Chloroplast protein import inhibition by a soluble factor from wheat germ lysate

Protein import into chloroplasts occurs post-translationally in vitro. The precursor proteins are generally synthesised in a reticulocyte lysate- or wheat germ lysate-derived system and imported out of this system into chloroplast. These complex soluble protein mixtures are likely to contain factors, which influence somehow the import competence and import efficiency. Here we describe a heat-stable soluble proteinaceaous factor, which inhibits protein import into chloroplasts in vitro. The inhibitor interacts directly with the precursor protein and renders it import incompetent. This mode of action is supported by two observations: firstly, binding of the precursor to the chloroplast surface is diminished in the presence of the inhibitor. Secondly, when chloroplasts were loaded with precursor proteins under conditions, which allow only binding but not import the inhibitor was unable to abolish the subsequent translocation step.     

Mitochondrial and chloroplast targeting sequences in tandem modify protein import specificity in plant organelles

Protein targeting to plant mitochondria and chloroplasts is usually very specific and involves targeting sequences located at the amino terminus of the precursor. We challenged the system by using combinations of mitochondrial and chloroplast targeting sequences attached to reporter genes. The sequences coding for the presequence of the mitochondrial F₁-ATPase -subunit and the transit peptide of the chloroplast chlorophyll a/b-binding protein, both from Nicotiana plumbaginifolia, were fused together in both combinations, then linked to the reporter genes, chloramphenicol acetyl transferase (CAT) and -glucuronidase (GUS), and introduced into tobacco. Analysis of CAT and GUS activities and proteins in the subcellular fractions revealed that the chloroplast transit peptide alone was not sufficient to target the reporter proteins to chloroplasts. However, when the mitochondrial -presequence was inserted downstream of the chloroplast sequence, import of CAT and GUS into chloroplasts was observed. Using the reciprocal system, the mitochondrial presequence alone was able to direct transport of CAT and, to a lesser extent, GUS to mitochondria; the GUS targeting to mitochondria was increased when the chloroplast targeting sequence was linked downstream of the mitochondrial presequence. Immuno-detection experiments using subcellular fractions confirmed the results observed by enzymatic assays. These results indicate the importance of the amino-terminal position of the targeting sequence in determining protein import specificity and are considered within the hypothesis of a co-translational protein import.     

Internal plastid-targeting signal found in a RubisCO small subunit protein of a chlorarachniophyte alga

In all plants and algae, most plastid proteins are encoded by the nuclear genome and, consequently, need to be transported into plastids across multiple membranes. In organisms with secondary plastids, which evolved by secondary endosymbioses, and are surrounded by three or four envelope membranes, precursors of nuclear-encoded plastid proteins generally have an N-terminal bipartite targeting sequence that consists of an endoplasmic reticulum (ER)-targeting signal peptide (SP) and a transit peptide-like (TPL) sequence. The bipartite targeting sequences have been demonstrated to be necessary and sufficient for targeting proteins into the plastids of many algal groups, including chlorarachniophytes. Here, we report a new type of targeting signal that is required for delivering a RubisCO small subunit (RbcS) protein into the secondary plastids of chlorarachniophyte algae. In this study, we analyzed the plastid-targeting ability of an RbcS pre-protein, using green fluorescent protein (GFP) as a reporter molecule in chlorarachniophyte cells. We demonstrate that the N-terminal bipartite targeting sequence of the RbcS pre-protein is not sufficient, and that a part of the mature protein is also necessary for plastid targeting. By deletion analyses of amino acids, we determined the approximate location of an internal plastid-targeting signal within the mature protein, which is involved in targeting the protein from the ER into the chlorarachniophyte plastids.     

羊草光合作用相关基因的克隆与分析

在构建了羊草叶片cDNA文库的基础上,利用M13载体通用引物筛选其亚文库,挑选阳性克隆进行测序,将测序结果在NCBI基因库中进行比对,得到一个Rubisco大亚基基因全长序列和Rubisco小亚基基因部分序列,并对其核苷酸及其编码的氨基酸序列进行分析。结果显示,Rubisco大亚基基因长度为1 796 bp,与禾本科大麦、小麦、野雀麦、粗山羊草、旱麦草、异形花草、黑麦等的核苷酸序列同源性达98%以上;羊草的Rubisco小亚基基因部分序列含有一个开放阅读框,其长度为186 bp,编码61个氨基酸,与禾本科的小麦、大麦、燕麦、黑麦以及扁穗雀麦Rubisco小亚基基因氨基酸序列的同源性分别为93%、93%、91%、91%、92%。羊草Rubisco基因的克隆与分析有利于进一步研究其光合作用效率。     

Homologous and heterologous protein import into mitochondria isolated from the green alga Chlamydomonas reinhardtii

We have established a homologous system for studying mitochondrial protein import in Chlamydomonas reinhardtii, using C. reinhardtii precursor proteins and mitochondria isolated from C. reinhardtii. The precursors of the F₁ ATP synthase subunit and the Rieske FeS protein were imported into mitochondria with high efficiency, while the F₁ subunit precursor was imported with much lower efficiency. The import of heterologous precursor proteins from higher plants was also less efficient. The precursor of the C. reinhardtii PsaF chloroplast protein was converted into a protease-protected form upon incubation with mitochondria. In vitro processing studies revealed that in contrast to the situation in higher plants, the processing of the precursors was catalysed by a soluble, matrix-located peptidase.     

The importance of the transit peptide and the transported protein for protein import into chloroplasts

Summary We compared the transport in vitro of fusion proteins of neomycin phosphotransferase II (NPTII) with either the transit peptide of the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase or the transit peptide and the 23 aminoterminal amino acids of the mature small subunit. The results showed that the transit peptide is sufficient for import of NPTII. However, transport of the fusion protein consisting of the transit peptide linked directly to NPTII was very inefficient. In contrast, the fusion protein containing a part of the mature SSU was imported with an efficiency comparable to that of the authentic SSU precursor. We conclude from these results that other features of the precursor protein in addition to the transit peptide are important for transport into chloroplasts. In order to identify functional regions in the transit peptide, we analyzed the transport of mutant fusion proteins. We found that the transport of fusion proteins with large deletions in the aminoterminal, or central part was drastically reduced. In contrast, duplication of a part of the transit peptide led to a marked increase in transport.     

Homologous protein import machineries in chloroplasts and cyanelles

The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria, especially in the presence of a peptidoglycan wall between the inner and outer envelope membranes. However, it is now clear that cyanelles are in fact primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario, cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high gene content of rhodoplasts and the peptidoglycan wall of cyanelles. This means that the import apparatuses of all primary plastids, i.e. those from glaucocystophytes, red algae, green algae and higher plants, should be homologous. If this is the case, then transit sequences should be similar and heterologous import experiments feasible. Thus far, heterologous in vitro import has been shown in one direction only: precursors from C. paradoxa were imported into isolated pea or spinach chloroplasts. Cyanelle transit sequences differ from chloroplast stroma targeting peptides in containing in their N-terminal domain an invariant phenylalanine residue which is shown here to be crucial for import. In addition, we now demonstrate that heterologous precursors are readily imported into isolated cyanelles, provided that the essential phenylalanine residue is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor/channel showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved, explaining the efficient heterologous import of native precursors from C. paradoxa.     

Isolation of Pea Thioredoxin f Precursor Protein and Characterization of its Biochemical Properties

Like many other soluble chloroplastic enzymes, thioredoxin f is nuclear-encoded and expressed as a precursor protein. After synthesis in the cytosol, it is imported into the chloroplast with subsequent cleavage of the transit sequence in the stroma. We report the expression and the partial purification of the recombinant precursor thioredoxin f protein. The prethioredoxin f was found to be located essentially in the insoluble Echerichia coli fraction, but could be renatured after urea treatment followed by dialysis. The renatured protein was active in the dithiothreitol- and thioredoxin-dependent activation of NADP malate dehydrogenase and also of fructose bisphosphatase and in the ferredoxin-thioredoxin-dependent fructose bisphosphatase activation. These data are discussed in relation with the known properties of mature thioredoxin f.     

Plasmodium falciparum apicoplast transit peptides are unstructured in vitro and during apicoplast import

Trafficking of soluble proteins to the apicoplast in Plasmodium falciparum is determined by an N-terminal transit peptide (TP) which is necessary and sufficient for apicoplast import. Apicoplast precursor proteins are synthesized at the rough endoplasmic reticulum, but are then specifically sorted from other proteins in the secretory pathway. The mechanism of TP recognition is presently unknown. Apicoplast TPs do not contain a conserved sequence motif; therefore, we asked whether they contain an essential structural motif. Using nuclear magnetic resonance to study a model TP from acyl carrier protein, we found a short, low-occupancy helix, but the TP was otherwise disordered. Using an in vivo localization assay, we blocked TP secondary structure by proline mutagenesis, but found robust apicoplast localization. Alternatively, we increased the helical content of the TP through mutation while maintaining established TP characteristics. Apicoplast import was disrupted in a helical mutant TP, but import was then restored by the further addition of a single proline. We conclude that structure in the TP interferes with apicoplast import, and therefore TPs are functionally disordered. These results provide an explanation for the amino acid bias observed in apicoplast TPs.     

Subcellular targeting of green fluorescent protein to plastids in transgenic rice plants provides a high-level expression system

In order to develop a high-level expression system in transgenic rice, we inserted a synthetic gene (sgfp) encoding a modified form of the green fluorescent protein (GFP) into two expression vectors, Act1-sgfp for an untargeted and rbcS-Tp-sgfp for a chloroplast targeted expression. Several fertile transgenic rice plants were produced by the Agrobacterium-mediated method. Confocal microscopic analyses demonstrated that, in cells expressing the Act1-sgfp, GFP fluorescence was localized within the cytoplasm and nucleoplasm whereas, in cells expressing the rbcS-Tp-sgfp fusion gene, the fluorescence was specifically targeted to chloroplasts and non-green plastids. The levels of sgfp expression were about 0.5% of the total soluble protein in mature leaf tissues of the Act1-sgfp transformed lines. In contrast, expression levels were markedly increased in mature leaf tissues of the rbcS-Tp-sgfp transformed lines, yielding about 10% of the total soluble protein. N-terminal sequencing of the localized GFPs revealed that the Tp-GFP fusion protein was correctly processed during import to non-green plastids, as well as to chloroplasts. Thus, our results demonstrate that GFP can be produced at high levels and localized in specific subcellular spaces of transgenic plants, providing a high-level expression system for general use in rice, an agronomically important cereal.     

拟南芥未知功能基因At3g61870编码蛋白的叶绿体定位研究

利用反向遗传学研究方法对1个预测的拟南芥叶绿体未知功能基因At3g61870编码蛋白进行了亚细胞定位研究.通过克隆At3g61870基因5′端长229 bp的DNA片段,与绿色荧光蛋白(GFP)基因构建重组表达载体pMON530-CP-TP-GFP,经农杆菌介导转化拟南芥.转基因植株的叶肉细胞经激光共聚焦显微镜观察,叶绿素自发荧光与GFP荧光共定位于叶绿体中.结果表明,未知功能基因At3g61870编码的蛋白质为叶绿体蛋白质.     

Complex protein targeting to dinoflagellate plastids

Protein trafficking pathways to plastids are directed by N-terminal targeting peptides. In plants this consists of a relatively simple transit peptide, while in organisms with secondary plastids (which reside within the endomembrane system) a signal peptide is appended to the transit peptide. Despite amino acid compositional differences between organisms, often due to nucleotide biases, the features of plastid targeting sequences are generally consistent within species. Dinoflagellate algae deviate from this trend. We have conducted an expressed sequence tag (EST) survey of the peridinin-plastid containing dinoflagellate Heterocapsa triquetra to identify and characterize numerous targeting presequences of plastid proteins encoded in the nucleus. Consistent with targeting systems present in other secondary plastid-containing organisms, these all possess a canonical signal peptide at their N termini, however two major classes of transit peptides occur. Both classes possess a common N-terminal portion of the transit peptide, but one class of transit peptides contains a hydrophobic domain that has been reported to act as a stop-transfer membrane anchor, temporarily arresting protein insertion into the endoplasmic reticulum. A second class of transit peptide lacks this feature. These two classes are represented approximately equally, and for any given protein the class is conserved across all dinoflagellate taxa surveyed to date. This dichotomy suggests that two mechanisms, perhaps even trafficking routes, may direct proteins to dinoflagellate plastids. A four-residue phenylalanine-based motif is also a consistent feature of H. triquetra transit peptides, which is an ancient feature predating red algae and galucophytes that was lost in green plastids.     

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

Peroxisomes are a family of organelles which have many unusual features. They can arise de novo from the endoplasmic reticulum by a still poorly characterized process, yet possess a unique machinery for the import of their matrix proteins. As peroxisomes lack DNA, their function, which is highly variable and dependent on developmental and/or environmental conditions, is determined by the post‐translational import of specific metabolic enzymes in folded or oligomeric states. The two classes of matrix targeting signals for peroxisomal proteins [PTS1 (peroxisomal targeting signal 1) and PTS2] are recognized by cytosolic receptors [PEX5 (peroxin 5) and PEX7 respectively] which escort their cargo proteins to, or possibly across, the peroxisome membrane. Although the membrane translocation mechanism remains unclear, it appears to be driven by thermodynamically favourable binding interactions. Recycling of the receptors from the peroxisome membrane requires ATP hydrolysis for two linked processes: ubiquitination of PEX5 (and the PEX7 co‐receptors in yeast) and the function of two peroxisome‐associated AAA (ATPase associated with various cellular activities) ATPases, which play a role in recycling or turnover of the ubiquitinated receptors. This review summarizes and integrates recent findings on peroxisome matrix protein import from yeast, plant and mammalian model systems, and discusses some of the gaps in our understanding of this remarkable protein transport system.     

Cytometric analysis of an epitope-tagged transit peptide bound to the chloroplast translocation apparatus

Chloroplast transit peptides are necessary and sufficient for the targeting and translocation of precursor proteins across the chloroplast envelope. However, the mechanism by which transit peptides engage the translocation apparatus has not been investigated. To analyse this interaction, we have developed a novel epitope-tagged transit peptide derived from the precursor of the small subunit of pea Rubisco. The recombinant transit peptide, His-S-SStp, contains a removable dual-epitope tag, His-S, at its N-terminus that permits both rapid purification via immobilized metal affinity chromatography and detection by blotting, flow cytometry and laser-scanning confocal microscopy. Unlike other chimeric precursors, which place the passenger protein C-terminal to the transit peptide, His-S-SStp bound to the translocation apparatus yet did not translocate across the chloroplast envelope. This early translocation intermediate allowed non-radioactive detection using fluorescent and chemiluminescent reporters. The physiological relevance of this interaction was confirmed by protein import competitions, sensitivity to pre- and post-import thermolysin treatment, photochemical cross-linking and organelle fractionation. The interaction was specific for the transit peptide since His-S alone did not engage the chloroplast translocation apparatus. Quantitation of the bound transit peptide was determined by flow cytometry, showing saturation of binding yet only slight ATP-dependence. The addition of GTP showed inhibition of the binding of His-S-SStp to the chloroplasts indicating an involvement of GTP in the formation of this early translocation intermediate. In addition, direct visualization of His-S-SStp and Toc75 by confocal microscopy revealed a patch-like labeling, suggesting a co-ordinate localization to discrete regions on the chloroplast envelope. These findings represent the first direct visualization of a transit peptide interacting with the chloroplast translocation apparatus. Furthermore, identification of a chloroplast-binding intermediate may provide a novel tool to dissect interactions between a transit peptide and the chloroplast translocation apparatus.     

The potato granule bound starch synthase chloroplast transit peptide directs recombinant proteins to plastids

The chloroplast targeting transit sequence from potato granule bound starch synthase (gbss) was used to direct the accumulation of recombinant proteins to the plastid stroma. The potato gbss transit sequence was fused to the N-terminus of the green fluorescent protein (GFP) and the Catharanthus roseus strictosidine synthase (Str1) enzyme. Fluorescence microscopy confirmed that the recombinant gbss-GFP fusion protein was exclusively targeted to the plastid stroma in tobacco suspension cells, demonstrating that the transit sequence was functional in vivo. The Str1 fusion protein accumulated to high levels in plastids isolated from transgenic plants. We conclude that the potato gbss transit sequence is functional and directs import of recombinant proteins into the chloroplast stroma.     

A novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts

Most chloroplast and mitochondrial precursor proteins are targeted specifically to either chloroplasts or mitochondria. However, there is a group of proteins that are dual targeted to both organelles. We have developed a novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The mitochondrial precursor of alternative oxidase, AOX was specifically targeted only to mitochondria. The chloroplastic precursor of small subunit of pea ribulose bisphosphate carboxylase/oxygenase, Rubisco, was mistargeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual import system. The dual targeted glutathione reductase GR precursor was targeted to both mitochondria and chloroplasts in both systems. The GR pre-sequence could support import of the mature Rubisco protein into mitochondria and chloroplasts in the single import system but only into chloroplasts in the dual import system. Although the GR pre-sequence could support import of the mature portion of the mitochondrial FAd subunit of the ATP synthase into mitochondria and chloroplasts, mature AOX protein was only imported into mitochondria under the control of the GR pre-sequence in both systems. These results show that the novel dual import system is superior to the single import system as it abolishes mistargeting of chloroplast precursors into pea mitochondria observed in a single organelle import system. The results clearly show that although the GR pre-sequence has dual targeting ability, this ability is dependent on the nature of the mature protein.