Arabidopsis thaliana Tic110, involved in chloroplast protein translocation, contains at least fourteen highly divergent heat-like repeated motifs

Endosymbiotic theory suggests that plastids originated from a photosynthetic bacterium that was engulfed by a primitive eukaryotic cell. In consequence, the chloroplast genome remains affected by this ancestral event, although it is reduced in size and the number of constituent genes. Most parts of the plastid genome have been transferred to the host cell nuclear genome and are nuclear-encoded. Thus, chloroplast proteins are synthesized in the cytosol as precursors with N-terminal extensions called transit peptides. The evolution of import machinery was required to transfer transit peptides to the stroma. Until the present, two protein complexes have been found to mediate the import process: the Toc (outer) and Tic (inner) envelope membrane translocons. The evolutionary origin of many Tic and Toc proteins has been established, but not for the Tic110 subunit. Tic110 binds signal peptides and serves as a scaffold for the recruitment of stromal components. In this study, we analyzed hydrophobic clusters, protein folds, and protein structure homology and we conclude that Tic110 is composed of fourteen repeated motifs related to HEAT-repeats. The explanation for the presence of such repeats in Tic110 is that membrane arrangement is found in separate domains and their probable function in the chloroplast import process is discussed.     

Genome analysis and its significance in four unicellular algae, Cyanidioshyzon merolae, Ostreococcus tauri, Chlamydomonas reinhardtii, and Thalassiosira pseudonana

Algae play a more important role than land plants in the maintenance of the global environment and productivity. Progress in genome analyses of these organisms means that we can now obtain information on algal genomes, global annotation and gene expression. The full genome information for several algae has already been analyzed. Whole genomes of the red alga Cyanidioshyzon merolae, the green algae Ostreococcus tauri and Chlamydomonas reinhardtii, and the diatom Thalassiosira pseudonana have been sequenced. Genome composition and the features of cells among the four algae were compared. Each alga maintains basic genes as photosynthetic eukaryotes and possesses additional gene groups to represent their particular characteristics. This review discusses and introduces the latest research that makes the best use of the particular features of each organism and the significance of genome analysis to study biological phenomena. In particular, examples of post-genome studies of organelle multiplication in C. merolae based on analyzed genome information are presented.     

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.     

Comparative genomic analysis of the Hsp70s from five diverse photosynthetic eukaryotes

We have identified 24 members of the DnaK subfamily of heat shock 70 proteins (Hsp70s) in the complete genomes of 5 diverse photosynthetic eukaryotes. The Hsp70s are a ubiquitous protein family that is highly conserved across all domains of life. Eukaryotic Hsp70s are found in a number of subcellular compartments in the cell: cytoplasm, mitochondrion (MT), chloroplast (CP), and endoplasmic reticulum (ER). Although the Hsp70s have been the subject of intense study in model organisms, very little is known of the Hsp70s from early diverging photosynthetic lineages. The sequencing of the complete genomes of Thalassiosira pseudonana (a diatom), Cyanidioschyzon merolae (a red alga), and 3 green algae (Chlamydomonas reinhardtii, Ostreococcus lucimarinus, Ostreococcus tauri) allow us to conduct comparative genomics of the Hsp70s present in these diverse photosynthetic eukaryotes. We have found that the distinct lineages of Hsp70s (MT, CP, ER, and cytoplasmic) each have different evolutionary histories. In general, evolutionary patterns of the mitochondrial and endoplasmic reticulum Hsp70s are relatively stable even among very distantly related organisms. This is not true of the chloroplast Hsp70s and we discuss the distinct evolutionary patterns between "green" and "red" plastids. Finally, we find that, in contrast to the angiosperms Arabidopsis thaliana and Oryza sativa that have numerous cytoplasmic Hsp70, the 5 algal species have only 1 cytoplasmic Hsp70 each. The evolutionary and functional implications of these differences are discussed.     

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.     

Cyanidioschyzon merolae genome. A tool for facilitating comparable studies on organelle biogenesis in photosynthetic eukaryotes

The ultrasmall unicellular red alga Cyanidioschyzon merolae lives in the extreme environment of acidic hot springs and is thought to retain primitive features of cellular and genome organization. We determined the 16.5-Mb nuclear genome sequence of C. merolae 10D as the first complete algal genome. BLASTs and annotation results showed that C. merolae has a mixed gene repertoire of plants and animals, also implying a relationship with prokaryotes, although its photosynthetic components were comparable to other phototrophs. The unicellular green alga Chlamydomonas reinhardtii has been used as a model system for molecular biology research on, for example, photosynthesis, motility, and sexual reproduction. Though both algae are unicellular, the genome size, number of organelles, and surface structures are remarkably different. Here, we report the characteristics of double membrane- and single membrane-bound organelles and their related genes in C. merolae and conduct comparative analyses of predicted protein sequences encoded by the genomes of C. merolae and C. reinhardtii. We examine the predicted proteins of both algae by reciprocal BLASTP analysis, KOG assignment, and gene annotation. The results suggest that most core biological functions are carried out by orthologous proteins that occur in comparable numbers. Although the fundamental gene organizations resembled each other, the genes for organization of chromatin, cytoskeletal components, and flagellar movement remarkably increased in C. reinhardtii. Molecular phylogenetic analyses suggested that the tubulin is close to plant tubulin rather than that of animals and fungi. These results reflect the increase in genome size, the acquisition of complicated cellular structures, and kinematic devices in C. reinhardtii.     

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。     

Physcomitrella patens as a model for the study of chloroplast protein transport: conserved machineries between vascular and non-vascular plants

A single general import pathway in vascular plants mediates the transport of precursor proteins across the two membranes of the chloroplast envelope, and at least four pathways are responsible for thylakoid protein targeting. While the transport systems in the thylakoid are related to bacterial secretion systems, the envelope machinery is thought to have arisen with the endosymbiotic event and to be derived, at least in part, from proteins present in the original endosymbiont. Recently the moss Physcomitrella patens has gained worldwide attention for its ability to undergo homologous recombination in the nuclear genome at rates unseen in any other land plants. Because of this, we were interested to know whether it would be a useful model system for studying chloroplast protein transport. We searched the large database of P. patens expressed sequence tags for chloroplast transport components and found many putative homologues. We obtained full-length sequences for homologues of three Toc components from moss. To our knowledge, this is the first sequence information for these proteins from non-vascular plants. In addition to identifying components of the transport machinery from moss, we isolated plastids and tested their activity in protein import assays. Our data indicate that moss and pea (Pisum sativum) plastid transport systems are functionally similar. These findings identify P. patens as a potentially useful tool for combining genetic and biochemical approaches for the study of chloroplast protein targeting.     

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.     

Tic21 is an essential translocon component for protein translocation across the chloroplast inner envelope membrane

An Arabidopsis thaliana mutant defective in chloroplast protein import was isolated and the mutant locus, cia5, identified by map-based cloning. CIA5 is a 21-kD integral membrane protein in the chloroplast inner envelope membrane with four predicted transmembrane domains, similar to another potential chloroplast inner membrane protein-conducting channel, At Tic20, and the mitochondrial inner membrane counterparts Tim17, Tim22, and Tim23. cia5 null mutants were albino and accumulated unprocessed precursor proteins. cia5 mutant chloroplasts were normal in targeting and binding of precursors to the chloroplast surface but were defective in protein translocation across the inner envelope membrane. Expression levels of CIA5 were comparable to those of major translocon components, such as At Tic110 and At Toc75, except during germination, at which stage At Tic20 was expressed at its highest level. A double mutant of cia5 At tic20-I had the same phenotype as the At tic20-I single mutant, suggesting that CIA5 and At Tic20 function similarly in chloroplast biogenesis, with At Tic20 functioning earlier in development. We renamed CIA5 as Arabidopsis Tic21 (At Tic21) and propose that it functions as part of the inner membrane protein-conducting channel and may be more important for later stages of leaf development.     

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.     

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.     

Differential accumulation of plastid preprotein translocon components during spruce (Picea abies L. Karst.) needle development

We demonstrate that basic components of the plastid protein-import apparatus originally found in pea, Toc34, Toc159, and Tic110, are also conserved in evolutionarily younger gymnosperms. We show that multiple isoforms of the preprotein receptor Toc34 differentially accumulate in various stages of needle development, while the amounts of Toc159 drastically decrease during chloroplast morphogenesis. Spruce Toc34 and Toc159 receptors are able to recognise and interact with the angiosperm precursor of the Rubisco small subunit. Young proplastids found in closed buds contain a highly elevated number of protein translocation complexes equipped with only two types of outer envelope receptors, Toc159 and a 30-kDa Toc34-related protein. Photosystem II (PSII) can already be assembled in a fully functional complex at this very early stage of needle development, suggesting that no additional receptor isoforms are needed for translocation of all necessary PSII components. We conclude that the accumulation of evolutionarily conserved plastid preprotein translocation components is differentially regulated during spruce needle development.     

Novel pathways for glycoprotein import into chloroplasts

Although the chloroplast contains its own genome, majority of its protein components are encoded by nuclear genes and must be imported post-translationally. In general, proteins synthesized by cytosolic ribosomes are post-translationally targeted to the chloroplast through interactions between their N-terminal transit sequence and protein translocon Toc/Tic complexes in the chloroplast membranes. An alternative pathway that mediates post-translational delivery of proteins to the chloroplast via the secretory pathway was recently described. This pathway provides new opportunities for complementation of the chloroplast protein maturation machinery with chaperones needing endoplasmic reticulum and/or Golgi typical maturations such as N-glycosylation for their biological activity or using chloroplasts as a storage compartment for glycoproteins.     

Stimulation of transit-peptide release and ATP hydrolysis by a cochaperone during protein import into chloroplasts

Three components of the chloroplast protein translocon, Tic110, Hsp93 (ClpC), and Tic40, have been shown to be important for protein translocation across the inner envelope membrane into the stroma. We show the molecular interactions among these three components that facilitate processing and translocation of precursor proteins. Transit-peptide binding by Tic110 recruits Tic40 binding to Tic110, which in turn causes the release of transit peptides from Tic110, freeing the transit peptides for processing. The Tic40 C-terminal domain, which is homologous to the C terminus of cochaperones Sti1p/Hop and Hip but with no known function, stimulates adenosine triphosphate hydrolysis by Hsp93. Hsp93 dissociates from Tic40 in the presence of adenosine diphosphate, suggesting that Tic40 functions as an adenosine triphosphatase activation protein for Hsp93. Our data suggest that chloroplasts have evolved the Tic40 cochaperone to increase the efficiency of precursor processing and translocation.     

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.     

An exogenous chloroplast genome for complex sequence manipulation in algae

We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii. Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus. Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.     

At least two Toc34 protein import receptors with different specificities are also present in spinach chloroplasts

The receptor components of the chloroplast protein import machinery, Toc34 and Toc159, are both encoded by small gene families in Arabidopsis thaliana. Recent results suggest that each member of these families preferentially interacts with different groups of precursor proteins. Here we address the question, whether multiple homologous Toc receptors are unique to Arabidopsis or whether they are a general phenomenon in plants. Indeed, in spinach we could identify at least two Toc34 proteins with different substrate specificities as demonstrated by competition and antibody inhibition experiments. In addition, an analysis of the available genomic data revealed the presence of at least two Toc34 homologs in six other plant species.