pea as a new component of this complex. Tic40 from pea is a homologue of a protein described earlier from Brassica napus as Cim/Com44 or the Toc36 subunit of the translocon at the outer envelope of chloroplasts, respectively (Wu, C., Seibert, F. S., and Ko, K. (1994) J. Biol. Chem. 269, 32264-32271; Ko, K., Budd, D., Wu, C., Seibert, F., Kourtz, L., and Ko, Z. W. (1995) J. Biol. Chem. 270, 28601-28608; Pang, P., Meathrel, K., and Ko, K. (1997) J. Biol. Chem. 272, 25623-25627). Tic40 can be covalently connected to Tic110 by the formation of a disulfide bridge under oxidizing conditions, indicating its close physical proximity to an established translocon component. The Tic40 protein is synthesized in the cytosol as a precursor with an N-terminal cleavable chloroplast targeting signal and imported into the organelle via the general import pathway. Immunoblotting and immunogold-labeling studies exclusively confine Tic40 to the chloroplastic inner envelope, in which it is anchored by a single putative transmembrane span.     

植物细胞中蛋白质向叶绿体转运的研究进展

植物细胞中叶绿体的功能主要依赖于叶绿体蛋白, 大部分叶绿体蛋白由核基因组编码, 在细胞质中合成并经过正确的分选后, 通过叶绿体外膜上的Toc复合体和/或内膜上的Tic复合体转运到叶绿体的不同部位。该文主要综述可能参与叶绿体蛋白分选的胞质因子以及Toc和Tic组分如何参与叶绿体蛋白转运的研究进展。     

Mechanism of protein import across the chloroplast envelope

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

Protein translocon at the Arabidopsis outer chloroplast membrane.

Chloroplasts are organelles essential for the photoautotrophic growth of plants. Their biogenesis from undifferentiated proplastids is triggered by light and requires the import of hundreds of different precursor proteins from the cytoplasm. Cleavable N-terminal transit sequences target the precursors to the chloroplast where translocon complexes at the outer (Toc complex) and inner (Tic complex) envelope membranes enable their import. In pea, the Toc complex is trimeric consisting of two surface-exposed GTP-binding proteins (Toc159 and Toc34) involved in precursor recognition and Toc75 forming an aequeous protein-conducting channel. Completion of the Arabidopsis genome has revealed an unexpected complexity of predicted components of the Toc complex in this plant model organism: four genes encode homologs of Toc159, two encode homologs of Toc34, but only one encodes a likely functional homolog of Toc75. The availability of the genomic sequence data and powerful molecular genetic techniques in Arabidopsis set the stage to unravel the mechanisms of chloroplast protein import in unprecedented depth.     

Tic20 and Tic22 Are New Components of the Protein Import Apparatus at the Chloroplast Inner Envelope Membrane

Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677–1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.     

Stable megadalton TOC–TIC supercomplexes as major mediators of protein import into chloroplasts

Preproteins are believed to be imported into chloroplasts through membrane contact sites where the translocon complexes of the outer (TOC) and inner (TIC) envelope membranes are assembled together. However, a single TOC–TIC supercomplex containing preproteins undergoing active import has not yet been directly observed. We optimized the blue native polyacrylamide gel electrophoresis (PAGE) (BN‐PAGE) system to detect and resolve megadalton (MD)‐sized complexes. Using this optimized system, the outer‐membrane channel Toc75 from pea chloroplasts was found in at least two complexes: the 880‐kD TOC complex and a previously undetected 1‐MD complex. Two‐dimensional BN‐PAGE immunoblots further showed that Toc75, Toc159, Toc34, Tic20, Tic56 and Tic110 were all located in the 880‐kD to 1.3‐MD region. During active preprotein import, preproteins were transported mostly through the 1‐MD complex and a smaller amount of preproteins was also detected in a complex of 1.25 MD. Antibody‐shift assays showed that the 1‐MD complex is a TOC–TIC supercomplex containing at least Toc75, Toc159, Toc34 and Tic110. Results from crosslinking and import with Arabidopsis chloroplasts suggest that the 1.25‐MD complex is also a supercomplex. Our data provide direct evidence supporting that chloroplast preproteins are imported through TOC–TIC supercomplexes, and also provide the first size estimation of these supercomplexes. Furthermore, unlike in mitochondria where translocon supercomplexes are only transiently assembled during preprotein import, in chloroplasts at least some of the supercomplexes are preassembled stable structures.     

Evolution of the general protein import pathway of plastids (review)

The evolutionary process that transformed a cyanobacterial endosymbiont into contemporary plastids involved not only inheritance but also invention. Because gram-negative bacteria lack a system for polypeptide import, the envelope translocon complex of the general protein import pathway was the most important invention of organelle evolution resulting in a pathway to import back into plastids those nuclear-encoded proteins supplemented with a transit peptide. Genome information of cyanobacteria, phylogenetically diverse plastids, and the nuclei of the first red alga, a diatom, and Arabidopsis thaliana allows us to trace back the evolutionary origin of the twelve currently known translocon components and to partly deduce their assembly sequence. Development of the envelope translocon was initiated by recruitment of a cyanobacterial homolog of the protein-import channel Toc75, which belongs to a ubiquitous and essential family of Omp85/D15 outer membrane proteins of gram-negative bacteria that mediate biogenesis of beta-barrel proteins. Likewise, three other translocon subunits (Tic20, Tic22, and Tic55) and several stromal chaperones have been inherited from the ancestral cyanobacterium and modified to take over the novel function of precursor import. Most of the remaining subunits seem to be of eukaryotic origin, recruited from pre-existing nuclear genes. The next subunits that joined the evolving protein import complex likely were Toc34 and Tic110, as indicated by the presence of homologous genes in the red alga Cyanidioschyzon merolae, followed by the stromal processing peptidase, members of the Toc159 receptor family, Toc64, Tic40, and finally some regulatory redox components (Tic62, Tic32), all of which were probably required to increase specificity and efficiency of precursor 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.     

Protein import into chloroplasts: new aspects of a well-known topic

Protein import into plant chloroplasts is a fascinating topic that is being investigated by many research groups. Since the majority of chloroplast proteins are synthesised as precursor proteins in the cytosol, they have to be posttranslationally imported into the organelle. For this purpose, most preproteins are synthesised with an N-terminal presequence, which is both necessary and sufficient for organelle recognition and translocation initiation. The import of preproteins is facilitated by two translocation machineries in the outer and inner envelope of chloroplasts, the Toc and Tic complexes, respectively. Translocation of precursor proteins across the envelope membrane has to be highly regulated to react to the metabolic requirements of the organelle. The aim of this review is to summarise the events that take place at the translocation machineries that are known so far. In addition, we focus in particular on alternative import pathways and the aspect of regulation of protein transport at the outer and inner envelope 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.     

The chloroplast protein import receptors Toc34 and Toc159 are phosphorylated by distinct protein kinases

The molecular composition of chloroplast outer and inner envelope translocons is fairly well established, but little is known about mechanisms and elements involved in import regulation. After synthesis in the cytosol, chloroplast targeted precursor proteins are recognized by outer envelope receptors Toc34 and Toc159. Phosphorylation plays an important role in regulation of Toc34 activity and preprotein binding. Using kinase renaturation assays, we have identified an ATP-dependent 98-kDa outer envelope kinase which is able to selectively phosphorylate Toc34 at a specific site. A 70-kDa outer envelope polypeptide phosphorylating Toc159 was identified by the same strategy. Antiserum against the 98-kDa kinase inhibits phosphorylation of Toc34, whereas labeling of Toc159 remains unaffected. Both kinases do not autophosphorylate in vitro and are unable to utilize myelin basic protein as substrate. We propose that distinct kinases are involved in regulation of chloroplast import via desensitization of preprotein receptors.     

The motors of protein import into chloroplasts

Chloroplast function is largely dependent on its resident proteins, most of which are encoded by the nuclear genome and are synthesized in cytosol. Almost all of these are imported through the translocons located in the outer and inner chloroplast envelope membranes. The motor protein that provides the driving force for protein import has been proposed to be Hsp93, a member of the Hsp100 family of chaperones residing in the stroma. Combining in vivo and in vitro approaches, recent publications have provided multiple lines of evidence demonstrating that a stromal Hsp70 system is also involved in protein import into this organelle. Thus it appears that protein import into chloroplasts is driven by two motor proteins, Hsp93 and Hsp70. A perspective on collaboration between these two chaperones is discussed.Key words: stromal Hsp70, chloroplast protein import, stromal motor complex, ATPase, Physcomitrella patens, Hsp93, Toc, Tic, transit peptide, translocationChloroplasts are plant and algal specific organelles where photosynthesis and many other cellular processes take place. Chloroplasts contain ∼3,000 proteins,^1,2 with about 100 encoded by the chloroplast genome. In other words, more than 90% of chloroplast proteins are encoded by nuclear genes, synthesized in the cytosol and post-translationally imported into plastids. Most imported proteins are synthesized as precursors with a cleavable N-terminal signal, called a transit peptide. Such precursors are recognized by receptors in the outer envelope membrane, translocated through translocons in the outer and inner envelope membranes of chloroplasts (Toc and Tic), and processed to either their mature- or intermediate-sized forms in the chloroplast stroma.^3–8 Thylakoid proteins are further transported to their final destinations via one of four pathways, the cpSec, cpSRP, cpTAT and spontaneous pathways.^9–11 It is believed that the precursors are translocated across the envelope membranes in at least partially unfolded conformations and that the import machinery possesses some degree of unfolding activity.¹²Three proteins make up the core Toc complex, Toc159, Toc34 and Toc75. The Toc159 and Toc34 proteins are receptors possessing GTPase activities and recognizing transit peptides. Toc75 is a ß-barrel protein that forms the protein-translocating channel across the outer envelope membrane.¹³ The Tic complex is also formed from multiple subunits. Tic110, Tic21 and Tic20 have each been suggested to function as the channel of the Tic complex.^14–16 A ternary complex containing the stroma-facing domain of Tic110, Tic40 and a stromal factor, Hsp93 (a member of the Hsp100 family, possessing two ATPase domains), interacts with incoming precursor proteins.^17–26 Hsp93 has been proposed to serve as the import motor.²⁷ Other Tic components include regulatory subunits Tic62, Tic55 and Tic32 that are purported to facilitate redox- and calcium/calmodulin-dependent precursor translocation across the inner envelope membrane (reviewed in ref. ³). Tic22 is a peripheral membrane protein associated with the inner envelope and exposed to the intermembrane space.²⁸ It is suggested that Tic22 connects the Toc and Tic translocons during protein import.     

Evolution of the general protein import pathway of plastids (Review)

The evolutionary process that transformed a cyanobacterial endosymbiont into contemporary plastids involved not only inheritance but also invention. Because Gram-negative bacteria lack a system for polypeptide import, the envelope translocon complex of the general protein import pathway was the most important invention of organelle evolution resulting in a pathway to import back into plastids those nuclear-encoded proteins supplemented with a transit peptide. Genome information of cyanobacteria, phylogenetically diverse plastids, and the nuclei of the first red alga, a diatom, and Arabidopsis thaliana allows us to trace back the evolutionary origin of the twelve currently known translocon components and to partly deduce their assembly sequence. Development of the envelope translocon was initiated by recruitment of a cyanobacterial homolog of the protein-import channel Toc75, which belongs to a ubiquitous and essential family of Omp85/D15 outer membrane proteins of Gram-negative bacteria that mediate biogenesis of β-barrel proteins. Likewise, three other translocon subunits (Tic20, Tic22, and Tic55) and several stromal chaperones have been inherited from the ancestral cyanobacterium and modified to take over the novel function of precursor import. Most of the remaining subunits seem to be of eukaryotic origin, recruited from pre-existing nuclear genes. The next subunits that joined the evolving protein import complex likely were Toc34 and Tic110, as indicated by the presence of homologous genes in the red alga Cyanidioschyzon merolae, followed by the stromal processing peptidase, members of the Toc159 receptor family, Toc64, Tic40, and finally some regulatory redox components (Tic62, Tic32), all of which were probably required to increase specificity and efficiency of precursor import.     

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.