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.     

In vivo analysis of the role of atTic20 in protein import into chloroplasts

The import of nucleus-encoded preproteins into plastids requires the coordinated activities of membrane protein complexes that facilitate the translocation of polypeptides across the envelope double membrane. Tic20 was identified previously as a component of the import machinery of the inner envelope membrane by covalent cross-linking studies with trapped preprotein import intermediates. To investigate the role of Tic20 in preprotein import, we altered the expression of the Arabidopsis Tic20 ortholog (atTic20) by antisense expression. Several antisense lines exhibited pronounced chloroplast defects exemplified by pale leaves, reduced accumulation of plastid proteins, and significant growth defects. The severity of the phenotypes correlated directly with the reduction in levels of atTic20 expression. In vitro import studies with plastids isolated from control and antisense plants indicated that the antisense plastids are defective specifically in protein translocation across the inner envelope membrane. These data suggest that Tic20 functions as a component of the protein-conducting channel at the inner envelope membrane.     

Effects of osmotic preconditioning on nuclear replication activity in seeds of pepper (Capsicum annuum)

Routing of cytosolically synthesized precursor proteins into chloroplasts is a specific process which involves a multitude of soluble and membrane components. In this review we wil1 focus on early events of the translocation pathway of nuclear coded plastidic precursor proteins and compare import routes for polypeptide of the outer chloroplast envelope to that of internal chloroplast compartments. A number of proteins housed in the chloroplast envelopes have been implied to be involved in the translocation process, but so far a certain function has not been assigned to any of these proteins. The only exception could be an envelope localized hsc 70 homologue which could retain the import competence of a precursor protein in transit into the organelle.     

Toc159- and Toc75-independent import of a transit sequence-less precursor into the inner envelope of chloroplasts

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.     

Mechanism of protein import across the chloroplast envelope

The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes.     

Stromal Hsp70 Is Important for Protein Translocation into Pea and Arabidopsis Chloroplasts

Hsp70 family proteins function as motors driving protein translocation into mitochondria and the endoplasmic reticulum. Whether Hsp70 is involved in protein import into chloroplasts has not been resolved. We show here Arabidopsis thaliana knockout mutants of either of the two stromal cpHsc70s, cpHsc70-1 and cpHsc70-2, are defective in protein import into chloroplasts during early developmental stages. Protein import was found to be affected at the step of precursor translocation across the envelope membranes. From solubilized envelope membranes, stromal cpHsc70 was specifically coimmunoprecipitated with importing precursors and stoichiometric amounts of Tic110 and Hsp93. Moreover, in contrast with receptors at the outer envelope membrane, cpHsp70 is important for the import of both photosynthetic and nonphotosynthetic proteins. These data indicate that cpHsc70 is part of the chloroplast translocon for general import and is important for driving translocation into the stroma. We further analyzed the relationship of cpHsc70 with the other suggested motor system, Hsp93/Tic40. Chloroplasts from the cphsc70-1 hsp93-V double mutant had a more severe import defect than did the single mutants, suggesting that the two proteins function in parallel. The cphsc70-1 tic40 double knockout was lethal, further indicating that cpHsc70-1 and Tic40 have an overlapping essential function. In conclusion, our data indicate that chloroplasts have two chaperone systems facilitating protein translocation into the stroma: the cpHsc70 system and the Hsp93/Tic40 system.     

Protein import into chloroplasts: new aspects of a well-known topic

Protein import into plant chloroplasts is a fascinating topic that is being investigated by many research groups. Since the majority of chloroplast proteins are synthesised as precursor proteins in the cytosol, they have to be posttranslationally imported into the organelle. For this purpose, most preproteins are synthesised with an N-terminal presequence, which is both necessary and sufficient for organelle recognition and translocation initiation. The import of preproteins is facilitated by two translocation machineries in the outer and inner envelope of chloroplasts, the Toc and Tic complexes, respectively. Translocation of precursor proteins across the envelope membrane has to be highly regulated to react to the metabolic requirements of the organelle. The aim of this review is to summarise the events that take place at the translocation machineries that are known so far. In addition, we focus in particular on alternative import pathways and the aspect of regulation of protein transport at the outer and inner envelope membrane.     

Analysis of the Interactions of Preproteins with the Import Machinery over the Course of Protein Import into Chloroplasts

We have investigated the interactions of two nuclear-encoded preproteins with the chloroplast protein import machinery at three stages in import using a label-transfer crosslinking approach. During energy-independent binding at the outer envelope membrane, preproteins interact with three known components of the outer membrane translocon complex, Toc34, Toc75, and Toc86. Although Toc75 and Toc86 are known to associate with preproteins during import, a role for Toc34 in preprotein binding previously had not been observed. The interaction of Toc34 with preproteins is regulated by the binding, but not hydrolysis of GTP. These data provide the first evidence for a direct role for Toc34 in import, and provide insights into the function of GTP as a regulator of preprotein recognition. Toc75 and Toc86 are the major targets of cross-linking upon insertion of preproteins across the outer envelope membrane, supporting the proposal that both proteins function in translocation at the outer membrane as well as preprotein recognition. The inner membrane proteins, Tic(21) and Tic22, and a previously unidentified protein of 14 kD are the major targets of crosslinking during the late stages in import. These data provide additional support for the roles of these components during protein translocation across the inner membrane. Our results suggest a defined sequence of molecular interactions that result in the transport of nuclear-encoded preproteins from the cytoplasm into the stroma of chloroplasts.     

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.     

Specific lipids influence the import capacity of the chloroplast outer envelope precursor protein translocon

Recent studies demonstrated that lipids influence the assembly and efficiency of membrane-embedded macromolecular complexes. Similarly, lipids have been found to influence chloroplast precursor protein binding to the membrane surface and to be associated with the Translocon of the Outer membrane of Chloroplasts (TOC). We used a system based on chloroplast outer envelope vesicles from Pisum sativum to obtain an initial understanding of the influence of lipids on precursor protein translocation across the outer envelope. The ability of the model precursor proteins p(OE33)titin and pSSU to be recognized and translocated in this simplified system was investigated. We demonstrate that transport across the outer membrane can be observed in the absence of the inner envelope translocon. The translocation, however, was significantly slower than that observed for chloroplasts. Enrichment of outer envelope vesicles with different lipids natively found in chloroplast membranes altered the binding and transport behavior. Further, the results obtained using outer envelope vesicles were consistent with the results observed for the reconstituted isolated TOC complex. Based on both approaches we concluded that the lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylinositol (PI) increased TOC-mediated binding and import for both precursor proteins. In contrast, enrichment in digalactosyldiacylglycerol (DGDG) improved TOC-mediated binding for pSSU, but decreased import for both precursor proteins. Optimal import occurred only in a narrow concentration range of DGDG.     

Identification of intermediates in the pathway of protein import into chloroplasts and their localization to envelope contact sites

We have used a hybrid precursor protein to study the pathway of protein import into chloroplasts. This hybrid (pS/protA) consists of the precursor to the small subunit of Rubisco (pS) fused to the IgG binding domains of staphylococcal protein A. The pS/protA is efficiently imported into isolated chloroplasts and is processed to its mature form (S/protA). In addition to the mature stromal form, two intermediates in the pathway of pS/protA import were identified at early time points in the import reaction. The first intermediate represents unprocessed pS/protA bound to the outer surface of the chloroplast envelope and is analogous to a previously characterized form of pS that is specifically bound to the chloroplast surface and can be subsequently translocated in the stroma (Cline, K., M. Werner-Washburne, T. H. Lubben, and K. Keegstra. 1985. J. Biol. Chem. 260:3691-3696.) The second intermediate represents a partially translocated form of the precursor that remains associated with the envelope membrane. This form is processed to mature S/protA, but remains susceptible to exogenously added protease in intact chloroplasts. We conclude that the envelope associated S/protA is spanning both the outer and inner chloroplast membranes en route to the stroma. Biochemical and immunochemical localization of the two translocation intermediates indicates that both forms are exposed at the surface of the outer membrane at sites where the outer and inner membrane are closely apposed. These contact zones appear to be organized in a reticular network on the outer envelope. We propose a model for protein import into chloroplasts that has as its central features two distinct protein conducting channels in the outer and inner envelope membranes, each gated open by a distinct subdomain of the pS signal sequence.     

Analysis of chloroplast transit peptide function using mutations in the carboxyl-terminal region

Protein import into chloroplasts requires a transit peptide, which interacts with the chloroplast transport apparatus and leads to translocation of the protein across the chloroplast envelope. While the amino acid sequences of many transit peptides are known, functional domains have been difficult to identify. Previous studies suggest that the carboxyl terminus of the transit peptide for ribulose bisphosphate carboxylase small subunit is important for both translocation across the chloroplast envelope and proper processing of the precursor protein. We dissected this region using in vitro mutagenesis, creating a set of mutants with small changes in primary structure predicted to cause alterations in secondary structure. The import behavior of the mutant proteins was assessed using isolated chloroplasts. Our results show that removal of a conserved arginine residue in this region results in impaired processing, but does not necessarily affect import rates. In contrast, substituting amino acids with low reverse turn or amphiphilic potential for other original residues affected import rate but not processing.     

Transport of proteins into chloroplasts

The import of cytoplasmically synthesized proteins into chloroplasts involves an interaction between at least two components; the precursor protein, and the import apparatus in the chloroplast envelope membrane. This review summarizes the information available about each of these components. Precursor proteins consist of an amino terminal transit peptide attached to a passenger protein. Transit peptides from various precurosrs are diverse with respect to length and amino acid sequence; analysis of their sequences has not revealed insight into their mode of action. A variety of foreign passenger proteins can be imported into chloroplasts when a transit peptide is present at the amino terminus. However, foreign passenger proteins are not imported as efficiently as natural passenger proteins, and some chimeric precursor proteins are not imported into chloroplasts at all. Therefore, the passenger protein, as well as the transit peptide, influences the import process. Import begins by binding of the precursor to the chloroplast surface. It has been suggested that this binding is mediated by a receptor, but evidence to support this hypothesis remains incomplete and a receptor protein has not yet been characterized. Protein translocation requires energy derived from ATP hydrolysis, although there are conflicting reports as to where hydrolysis occurs and it is unclear how this energy is utilized. The mechanism(s) whereby proteins are translocated across either the two envelope membranes or the thylakoid membrane is not known.Abbreviations EPSP 5-enolpyruvyulshikimate-3-phosphate - LHCP Chlorophyll a/b binding protein of the light-harvesting complex - NPT-II Neomycin phosphotransferase II - PC Plastocyanin - Pr Precursor - Rubisco Ribulose-1,5,-bisphosphate carboxylase/oxygenase - SS Small subunit of Rubisco     

Methotrexate does not block import of a DHFR fusion protein into chloroplasts

Protein import into chloroplasts requires the movement of a precursor protein across the envelope membranes. The conformation of a precursor as it passes from the aqueous medium across the hydrophobic membranes is not known in detail. To address this problem we examined precursor conformation during translocation using the chimeric precursor PCDHFR, which contains the plastocyanin (PC) transit peptide in front of mouse cytosolic dihydrofolate reductase (DHFR). The chimeric protein is targeted to chloroplasts and is competent for import. The conformation of PCDHFR can be stabilized by complexing with methotrexate, an analogue of the substrate of DHFR. Methotrexate strongly inhibits DHFR import into yeast mitochondria (M. Eilers and G. Schatz, Nature 322 (1986) 228–232), presumably because the precursor must unfold to cross the membrane and it cannot do so when complexed with methotrexate. We show here that methotrexate does not block PCDHFR import into chloroplasts. Methotrexate does slow the rate of import, and protects DHFR from degradation once inside chloroplasts. The processed protein is localized in the stroma, indicating that import into thylakoids is impeded. Protease sensitivity assays indicate that the complex of precursor protein with methotrexate changes in conformation during the translocation across the envelope.     

Isolation and characterization of a cDNA clone encoding a cognate 70-kDa heat shock protein of the chloroplast envelope.

The translocation of proteins into the endoplasmic reticulum, the mitochondrion, and the chloroplast has recently been shown to involve homologues of the highly conserved 70-kDa heat shock protein (HSP70) family. In this study, we have isolated and sequenced a full-length cDNA clone encoding a cognate 70-kDa heat shock protein of the spinach chloroplast envelope (SCE70). The cDNA insert is 2,535 base pairs long and codes for 653 amino acid residues of a protein with a predicted molecular mass of 71,731 daltons. The deduced amino acid sequence shows a high degree of homology with HSP70 proteins from other organisms. Southern genomic and RNA analyses reveal different hybridization patterns than that observed for a heat-inducible 70-kDa protein gene. The protein synthesized from the SCE70 cDNA insert co-migrates with a 70-kDa polypeptide of the chloroplast envelope following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis and import studies indicate that SCE70 is associated with the chloroplast outer envelope. The import data suggest that SCE70 is targeted to the envelope membrane via a pathway different from other plastidic precursors but similar to that recently reported for outer envelope proteins SOE1 and OM14.     

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.     

A constituent of the chloroplast import complex represents a new type of GTP-binding protein

The 34 kDa polypeptide of the outer envelope membranes from pea chloroplasts (OEP 34) is a major constituent of this membrane. OEP 34 is detected on polyacrylamide gels under non-reducing condition in association with OEP 75, the putative protein translocation pore. An antiserum against OEP 34 is able to co-immunoprecipitate the precursor of Rubisco small subunit from a partially purified import complex of chloroplast outer envelope membranes. A full-length cDNA clone coding for pea OEP 34 has been isolated. Analysis of the deduced amino acid sequence revealed typical and conserved sequence motifs found in GTP-binding proteins, making it a new and unique member of this superfamily. OEP 34 behaves as an integral constituent of the outer chloroplast envelope, which is anchored by its C-terminus into the membrane, while the majority of the protein projects into the cytoplasm. OEP 34 does not possess a cleavable N-terminal transit sequence but it is targeted to the chloroplasts and integrated into the outer membranes by internal sequence information which seems to be present in the C-terminal membrane anchor region. Productive integration of OEP 34 into the outer envelope requires, in contrast to other OEPs, protease-sensitive chloroplast surface components and is stimulated by ATR. The GTP binding specificity of OEP 34 is demonstrated by photo-affinity labelling in the presence of [α-³²P]GTP. Overexpressed and purified OEP 34 possesses endogenous GTPase activity. These results indicate a possible regulatory function of OEP 34 in protein translocation into chloroplasts.     

Calcium regulation of chloroplast protein import

The majority of chloroplast proteins is nuclear-encoded and therefore synthesized on cytosolic ribosomes. In order to enter the chloroplast, these proteins have to cross the double-membrane surrounding the organelle. This is achieved by means of two hetero-oligomeric protein complexes in the outer and inner envelope, the Toc and Tic translocon. The process of chloroplast import is highly regulated on both sides of the envelope membranes. Our studies indicate the existence of an undescribed mode of control for this process so far, at the same time providing further evidence that the chloroplast is integrated into the calcium-signalling network of the cell. In pea chloroplasts, the calmodulin inhibitor Ophiobolin A as well as the calcium ionophores A23187 and Ionomycin affect the translocation of those chloroplast proteins that are imported with an N-terminal cleavable presequence. Import of these proteins is inhibited in a concentration-dependent manner. Addition of external calmodulin or calcium can counter the effect of these inhibitors. Translocation of chloroplast proteins that do not possess a cleavable transit peptide, that is outer envelope proteins or the inner envelope protein Tic32, is not affected. These results suggest that the import of a certain subset of chloroplast proteins is regulated by calcium. Our studies furthermore indicate that this regulation occurs downstream of the Toc translocon either within the intermembrane space or at the inner envelope translocon. A potential promoter of the calcium regulation is calmodulin, a protein well known as part of the plant's calcium signalling 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.     

New Insights into the Protein Import Machinery of the Chloroplast's Outer Envelope

A large number of plastid localized proteins are post-translationally imported as precursor proteins from the cytosol into the organelle. Recognition and translocation is accomplished by a subset of chloroplast envelope proteins, which were identified by different but complementary methods. The o uter e nvelope p roteins OEP 86, OEP 75, OEP 70 (a heat shock cognate 70 homologue) and OEP 34 are clearly involved in the import event and can be isolated as one functionally active translocation unit. For three of these proteins cDNA clones have been very recently obtained, namely OEP 86, OEP 75 and OEP 34. OEP 86 seems to be a precursor protein receptor which could be regulated by GTP binding and ATP-dependent phosphorylation-dephosphorylation. OEP 75 is part of the translocation pore traversing the membrane in multiple β-sheets. OEP 34 is tightly associated with OEP 75. It represents a new type of GTP-binding protein which possesses endogenous GTPase activity. Multiple GTP binding and hydrolysis cycles as well as protein phosphorylation-dephosphorylation events might, therefore, regulate the interaction of a precursor protein with the translocation machinery of the outer envelope, making it very distinct from the mitochondrial outer membrane system. Further proteins of the inner envelope membrane, namely IEP 97 and IEP 36, have been implied to function in the translocation event. These recent data allow not only identification of the players in the game but also speculation about mechanisms and regulation of translocation.