Mechanisms of protein import and routing in chloroplasts

The vast majority of the approximately 3000 different proteins required to build a fully functional chloroplast are encoded by the nuclear genome and translated on cytosolic ribosomes. As chloroplasts are each surrounded by a double-membrane system, or envelope, sophisticated mechanisms are necessary to mediate the import of these nucleus-encoded proteins into chloroplasts. Once inside the organelle, many chloroplast proteins engage one of four additional protein sorting mechanisms that direct targeting to the internal thylakoid membrane system.     

Import pathways of chloroplast interior proteins and the outer-membrane protein OEP14 converge at Toc75

Most chloroplast outer-membrane proteins are synthesized at their mature size without cleavable targeting signals. Their insertion into the outer membrane is insensitive to thermolysin pretreatment of chloroplasts and does not require ATP. It has therefore been assumed that insertion of outer-membrane proteins proceeds through a different pathway from import into the interior of chloroplasts, which requires a thermolysin-sensitive translocon complex and ATP. Here, we show that a model outer-membrane protein, OEP14, competed with the import of a chloroplast interior protein, indicating that the two import pathways partially overlapped. Cross-linking studies showed that, during insertion, OEP14 was associated with Toc75, a thermolysin-resistant component of the outer-membrane protein-conducting channel that mediates the import of interior-targeted precursor proteins. Whereas almost no OEP14 inserted into protein-free liposomes, OEP14 inserted into proteoliposomes containing reconstituted Toc75 with a high efficiency. Taken together, our data indicate that Toc75 mediates OEP14 insertion, and therefore plays a dual role in the targeting of proteins to the outer envelope membrane and interior of chloroplasts.     

A heat shock protein localized to chloroplasts is a member of a eukaryotic superfamily of heat shock proteins.

We have isolated cDNA clones from soybean and pea that specify nuclear-encoded heat shock proteins (HSPs) which localize to chloroplasts. The mRNAs for these HSPs are undetectable at control temperatures, but increase approximately 150-fold during a 2-h heat shock. Hybridization-selection followed by in vitro translation demonstrates that these HSPs are synthesized as precursor proteins which are processed by the removal of 5-6.5 kd during import into isolated chloroplasts. The nucleotide sequence of the cDNAs shows the derived amino acid sequences of the mature pea and soybean proteins are 79% identical. While the predicted transit peptide encoded by the pea cDNA has some characteristics typical of transit sequences, including high Ser content, multiple basic residues and no acidic residues, it lacks two domains proposed to be important for import and maturation of other chloroplast proteins. The carboxy-terminal region of the chloroplast HSP has significant homology to cytoplasmic HSPs from soybean and other eukaryotes. We hypothesize that the chloroplast HSP shares a common structural and functional domain with low mol. wt HSPs which localize to other parts of the cell, and may have evolved from a nuclear gene.     

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.     

Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts

Precursor forms of chloroplast proteins synthesized in cell-free translation systems can be imported posttranslationally into isolated, intact chloroplasts. Radiochemically pure precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase and to the light-harvesting chlorophyll a/b protein have been prepared by in vitro translation of hybrid-selected mRNA and used to study this import process. If chloroplasts are pretreated with the uncoupler nigericin, import does not occur, but the precursors bind to the chloroplast surface. Reincubation of the precursor-chloroplast complex in the presence of ATP results in import of bound precursors. The binding appears to be mediated by proteins of the outer chloroplast envelope membrane because pretreatment of chloroplasts with protease inhibits their ability to bind as well as to import precursors. These results indicate that at least a portion of the observed binding is to functional receptor proteins involved in the import process.     

The effect of amino acid-modifying reagents on chloroplast protein import and the formation of early import intermediates

In order to identify functionally important amino acid residues in the chloroplast protein import machinery, chloroplasts were preincubated with amino-acid-modifying reagents and then allowed to import or form early import intermediates with precursor proteins. Incubation of chloroplasts with N-ethyl maleimide, diethyl pyrocarbonate, phenylglyoxal, 4,4'-di-isothiocyanatostilbene 2,2'-disulphonic acid (DIDS), dicyclohexylcarbodiimide (DCCD), and 1-ethyl- 3-dimethylaminopropylcarbodiimide (EDC) inhibited both import and formation of early import intermediates with precursor proteins by chloroplasts. This suggests that one or more of the binding components of the chloroplast protein import machinery contains functionally important solvent-exposed cysteine, histidine, arginine, and aspartate/glutamate residues, as well as functionally important lysine and aspartate/ glutamate residues in a hydrophobic environment.     

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.     

The plastid-encoded protein Orf2971 is required for protein translocation and chloroplast quality control

Photosynthesis and the biosynthesis of many important metabolites occur in chloroplasts. In these semi-autonomous organelles, the chloroplast genome encodes approximately 100 proteins. The remaining chloroplast proteins, close to 3,000, are encoded by nuclear genes whose products are translated in the cytosol and imported into chloroplasts. However, there is still no consensus on the composition of the protein import machinery including its motor proteins and on how newly imported chloroplast proteins are refolded. In this study, we have examined the function of orf2971, the largest chloroplast gene of Chlamydomonas reinhardtii. The depletion of Orf2971 causes the accumulation of protein precursors, partial proteolysis and aggregation of proteins, increased expression of chaperones and proteases, and autophagy. Orf2971 interacts with the TIC (translocon at the inner chloroplast envelope) complex, catalyzes ATP (adenosine triphosphate) hydrolysis, and associates with chaperones and chaperonins. We propose that Orf2971 is intimately connected to the protein import machinery and plays an important role in chloroplast protein quality control.

Repression of Orf2971 induces accumulation of chloroplast precursor proteins and impaired chloroplast quality indicating that Orf2971 is required for protein import and chloroplast quality control.

IN A NUTSHELL Background: The chloroplast is an important bioreactor as well as a photosynthetic site. Approximately 3,000 plastid proteins encoded in the nucleus are translocated into the chloroplast envelope via the TOC (translocon at the outer chloroplast envelope) and TIC machineries. Most nucleus-encoded preproteins that enter the plastid are unfolded as they traverse the TOC–TIC import complexes. To prevent these unfolded or misfolded proteins from causing chloroplast damage, a quality control mechanism comprising molecular chaperones and proteases ensures that all polypeptides entering chloroplasts are either correctly folded or degraded. However, there is still no consensus on the TIC complex’s components, motor proteins, or mechanism for refolding proteins entering the chloroplast. Question: What is the precise function of each of the proteins in the TIC complex? What is the composition of the chloroplast protein import machinery motor? How are the newly imported chloroplast proteins refolded and assembled into functional complexes? Findings: We found that Orf2971, encoded by the largest gene in the Chlamydomonas reinhardtii chloroplast genome and proposed to be an ortholog of Ycf2, is directly associated with the protein import machinery and plays a crucial role in ensuring the quality of proteins targeted to the chloroplast. Orf2971 deficiency induces protein precursor accumulation, partial proteolysis and protein aggregation, increased expression of chaperones and proteases, and autophagy. We hypothesize that Orf2971 is intimately linked to the protein import machinery and plays a critical role in chloroplast protein quality control. Next steps: The next challenge is to identify the sorting components associated with this complex on the stromal side. Furthermore, additional experimental evidence is required to investigate the relationship between different import machineries, including the analysis of the accumulation of precursor proteins in the various import mutants.     

Isolated plant mitochondria import chloroplast precursor proteins in vitro with the same efficiency as chloroplasts.

Most chloroplast and mitochondrial proteins are synthesized with N-terminal presequences that direct their import into the appropriate organelle. In this report we have analyzed the specificity of standard in vitro assays for import into isolated pea chloroplasts and mitochondria. We find that chloroplast protein import is highly specific because mitochondrial proteins are not imported to any detectable levels. Surprisingly, however, pea mitochondria import a range of chloroplast protein precursors with the same efficiency as chloroplasts, including those of plastocyanin, the 33-kDa photosystem II protein, Hcf136, and coproporphyrinogen III oxidase. These import reactions are dependent on the Deltaphi across the inner mitochondrial membrane, and furthermore, marker enzyme assays and Western blotting studies exclude any import by contaminating chloroplasts in the preparation. The pea mitochondria specifically recognize information in the chloroplast-targeting presequences, because they also import a fusion comprising the presequence of coproporphyrinogen III oxidase linked to green fluorescent protein. However, the same construct is targeted exclusively into chloroplasts in vivo indicating that the in vitro mitochondrial import reactions are unphysiological, possibly because essential specificity factors are absent in these assays. Finally, we show that disruption of potential amphipathic helices in one presequence does not block import into pea mitochondria, indicating that other features are recognized.     

Chloroplast precursor proteins compete to form early import intermediates in isolated pea chloroplasts

In order to ascertain whether there is one site for the import of precursor proteins into chloroplasts or whether different precursor proteins are imported via different import machineries, chloroplasts were incubated with large quantities of the precursor of the 33 kDa subunit of the oxygen-evolving complex (pOE33) or the precursor of the light-harvesting chlorophyll a/b-binding protein (pLHCP) and tested for their ability to import a wide range of other chloroplast precursor proteins. Both pOE33 and pLHCP competed for import into chloroplasts with precursors of the stromally-targeted small subunit of Rubisco (pSSu), ferredoxin NADP(+) reductase (pFNR) and porphobilinogen deaminase; the thylakoid membrane proteins LHCP and the Rieske iron-sulphur protein (pRieske protein); ferrochelatase and the gamma subunit of the ATP synthase (which are both associated with the thylakoid membrane); the thylakoid lumenal protein plastocyanin and the phosphate translocator, an integral membrane protein of the inner envelope. The concentrations of pOE33 or pLHCP required to cause half-maximal inhibition of import ranged between 0.2 and 4.9 microM. These results indicate that all of these proteins are imported into the chloroplast by a common import machinery. Incubation of chloroplasts with pOE33 inhibited the formation of early import intermediates of pSSu, pFNR and pRieske protein.     

AKR2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesis

In plant cells, chloroplasts have essential roles in many biochemical reactions and physiological responses. Chloroplasts require numerous protein components, but only a fraction of these proteins are encoded by the chloroplast genome. Instead, most are encoded by the nuclear genome and imported into chloroplasts from the cytoplasm post-translationally. Membrane proteins located in the chloroplast outer envelope membrane (OEM) have a critical function in the import of proteins into the chloroplast. However, the biogenesis of chloroplast OEM proteins remains poorly understood. Here, we report that an Arabidopsis ankyrin repeat protein, AKR2A, plays an essential role in the biogenesis of the chloroplast OEM proteins. AKR2A binds to chloroplast OEM protein targeting signals, as well as to chloroplasts. It also displays chaperone activity towards chloroplast OEM proteins, and facilitates the targeting of OEP7 to chloroplasts in vitro. AKR2A RNAi in plants with an akr2b knockout background showed greatly reduced levels of chloroplast proteins, including OEM proteins, and chloroplast biogenesis was also defective. Thus, AKR2A functions as a cytosolic mediator for sorting and targeting of nascent chloroplast OEM proteins to the chloroplast.     

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.     

A systemic proteomic analysis of <Emphasis Type="Italic">Populus</Emphasis> chloroplast by using shotgun method

The chloroplast is one of the most important organelles in plants. Proteomic investigations of chloroplasts have been undertaken for many herb plant species, but to date no such investigation has been reported for woody plant chloroplasts. In the present study we initiated a systematic proteomic study of Populus chloroplasts using a shotgun proteomic method. After isolation of chloroplasts and tryptic digestion of the proteins, the protein fragments were separated via HPLC using an SCX column, and the peptides were analyzed by LC-MS/MS; 119 proteins were successfully identified. Based on annotation information in the UniProtKB/Swiss-Prot database, these proteins were identified as being localized in the chloroplast thylakoid membrane, chloroplast stroma, chloroplast thylakoid lumen, and plastoglobules. Over 50% of all identified proteins were confirmed as chloroplast thylakoid proteins, and 85 are encoded by the chloroplast genome with the remaining proteins encoded by the nuclear genome. Based on functional annotation, these proteins were classified into four functional categories, including photosynthesis, redox regulation and stress, primary and secondary metabolism, transport and signaling. These data provide a valuable basis for further studies on photosynthesis in poplar species.     

茶树叶绿体及其蛋白的分离研究

茶树叶片叶绿体的有效分离纯化是进行茶树叶绿体代谢组学和蛋白质组学研究的基础.本文以茶树鲜叶为材料,通过叶绿体得率、希尔反应等纯度和完整度指标,比较了Percoll密度梯度离心法和蔗糖密度梯度离心法对叶绿体分离纯化的效果;通过蛋白质含量和SDS-PAGE电泳图谱,比较了涨破法和冻融法对叶绿体蛋白的提取效果.结果发现Per...     

The M domain of atToc159 plays an essential role in the import of proteins into chloroplasts and chloroplast biogenesis

Toc159, a protein located in the outer envelope membrane and the cytosol, is an important component of the receptor complex for nuclear-encoded chloroplast proteins. We investigated the molecular mechanism of protein import into chloroplasts by atToc159 using the ppi2 mutant, which has a T-DNA insertion at atToc159, shows an albino phenotype, and does not survive beyond the seedling stage due to a defect in protein import into chloroplasts. First we established that transiently expressing atToc159 in protoplasts obtained from the white leaf tissues of ppi2 plants complements the protein import defect into chloroplasts. Using this transient expression approach and a series of deletion mutants, we demonstrated that the C-terminal membrane-anchored (M) domain is targeted to the chloroplast envelope membrane in ppi2 protoplasts, and is sufficient to complement the defect in protein import. The middle GTPase (G) domain plays an additional critical role in protein import: the atToc159[S/N] and atToc159[D/L] mutants, which have a mutation at the first and second GTP-binding motifs, respectively, do not support protein import into chloroplasts. Leaf cells of transgenic plants expressing the M domain in a ppi2 background contained nearly fully developed chloroplasts with respect to size and density of thylakoid membranes, and displayed about half as much chlorophyll as wild-type cells. In transgenic plants, the isolated M domain localized to the envelope membrane of chloroplasts but not the cytosol. Based on these results, we propose that the M domain is the minimal structure required to support protein import into chloroplasts, while the G domain plays a regulatory role.     

Tic22 is targeted to the intermembrane space of chloroplasts by a novel pathway.

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.     

Molecular chaperones involved in chloroplast protein import.

Transport of cytoplasmically synthesized precursor proteins into chloroplasts, like the protein transport systems of mitochondria and the endoplasmic reticulum, appears to require the action of molecular chaperones. These molecules are likely to be the sites of the ATP hydrolysis required for precursor proteins to bind to and be translocated across the two membranes of the chloroplast envelope. Over the past decade, several different chaperones have been identified, based mainly on their association with precursor proteins and/or components of the chloroplast import complex, as putative factors mediating chloroplast protein import. These factors include cytoplasmic, chloroplast envelope-associated and stromal members of the Hsp70 family of chaperones, as well as stromal Hsp100 and Hsp60 chaperones and a cytoplasmic 14-3-3 protein. While many of the findings regarding the action of chaperones during chloroplast protein import parallel those seen for mitochondrial and endoplasmic reticulum protein transport, the chloroplast import system also has unique aspects, including its hypothesized use of an Hsp100 chaperone to drive translocation into the organelle interior. Many questions concerning the specific functions of chaperones during protein import into chloroplasts still remain that future studies, both biochemical and genetic, will need to address.     

From nuclear genes to chloroplast localized proteins

There is broad evidence that an endosymbiotic uptake of a cyanobacterial-type organism was the point of origin for the evolution of chloroplasts. During organelle evolution extensive gene transfer from the symbiont to the host genome occurred, which raises the question of how these gene products, namely proteins, which are still functional in chloroplasts, find their way back ‘home’. Nuclear-encoded proteins enter plastids via a complex import machinery that requires the coordinate interplay of a variety of soluble and membrane-bound factors on the cytosolic site as well as on the stromal side of the chloroplast envelope membranes. We define that the process called ‘import of chloroplast precursor proteins’ begins with the release of the polypeptide from the ribosomes and binding to cytosolic factors, such as a guidance complex, which accompanies (chaperones) proteins to chloroplasts. The translocation across the envelope membranes engages distinct translocation machineries at the outer and the inner envelope membranes. Additionally subsequent sorting events to different subcompartments within the plastids are operated by a number of distinct pathways, all of which seem to involve multiple subunits, which are largely of bacterial (symbiotic) origin. The evolutionary history of proteins mediating the import of chloroplast constituents across the envelope membranes seems more diverse. Since cyanobacteria lack a protein import pathway, it is not surprising that only a few subunits of the chloroplast translocon seem to be of symbiotic origin while others seem to be eukaryotic additions.     

Cryopreservation of chloroplasts and thylakoids for studies of protein import and integration

A method is presented for preservation of isolated intact chloroplasts and isolated thylakoids for use in chloroplast protein import and thylakoid protein integration studies. Chloroplasts of pea (Pisum sativum) were preserved by storage in liquid nitrogen in the presence of a cryoprotective agent. Dimethyl sulfoxide was the most effective of several cryoprotectants examined. Approximately 65 to 70% of chloroplasts stored in liquid nitrogen in the presence of dimethyl sulfoxide remained intact upon thawing and were fully functional for the import of precursor proteins. Imported proteins were correctly localized within these chloroplasts, a process that for two of the proteins tested involved transport into the thylakoids. Lysate obtained from preserved chloroplasts was functional for protein integration assays. Preserved chloroplasts retained import and localization capability for up to 6 months of storage. Thylakoids were preserved by a modification of a method previously described (Farkas DL, Malkin S [1979] Plant Physiol 64: 942-947) for preservation of photosynthetic competence. Preserved thylakoids were nearly as active for protein integration studies as freshly prepared thylakoids. The ability to store chloroplasts and subfractions for extended periods will facilitate investigations of plastid protein biogenesis.     

AtToc90, a New GTP-Binding Component of the Arabidopsis Chloroplast Protein Import Machinery

AtToc159 is a GTP-binding chloroplast protein import receptor. In vivo, atToc159 is required for massive accumulation of photosynthetic proteins during chloroplast biogenesis. Yet, in mutants lacking atToc159 photosynthetic proteins still accumulate, but at strongly reduced levels whereas non-photosynthetic proteins are imported normally: This suggests a role for the homologues of atToc159 (atToc132, -120 and -90). Here, we show that atToc90 supports accumulation of photosynthetic proteins in plastids, but is not required for import of several constitutive proteins. Part of atToc90 associates with the chloroplast surface in vivo and with the Toc-complex core components (atToc75 and atToc33) in vitro suggesting a function in chloroplast protein import similar to that of atToc159. As both proteins specifically contribute to the accumulation of photosynthetic proteins in chloroplasts they may be components of the same import pathway.