Protein import into chloroplasts

Most chloroplastic proteins are encoded in the nucleus, synthesized on cytosolic ribosomes and subsequently imported into the organelle. In general, proteins destined for the chloroplast are synthesized as precursor proteins with a cleavable N-terminal presequence that mediates routing to the inside of the chloroplast. These precursor proteins have to be targeted to the correct organellar membrane surface after their release from the ribosome and furthermore they have to be maintained in a conformation suitable for translocation across the two envelope membranes. Recognition and import of most chloroplastic precursor proteins are accomplished by a jointly used translocation apparatus. Different but complementary studies of several groups converged recently in the identification of the outer envelope proteins OEP86, OEP75, OEP70 (a Hsp 70-related protein), OEP34, and of the inner envelope protein IEP110 as components of this translocation machinery. None of these proteins, except for OEP70, shows any homology to components of other protein translocases. The plastid import machinery thus seems to be an original development in evolution. Following translocation into the organelle, chloroplastic proteins are sorted to their suborganellar destination, i.e., the inner envelope membrane, the thylakoid membrane, and the thylakoid lumen. This structural and evolutionary complexity of chloroplasts is reflected by a variety of routing mechanisms by which proteins reach their final location once inside the organelle. This review will focus on recent advances in the identification of components of the chloroplastic protein import machinery, and new insights into the pathways of inter-and intraorganellar sorting.     

Insights into the Clp/HSP100 chaperone system from chloroplasts of Arabidopsis thaliana

HSP100 proteins are molecular chaperones involved in protein quality control. They assist in protein (un)folding, prevent aggregation, and are thought to participate in precursor translocation across membranes. Caseinolytic proteins ClpC and ClpD from plant chloroplasts belong to the HSP100 family. Their role has hitherto been investigated by means of physiological studies and reverse genetics. In the present work, we employed an in vitro approach to delve into the structural and functional characteristics of ClpC2 and ClpD from Arabidopsis thaliana (AtClpC2 and AtClpD). They were expressed in Escherichia coli and purified to near-homogeneity. The proteins were detected mainly as dimers in solution, and, upon addition of ATP, the formation of hexamers was observed. Both proteins exhibited basal ATPase activity (K(m), 1.42 mm, V(max), 0.62 nmol/(min × μg) for AtClpC2 and K(m) ～19.80 mm, V(max) ～0.19 nmol/(min × μg) for AtClpD). They were able to reactivate the activity of heat-denatured luciferase (～40% for AtClpC2 and ～20% for AtClpD). The Clp proteins tightly bound a fusion protein containing a model transit peptide. This interaction was detected by binding assays, where the chaperones were selectively trapped by the transit peptide-containing fusion, immobilized on glutathione-agarose beads. Association of HSP100 proteins to import complexes with a bound transit peptide-containing fusion was also observed in intact chloroplasts. The presented data are useful to understand protein quality control and protein import into chloroplasts in plants.     

Protein import into chloroplasts

The structural complexity of chloroplasts is reflected in their intriguing protein-targeting system. Not only must nucleus-encoded proteins be targeted to the chloroplast, but also, once inside the chloroplast, these polypeptides must be directed to their final destination in one of six intrachloroplastic compartments. Although the details of this process remain elusive, many recent advances have improved our vantage point for examining this system.     

Protein import into chloroplasts

Protein import into chloroplasts is a highly regulated process. The activity of the major import receptors is regulated by protein phosphorylation, as well as by GTP binding and hydrolysis. Complete translocation into the organelle could depend on its redox status, as sensed by the Tic complex. A further possibility is that, upon phosphorylation, precursor proteins form a highly import-competent guidance complex in the cytosol. Hence, several levels of regulation seem to coexist.     

Protein import into cyanelles and complex chloroplasts

Higher-plant, green and red algal chloroplasts are surrounded by a double membrane envelope. The glaucocystophyte plastid (cyanelle) has retained a prokaryotic cell wall between the two envelope membranes. The complex chloroplasts of Euglena and dinoflagellates are surrounded by three membranes while the complex chloroplasts of chlorarachniophytes, cryptomonads, brown algae, diatoms and other chromophytes, are surrounded by 4 membranes. The peptidoglycan layer of the cyanelle envelope and the additional membranes of complex chloroplasts provide barriers to chloroplast protein import not present in the simpler double membrane chloroplast envelope. Analysis of presequence structure and in vitro import experiments indicate that proteins are imported directly from the cytoplasm across the two envelope membranes and peptidoglycan layer into cyanelles. Protein import into complex chloroplasts is however fundamentally different. Analysis of presequence structure and in vitro import into microsomal membranes has shown that translocation into the ER is the first step for protein import into complex chloroplasts enclosed by three or four membranes. In vivo pulse chase experiments and immunoelectronmicroscopy have shown that in Euglena, proteins are transported from the ER to the Golgi apparatus prior to import across the three chloroplast membranes. Ultrastructural studies and the presence of ribosomes on the outermost of the four envelope membranes suggests protein import into 4 membrane-bounded complex chloroplasts is directly from the ER like outermost membrane into the chloroplast. The fundamental difference in import mechanisms, post-translational direct chloroplast import or co-translational translocation into the ER prior to chloroplast import, appears to reflect the evolutionary origin of the different chloroplast types. Chloroplasts with a two-membrane envelope are thought to have evolved through the primary endosymbiotic association between a eukaryotic host and a photosynthetic prokaryote while complex chloroplasts are believed to have evolved through a secondary endosymbiotic association between a heterotrophic or possibly phototrophic eukaryotic host and a photosynthetic eukaryote.     

Protein import into chloroplasts.

Chloroplasts have evolved an elaborate system of membrane and soluble subcompartments to organize and regulate photosynthesis and essential aspects of amino acid and lipid metabolism. The biogenesis and maintenance of organellar architecture rely on protein subunits encoded by both nuclear and plastid genomes. Import of nuclear-encoded proteins is mediated by interactions between the intrinsic N-terminal transit sequence of the nuclear-encoded preprotein and a common import machinery at the chloroplast envelope. Recent investigations have shown that there are two unique membrane-bound translocation systems, in the outer and inner envelope membranes, which physically associate during import to transport preproteins from the cytoplasm to the internal stromal compartment. This review discusses current understanding of these translocation systems and models for the way in which they might function.     

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.     

拟南芥未知功能基因At4g22890编码蛋白的叶绿体定位

蛋白质的亚细胞定位信息对于深入了解该蛋白质的功能具有重要意义。本文对一个预测的拟南芥叶绿体未知功能基因At4g22890编码蛋白进行了叶绿体定位研究。我们克隆了该基因5′端长208bp的DNA片段,与绿色荧光蛋白(GFP)基因构建重组表达载体pMON530-cTP-GFP,经农杆菌介导转化拟南芥。转基因植株经激光共聚焦显微镜观察,GFP荧光仅在叶绿体中观察到,表明所克隆的DNA序列编码的多肽能够将At4g22890编码蛋白质引导进入叶绿体,由此推测该蛋白质为叶绿体蛋白质。     

Protein import into chloroplasts involves redox-regulated proteins

Pre-protein translocation into chloroplasts is accomplished by two distinct translocation machineries in the outer and inner envelope, respectively. We have isolated the translocon at the inner envelope membrane (Tic complex) by blue-native PAGE and describe a new Tic subunit, Tic62. Tic62, together with Tic110 and Tic55, forms a core translocation unit. The N-terminus of Tic62 shows strong homologies to NAD(H) dehydrogenases in eukaryotes and to Ycf39-like proteins present in cyanobacteria and non-green algae. The stromal-facing C-terminus of Tic62 contains a novel, repetitive module that interacts with a ferredoxin-NAD(P)(+) oxidoreductase. Ferredoxin-NAD(P)(+) oxidoreductase catalyses the final electron transfer of oxygenic photosynthesis from ferredoxin to NAD(P). Substrates that interfere with either NAD binding, such as deamino-NAD, or influence the ratio of NAD(P)/NAD(P)H, such as ruthenium hexamine trichloride, modulate the import characteristics of leaf-specific ferredoxin-NAD(P)(+) oxidoreductase isologues differently. We conclude that the Tic complex can regulate protein import into chloroplasts by sensing and reacting to the redox state of the organelle.     

Protein import: the hitchhikers guide into chloroplasts

Plastids originated from an endosymbiotic event between an early eukaryotic host cell and an ancestor of today's cyanobacteria. During the events by which the engulfed endosymbiont was transformed into a permanent organelle, many genes were transferred from the plastidal genome to the nucleus of the host cell. Proteins encoded by these genes are synthesised in the cytosol and subsequently translocated into the plastid. Therefore they contain an N-terminal cleavable transit sequence that is necessary for translocation. The sequence is plastid-specific, thus preventing mistargeting into other organelles. Receptors embedded into the outer envelope of the plastid recognise the transit sequences, and precursor proteins are translocated into the chloroplast by a proteinaceous import machinery located in both the outer and inner envelopes. Inside the stroma the transit sequences are cleaved off and the proteins are further routed to their final locations within the plastid.     

Regions in the transit peptide of SSU essential for transport into chloroplasts

Summary Deletion mutations, 3–19 amino acids in size, were introduced into the transit peptide (57 amino acids) of a small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase from pea. Transport of the authentic small subunit precursor (pSSU) and of the mutant pSSUs by isolated chloroplasts of pea was examined. We show that the transit peptide contains two different, separated functional regions. A deletion mutation in the central region of the transit peptide, a region purported to be important for function, barely affected transport. Changes in the amino-terminal region of the transit peptide drastically reduced transport. Processing of mutants affected in either the amino-terminal or central portion of the transit peptide appeared normal. A deletion mutation at the carboxy-terminus of the transit peptide interfered with both transport and processing. From the aberrant processing we suggest that pSSU is matured in more than one step, and that the maturation signal is located within the carboxy-terminal 16 amino acids. The methionine residue at the evolutionarily conserved cleavage site (cysteine-methionine) between the transit peptide and the mature protein is not essential for processing.     

The amyloplast-targeting transit peptide of the waxy protein of maize also mediates protein transport in vitro into chloroplasts

Summary The transit peptide of the waxy protein of maize which in the maize plant targets this protein only into amyloplasts was used for in vitro protein transport experiments with isolated amyloplasts from maize and chloroplasts from maize, pea and potato. In the presence of both intact and disrupted amyloplasts an artificial preprotein (TP30), consisting of the waxy transit peptide plus the first 34 amino acids of the mature waxy protein fused in-frame to the -glucuronidase of Escherichia coli, is processed to the size expected when the transit peptide is cleaved off. The chloroplasts studied show in vitro import and correct processing of both TP30 and the authentic waxy protein, but not of the -glucuronidase without the waxy transit peptide. The in vitro import of TP30 into chloroplasts is almost as efficient as that of the precursor of the small subunit of pea ribulose-1,5-bisphosphate carboxylase, a nuclear-encoded chloroplast protein, whereas the waxy protein accumulates to a lesser extent in the chloroplasts. Since the amino-terminal transit peptides of TP30 and the waxy precursor are the same, this difference must be due to the mature part of the waxy protein. One possible explanation is the observed instability of the waxy protein in the presence of chloroplasts.     

Quantitative Analysis of the Chloroplast Molecular Chaperone ClpC/Hsp93 in Arabidopsis Reveals New Insights into Its Localization,Interaction with the Clp Proteolytic Core,and Functional Importance

The molecular chaperone ClpC/Hsp93 is essential for chloroplast function in vascular plants. ClpC has long been held to act both independently and as the regulatory partner for the ATP-dependent Clp protease, and yet this and many other important characteristics remain unclear. In this study, we reveal that of the two near-identical ClpC paralogs (ClpC1 and ClpC2) in Arabidopsis chloroplasts, along with the closely related ClpD, it is ClpC1 that is the most abundant throughout leaf maturation. An unexpectedly large proportion of both chloroplast ClpC proteins (30% of total ClpC content) associates to envelope membranes in addition to their stromal localization. The Clp proteolytic core is also bound to envelope membranes, the amount of which is sufficient to bind to all the similarly localized ClpC. The role of such an envelope membrane Clp protease remains unclear although it appears uninvolved in preprotein processing or Tic subunit protein turnover. Within the stroma, the amount of oligomeric ClpC protein is less than that of the Clp proteolytic core, suggesting most if not all stromal ClpC functions as part of the Clp protease; a proposal supported by the near abolition of Clp degradation activity in the clpC1 knock-out mutant. Overall, ClpC appears to function primarily within the Clp protease, as the principle stromal protease responsible for maintaining homeostasis, and also on the envelope membrane where it possibly confers a novel protein quality control mechanism for chloroplast preprotein import.     

两个拟南芥未知功能蛋白的亚细胞定位研究

蛋白质的亚细胞定位对于深入了解该蛋白质所行使的生理功能具有重要意义。经生物信息学预测,两个拟南芥未知功能基因At4g16410与Atl gI8060编码蛋白含有叶绿体定位信息。我们分别克隆了这两个基因5’端长199bp与220bp的DNA片段,与绿色荧光蛋白（GFP）基因构建重组表达载体pMON530-cTP1-GFP与pMON530-cTP2-GFP,经农杆菌介导转化拟南芥。两种转基因植株经激光共聚焦显微镜观察,GFP荧光仅在叶绿体中观察到,表明所克隆的两段DNA序列编码的多肽能够将At4gl6410与Atlgl8060编码蛋白质引导进入叶绿体,确定这两个蛋白质均为叶绿体蛋白质。     

Nucleoside diphosphate kinase from pea chloroplasts: purification,cDNA cloning and import into chloroplasts

Nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) was enriched 1900-fold from purified pea (Pisum sativum L. cv. Golf.) chloroplasts. The active enzyme preparation contained two polypeptides of apparent molecular weight 18.5 kDa and 17.4kDa. Both proteins were enzymatically active and were recognized by an antiserum raised against NDPK from spinach chloroplasts, suggesting the existence of two isoforms in pea chloroplasts. The N-terminal protein sequence data were obtained for both polypeptides and compared with the nucleotide sequence of a cDNA clone isolated from a pea cDNA library. The analysis revealed that the two NDPK forms are encoded for by one mRNA, indicating that the lower-molecular-weight form could represent a proteolytic breakdown product of the 18.5-kDa NDPK. The pea chloroplastic NDPK is made as a larger precursor protein which is imported into chloroplasts. The NDPK precursor is then processed by the stromal processing peptidase to yield the 18.5-kDa form.Abbreviations NDPK nucleoside diphosphate kinase - preNDPK precursor NDPK - ps-NDPK cDNA coding for Pisum sativum NDPK II We thank Dr. Schmidt, University Göttingen, Germany, for doing the protein sequencing. This work was supported in part by grants from the Deutsche Forschungsgemeinschaft.     

Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif

Plastids of diatoms and related algae evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into a eukaryotic host cell and its subsequent reduction into an organelle. As a result diatom plastids are surrounded by four membranes. Protein targeting of nucleus encoded plastid proteins across these membranes depends on N-terminal bipartite presequences consisting of a signal and a transit peptide-like domain. Diatoms and cryptophytes share a conserved amino acid motif of unknown function at the cleavage site of the signal peptides (ASAFAP), which is particularly important for successful plastid targeting. Screening genomic databases we found that in rare cases the very conserved phenylalanine within the motif may be replaced by tryptophan, tyrosine or leucine. To test such unusual presequences for functionality and to better understand the role of the motif and putative receptor proteins involved in targeting, we constructed presequence:GFP fusion proteins with or without modifications of the “ASAFAP”-motif and expressed them in the diatom Phaeodactylum tricornutum. In this comprehensive mutational analysis we found that only the aromatic amino acids phenylalanine, tryptophan, tyrosine and the bulky amino acid leucine at the +1 position of the predicted signal peptidase cleavage site allow plastid import, as expected from the sequence comparison of native plastid targeting presequences of P. tricornutum and the cryptophyte Guillardia theta. Deletions within the signal peptide domains also impaired plastid import, showing that the presence of F at the N-terminus of the transit peptide together with a cleavable signal peptide is crucial for plastid import. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. Gruber and S. Vugrinec contributed equally to this work.     

Protein translocation into peroxisomes by ring-shaped import receptors

Folded and functional proteins destined for translocation from the cytosol into the peroxisomal matrix are recognized by two different peroxisomal import receptors, Pex5p and Pex7p. Both cargo-loaded receptors dock on the same translocon components, followed by cargo release and receptor recycling, as part of the complete translocation process. Recent structural and functional evidence on the Pex5p receptor has provided insight on the molecular requirements of specific cargo recognition, while the remaining processes still remain largely elusive. Comparison of experimental structures of Pex5p and a structural model of Pex7p reveal that both receptors are built by ring-like arrangements with cargo binding sites, central to the respective structures. Although, molecular insight into the complete peroxisomal translocon still remains to be determined, emerging data allow to deduce common molecular principles that may hold for other translocation systems as well.     

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.     

The transition of early translocation intermediates in chloroplasts is accompanied by the movement of the targeting signal on the precursor protein

During protein import into chloroplasts, precursor proteins are docked to these organelles under stringent energy conditions to form early translocation intermediates. Depending on the temperature and the requirement for ATP, different types of early-intermediates are present, for which the extent of precursor protein translocation differs [H. Inoue, M. Akita, J. Biol. Chem. 283 (2008) 7491–7502]. However, it has not been determined whether the environment surrounding the precursor differs for each intermediate. We therefore employed a site-specific photo-crosslinking strategy in our current study to capture any components in close proximity to the targeting signal of the precursors within the early-intermediates. Various crosslinked products, one of which contains Toc75, were identified. The appearance of these products was found to be dependent on the position of the precursor upon modification by the crosslinker and also the intermediate state. This indicated that the transition of early translocation intermediates is accompanied with the movement of the targeting signal within the early-intermediates.