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1.
Gutensohn M Fan E Frielingsdorf S Hanner P Hou B Hust B Klösgen RB 《Journal of plant physiology》2006,163(3):333-347
The chloroplast is an organelle of prokaryotic origin that is situated in an eukaryotic cellular environment. As a result of this formerly endosymbiotic situation, the chloroplast houses a unique set of protein transport machineries. Among those are evolutionarily young transport pathways which are responsible for the import of the nuclear-encoded proteins into the organelle as well as ancient pathways operating in the 'export' of proteins from the stroma (the former cyanobacterial cytosol) across the thylakoid membrane into the thylakoid lumen. In this review, we have tried to address the main features of these various transport pathways. 相似文献
2.
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. 相似文献
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4.
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. 相似文献
5.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2019,1867(6):627-636
The GTPases Toc159 and Toc34 of the translocon of the outer envelope of chloroplasts (TOC) are involved in recognition and transfer of precursor proteins at the cytosolic face of the organelle. Both proteins engage multiple interactions within the translocon during the translocation process, including dimeric states of their G-domains. The units of the Toc34 homodimer are involved in the recognition of the transit peptide representing the translocation signal of precursor proteins. This substrate recognition is part of the regulation of the GTPase cycle of Toc34. The Toc159 monomer and the Toc34 homodimer recognize the transit peptide of the small subunit of Rubisco at the N- and at the C-terminal region, respectively. Analysis of the transit peptide interaction by crosslinking shows that the heterodimer between both G-domains binds pSSU most efficiently. While substrate recognition by Toc34 homodimer was shown to regulate nucleotide exchange, we provide evidence that the high activation energy of the GTPase Toc159 is lowered by substrate recognition. The nucleotide affinity of Toc34G homodimer and Toc159G monomer are distinct, Toc34G homodimer recognizes GDP and Toc159G GTP with highest affinity. Moreover, the analysis of the nucleotide association rates of the monomeric and dimeric receptor units suggests that the heterodimer has an arrangement distinct from the homodimer of Toc34. Based on the biochemical parameters determined we propose a model for the order of events at the cytosolic side of TOC. The molecular processes described by this hypothesis range from transit peptide recognition to perception of the substrate by the translocation channel. 相似文献
6.
Becker T Hritz J Vogel M Caliebe A Bukau B Soll J Schleiff E 《Molecular biology of the cell》2004,15(11):5130-5144
Translocation of proteins across membranes is essential for the biogenesis of each cell and is achieved by proteinaceous complexes. We analyzed the translocation complex of the intermembrane space from chloroplasts and identified a 12-kDa protein associated with the Toc machinery. Toc12 is an outer envelope protein exposing a soluble domain into the intermembrane space. Toc12 contains a J-domain and stimulates the ATPase activity of DnaK. The conformational stability and the ability to stimulate Hsp70 are dependent on a disulfide bridge within the loop region of the J-domain, suggesting a redox-regulated activation of the chaperone. Toc12 is associated with Toc64 and Tic22. Its J-domain recruits the Hsp70 of outer envelope membrane to the intermembrane space translocon and facilitates its interaction to the preprotein. 相似文献
7.
Toc159- and Toc75-independent import of a transit sequence-less precursor into the inner envelope of chloroplasts 总被引:1,自引:0,他引:1
Miras S Salvi D Piette L Seigneurin-Berny D Grunwald D Reinbothe C Joyard J Reinbothe S Rolland N 《The Journal of biological chemistry》2007,282(40):29482-29492
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. 相似文献
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9.
Chloroplasts import post-translationally most of their constituent polypeptides via two distinct translocon units located in the outer and inner envelope. The protein import channel of the translocon of the outer envelope of chloroplasts, Toc75, is the most abundant protein in that membrane. We identify a novel Toc75 homologous protein, atToc75-V, a prominent protein that is clearly localized in the chloroplastic outer envelope. Phylogenetic analysis indicates that Toc75-V is more closely related to its prokaryotic ancestors than to Toc75 from plants. The presence of a second translocation channel suggests that alternative, previously unrecognized import routes into chloroplasts might exist. 相似文献
10.
Tic22 previously was identified as a component of the general import machinery that functions in the import of nuclear-encoded proteins into the chloroplast. Tic22 is peripherally associated with the outer face of the inner chloroplast envelope membrane, making it the first known resident of the intermembrane space of the envelope. We have investigated the import of Tic22 into isolated chloroplasts to define the requirements for targeting of proteins to the intermembrane space. Tic22 is nuclear-endoded and synthesized as a preprotein with a 50-amino acid N-terminal presequence. The analysis of deletion mutants and chimerical proteins indicates that the precursor of Tic22 (preTic22) presequence is necessary and sufficient for targeting to the intermembrane space. Import of preTic22 was stimulated by ATP and required the presence of protease-sensitive components on the chloroplast surface. PreTic22 import was not competed by an excess of an authentic stromal preprotein, indicating that targeting to the intermembrane space does not involve the general import pathway utilized by stromal preproteins. On the basis of these observations, we conclude that preTic22 is targeted to the intermembrane space of chloroplasts by a novel import pathway that is distinct from known pathways that target proteins to other chloroplast subcompartments. 相似文献
11.
The import of nuclear-encoded preproteins is necessary to maintain chloroplast function. The recognition and transfer of most precursor proteins across the chloroplast envelopes are facilitated by two membrane-inserted protein complexes, the translocons of the chloroplast outer and inner envelope (Toc and Tic complexes, respectively). Several signals have been invoked to regulate the import of preproteins. In our study, we were interested in redox-based import regulation mediated by two signals: regulation based on thiols and on the metabolic NADP+/NADPH ratio. We sought to identify the proteins participating in the regulation of these transport pathways and to characterize the preprotein subgroups whose import is redox-dependent. Our results provide evidence that the formation and reduction of disulfide bridges in the Toc receptors and Toc translocation channel have a strong influence on import yield of all tested preproteins that depend on the Toc complex for translocation. Furthermore, the metabolic NADP+/NADPH ratio influences not only the composition of the Tic complex, but also the import efficiency of most, but not all, preproteins tested. Thus, several Tic subcomplexes appear to participate in the translocation of different preprotein subgroups, and the redox-active compo- nents of these complexes likely play a role in regulating transport. 相似文献
12.
Most chloroplastic proteins are nuclear-encoded and must be transported into the organelle post-translationally. Proteinaceous components in the outer and inner envelope membranes of chloroplasts responsible for this import process were originally identified from pea seedlings. We sought to determine whether these proteins are conserved among different plant species other than pea and among different plastid types. We analyzed plant EST databases and found the presence of homologues to pea chloroplastic protein translocation components, Tic110 and Toc75, in both monocot and dicot species. Because these clones were obtained from various tissues, their presence in different types of plastids is proposed. Protein extracts were prepared from several plant species and from different plant tissues, and then probed with antisera raised against pea Tic110 and Toc75. The results support the idea that translocation components originally found in pea chloroplasts are conserved among different plant species and are present in various plastid types. 相似文献
13.
Kasmati AR Töpel M Patel R Murtaza G Jarvis P 《The Plant journal : for cell and molecular biology》2011,66(5):877-889
The Tic20 protein was identified in pea (Pisum sativum) as a component of the chloroplast protein import apparatus. In Arabidopsis, there are four Tic20 homologues, termed atTic20‐I, atTic20‐IV, atTic20‐II and atTic20‐V, all with predicted topological similarity to the pea protein (psTic20). Analysis of Tic20 sequences from many species indicated that they are phylogenetically unrelated to mitochondrial Tim17‐22‐23 proteins, and that they form two evolutionarily conserved subgroups [characterized by psTic20/atTic20‐I/IV (Group 1) and atTic20‐II/V (Group 2)]. Like psTic20, all four Arabidopsis proteins have a predicted transit peptide consistent with targeting to the inner envelope. Envelope localization of each one was confirmed by analysis of YFP fusions. RT‐PCR and microarray data revealed that the four genes are expressed throughout development. To assess the functional significance of the genes, T‐DNA mutants were identified. Homozygous tic20‐I plants had an albino phenotype that correlated with abnormal chloroplast development and reduced levels of chloroplast proteins. However, knockouts for the other three genes were indistinguishable from the wild type. To test for redundancy, double and triple mutants were studied; apart from those involving tic20‐I, none was distinguishable from the wild type. The tic20‐I tic20‐II and tic20‐I tic20‐V double mutants were albino, like the corresponding tic20‐I parent. In contrast, tic20‐I tic20‐IV double homozygotes could not be identified, due to gametophytic and embryonic lethality. Redundancy between atTic20‐I and atTic20‐IV was confirmed by complementation analysis. Thus, atTic20‐I and atTic20‐IV are the major functional Tic20 isoforms in Arabidopsis, with partially overlapping roles. While the Group 2 proteins have been conserved over approximately 1.2 billion (1.2 × 109) years, they are not essential for normal development. 相似文献
14.
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. 相似文献
15.
Machettira AB Gross LE Sommer MS Weis BL Englich G Tripp J Schleiff E 《Plant molecular biology》2011,77(4-5):381-390
Tic20 is a central, membrane-embedded component of the precursor protein translocon of the inner envelope of chloroplasts (TIC). In Arabidopsis thaliana, four different isoforms of Tic20 exist. They are annotated as atTic20-I, -II, -IV and -V and form two distinct phylogenetic subfamilies in embryophyta. Consistent with atTic20-I being the only essential isoform for chloroplast development, we show that the protein is exclusively targeted to the chloroplasts inner envelope. The same result is observed for atTic20-II. In contrast, atTic20-V is localized in thylakoids and atTic20-IV dually localizes to chloroplasts and mitochondria. These results together with the previously established expression profiles explain the recently described phenotypes of Tic20 knockout plants and point towards a functional diversification of these proteins within the family. For all Tic20 proteins a 4-helix topology is proposed irrespective of the targeted membrane, which in part could be confirmed in vivo by application of a self-assembling GFP-based topology approach. By the same approach we show that the inner envelope localized Tic20 proteins expose their C-termini to the chloroplast stroma. This localization would be consistent with the positive inside rule considering a stromal translocation intermediate as discussed. 相似文献
16.
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. 相似文献
17.
Initial binding of preproteins involving the Toc159 receptor can be bypassed during protein import into chloroplasts
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Two integral outer envelope GTPases, Toc34 and Toc86, are proposed to regulate the recognition and translocation of nuclear-encoded preproteins during the early stages of protein import into chloroplasts. Defining the precise roles of Toc86 and Toc34 has been complicated by the inability to distinguish their GTPase activities. Furthermore, the assignment of Toc86 function is rendered equivocal by recent reports suggesting that the standard protocol for the isolation of chloroplasts results in significant proteolysis of Toc86 (B. Bolter, T. May, J. Soll [1998] FEBS Lett 441: 59-62; G. Schatz [1998] Nature 395: 439-440). We demonstrate that Toc86 corresponds to a native protein of 159 kD in pea (Pisum sativum), designated Toc159. We take advantage of the proteolytic sensitivity of Toc159 to selectively remove its 100-kD cytoplasmic GTPase domain and thereby distinguish its activities from other import components. Proteolysis eliminates detectable binding of preproteins at the chloroplast surface, which is consistent with the proposed role of Toc159 as a receptor component. Remarkably, preprotein translocation across the outer membrane can occur in the absence of the Toc159 cytoplasmic domain, suggesting that binding can be bypassed. Translocation remains sensitive to GTP analogs in the absence of the Toc159 GTP-binding domain, providing evidence that Toc34 plays a key role in the regulation of translocation by GTP. 相似文献
18.
Toc64/OEP64 is not essential for the efficient import of proteins into chloroplasts in Arabidopsis thaliana 总被引:1,自引:0,他引:1
Aronsson H Boij P Patel R Wardle A Töpel M Jarvis P 《The Plant journal : for cell and molecular biology》2007,52(1):53-68
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. 相似文献
19.
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. 相似文献
20.
The chloroplast protein translocation complexes of Chlamydomonas reinhardtii: a bioinformatic comparison of Toc and Tic components in plants, green algae and red algae
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The recently completed genome of Chlamydomonas reinhardtii was surveyed for components of the chloroplast protein translocation complexes. Putative components were identified using reciprocal BlastP searches with the protein sequences of Arabidopsis thaliana as queries. As a comparison, we also surveyed the new genomes of the bryophyte Physcomitrella patens, two prasinophyte green algae (Ostreococcus lucimarinus and Ostreococcus tauri), the red alga Cyanidioschizon merolae, and several cyanobacteria. Overall, we found that the components of the import pathway are remarkably well conserved, particularly among the Viridiplantae lineages. Specifically, C. reinhardtii contained almost all the components found in A. thaliana, with two exceptions. Missing from C. reinhardtii are the C-terminal ferredoxin-NADPH-reductase (FNR) binding domain of Tic62 and a full-length, TPR-bearing Toc64. Further, the N-terminal domain of C. reinhardtii Toc34 is highly acidic, whereas the analogous region in C. reinhardtii Toc159 is not. This reversal of the vascular plant model may explain the similarity of C. reinhardtii chloroplast transit peptides to mitochondrial-targeting peptides. Other findings from our genome survey include the absence of Tic22 in both Ostreococcus genomes; the presence of only one Toc75 homolog in C. merolae; and, finally, a distinctive propensity for gene duplication in P. patens. 相似文献