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.     

Toc, Tic, and chloroplast protein import.

The vast majority of chloroplast proteins are synthesized in precursor form on cytosolic ribosomes. Chloroplast precursor proteins have cleavable, N-terminal targeting signals called transit peptides. Transit peptides direct precursor proteins to the chloroplast in an organelle-specific way. They can be phosphorylated by a cytosolic protein kinase, and this leads to the formation of a cytosolic guidance complex. The guidance complex--comprising precursor, hsp70 and 14-3-3 proteins, as well as several unidentified components--docks at the outer envelope membrane. Translocation of precursor proteins across the envelope is achieved by the joint action of molecular machines called Toc (translocon at the outer envelope membrane of chloroplasts) and Tic (translocon at the inner envelope membrane of chloroplasts), respectively. The action of the Toc/Tic apparatus requires the hydrolysis of ATP and GTP at different levels, indicating energetic requirements and regulatory properties of the import process. The main subunits of the Toc and Tic complexes have been identified and characterized in vivo, in organello and in vitro. Phylogenetic evidence suggests that several translocon subunits are of cyanobacterial origin, indicating that today's import machinery was built around a prokaryotic core.     

Toc,tic, and chloroplast protein import

The vast majority of chloroplast proteins are synthesized in precursor form on cytosolic ribosomes. Chloroplast precursor proteins have cleavable, N-terminal targeting signals called transit peptides. Transit peptides direct precursor proteins to the chloroplast in an organelle-specific way. They can be phosphorylated by a cytosolic protein kinase, and this leads to the formation of a cytosolic guidance complex. The guidance complex--comprising precursor, hsp70 and 14-3-3 proteins, as well as several unidentified components--docks at the outer envelope membrane. Translocation of precursor proteins across the envelope is achieved by the joint action of molecular machines called Toc (translocon at the outer envelope membrane of chloroplasts) and Tic (translocon at the inner envelope membrane of chloroplasts), respectively. The action of the Toc/Tic apparatus requires the hydrolysis of ATP and GTP at different levels, indicating energetic requirements and regulatory properties of the import process. The main subunits of the Toc and Tic complexes have been identified and characterized in vivo, in organello and in vitro. Phylogenetic evidence suggests that several translocon subunits are of cyanobacterial origin, indicating that today's import machinery was built around a prokaryotic core.     

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.     

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.     

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.     

Plastid division coordination across a double-membraned structure

Chloroplasts still retain components of the bacterial cell division machinery and research over the past decade has led to an understanding of how these stromal division proteins assemble and function as a complex chloroplast division machinery. However, during evolution plant chloroplasts have acquired a number of cytosolic division proteins, indicating that unlike the cyanobacterial ancestors of plastids, chloroplast division in higher plants require a second division machinery located on the chloroplast outer envelope membrane. Here we review the current understanding of the stromal and cytosolic plastid division machineries and speculate how two protein machineries coordinate their activities across a double-membraned structure.     

The Tic20 gene family: Phylogenetic analysis and evolutionary considerations

Tic20 is a polytopic protein of the inner envelope membrane of chloroplasts, and it is proposed to act as a translocation channel during chloroplast protein import. By analyzing 29 sequences from diverse organisms, it was evident that Tic20-related proteins form two distinct clades, termed Group 1 and Group 2. The former group includes canonical Tic20 proteins that are essential for chloroplast development, while members of the latter are of unknown function. An increased evolutionary rate, in connection with adaptation to terrestrial life, was detected in Group 1. Interestingly, the sub-cellular (genomic) localization of genes coding for Group 1 proteins differs between evolutionary lineages.Key words: Arabidopsis, chloroplast protein import, plastids, protein targeting, Tic20     

Toc64, a new component of the protein translocon of chloroplasts

A subunit of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) of 64 kD is described, Toc64. Toc64 copurifies on sucrose density gradients with the isolated Toc complex. Furthermore, it can be cross-linked in intact chloroplasts to a high molecular weight complex containing both Toc and Tic subunits and a precursor protein. The 0 A cross-linker CuCl(2) yields the reversible formation of disulfide bridge(s) between Toc64 and the established Toc complex subunits in purified outer envelope membranes. Toc64 contains three tetratricopeptide repeat motifs that are exposed at the chloroplast cytosol interface. We propose that Toc64 functions early in preprotein translocation, maybe as a docking protein for cytosolic cofactors of the protein import into chloroplasts.     

A gateway to chloroplasts - protein translocation and beyond

The biogenesis of chloroplasts requires the coordinated interplay with the nucleus and the cytoplasm. The majority of chloroplast proteins are encoded by the nuclear genome and must be faithfully and efficiently delivered to the organelle upon completion of translation in the cytosol. This high-fidelity targeting is accomplished by specific chloroplast targeting signal peptides. Several cytoplasmic factors recognise, modify, and bind this targeting sequence, and deliver the preproteins to the chloroplast translocation machinery. The multisubunit translocation complex at the outer envelope contains receptor proteins, a translocation channel, and accessory subunits. Complete import into the stroma utilizes both outer and inner envelope translocons and molecular chaperones in the intermembrane space and in the stroma. The entire import process appears to be regulated by phosphorylation, nucleotide binding, and hydrolysis. Recent evidence indicates that several subunits of the chloroplast import machinery may have evolved from cyanobacterial ancestors.     

Interaction of plant mitochondrial and chloroplast signal peptides with the Hsp70 molecular chaperone.

Most mitochondrial and chloroplast proteins are synthesized on cytosolic polyribosomes as precursor proteins, with an N-terminal signal sequence that targets the precursor to the correct organelle. In mitochondria, the chaperone Hsp70 functions as a molecular motor, pulling the precursor across the mitochondrial membranes; 97.0% of plant mitochondrial presequences contain an Hsp70 binding site. In chloroplasts, the outer envelope, intermembrane space and a stromal Hsp70 are thought to participate in protein import; 82.5% of chloroplast transit peptides have an Hsp70 binding site. The interaction of signal peptides with Hsp70 during the import process is supported by biochemical and bioinformatic studies.     

In vivo studies on the roles of two closely related Arabidopsis Tic20 proteins, AtTic20-I and AtTic20-IV

Protein translocation across the inner envelope of plastids is mediated by the TIC (translocon at the inner envelope membrane of chloroplasts) protein translocation machinery. Tic20 has been shown to function as a central component of TIC machinery. The Arabidopsis genome encodes four Tic20 homologous proteins, AtTic20-I, AtTic20-II, AtTIC20-IV and AtTic20-V, among which only AtTic20-I has been extensively characterized and demonstrated to be essential for protein import into chloroplasts. AtTic20-I is more closely related to AtTic20-IV than to AtTic20-II or AtTic20-V, whereas AtTic20-II and AtTic20-V show higher similarities to each other than to AtTic20-I or AtTic20-IV. Here, we show that AtTic20-IV is expressed mainly in roots whereas AtTic20-I is more abundant in shoots than in roots. Although AtTic20-IV is dispensable for viability in the wild-type background, interestingly, expression of AtTic20-IV is markedly elevated in both shoots and roots in the tic20-I knockout mutant that exhibits severe albino and seedling-lethal phenotypes. The albino tic20-I seedlings do not accumulate any of the photosynthetic proteins analyzed, but the plastids can still import non-photosynthetic housekeeping proteins. This residual import ability of the tic20-I mutant can be attributed to partial compensation by the elevated expression of AtTic20-IV, since a double knockout mutant of AtTic20-I and AtTic20-IV exhibits more severe embryonic lethality. Further overexpression of AtTic20-IV in the tic20-I mutant can only marginally rescue the accumulation of photosynthetic proteins in the albino seedlings. These data demonstrate an absolute requirement of at least one of the two closely related Tic20 proteins in protein translocation across the inner envelope of plastids and also suggest their distinct substrate preferences.     

Filling the Gap, Evolutionarily Conserved Omp85 in Plastids of Chromalveolates

Chromalveolates are a diverse group of protists that include many ecologically and medically relevant organisms such as diatoms and apicomplexan parasites. They possess plastids generally surrounded by four membranes, which evolved by engulfment of a red alga. Today, most plastid proteins must be imported, but many aspects of protein import into complex plastids are still cryptic. In particular, how proteins cross the third outermost membrane has remained unexplained. We identified a protein in the third outermost membrane of the diatom Phaeodactylum tricornutum with properties comparable to those of the Omp85 family. We demonstrate that the targeting route of P. tricornutum Omp85 parallels that of the translocation channel of the outer envelope membrane of chloroplasts, Toc75. In addition, the electrophysiological properties are similar to those of the Omp85 proteins involved in protein translocation. This supports the hypothesis that P. tricornutum Omp85 is involved in precursor protein translocation, which would close a gap in the fundamental understanding of the evolutionary origin and function of protein import in secondary plastids.     

Genome-based reconstruction of the protein import machinery in the secondary plastid of a chlorarachniophyte alga

Most plastid proteins are encoded by their nuclear genomes and need to be targeted across multiple envelope membranes. In vascular plants, the translocons at the outer and inner envelope membranes of chloroplasts (TOC and TIC, respectively) facilitate transport across the two plastid membranes. In contrast, several algal groups harbor more complex plastids, the so-called secondary plastids, which are surrounded by three or four membranes, but the plastid protein import machinery (in particular, how proteins cross the membrane corresponding to the secondary endosymbiont plasma membrane) remains unexplored in many of these algae. To reconstruct the putative protein import machinery of a secondary plastid, we used the chlorarachniophyte alga Bigelowiella natans, whose plastid is bounded by four membranes and still possesses a relict nucleus of a green algal endosymbiont (the nucleomorph) in the intermembrane space. We identified nine homologs of plant-like TOC/TIC components in the recently sequenced B. natans nuclear genome, adding to the two that remain in the nucleomorph genome (B. natans TOC75 [BnTOC75] and BnTIC20). All of these proteins were predicted to be localized to the plastid and might function in the inner two membranes. We also show that the homologs of a protein, Der1, that is known to mediate transport across the second membrane in the several lineages with secondary plastids of red algal origin is not associated with plastid protein targeting in B. natans. How plastid proteins cross this membrane remains a mystery, but it is clear that the protein transport machinery of chlorarachniophyte plastids differs from that of red algal secondary plastids.     

Conservative sorting in a primitive plastid. The cyanelle of Cyanophora paradoxa

Higher plant chloroplasts possess at least four different pathways for protein translocation across and protein integration into the thylakoid membranes. It is of interest with respect to plastid evolution, which pathways have been retained as a relic from the cyanobacterial ancestor ('conservative sorting'), which ones have been kept but modified, and which ones were developed at the organelle stage, i.e. are eukaryotic achievements as (largely) the Toc and Tic translocons for envelope import of cytosolic precursor proteins. In the absence of data on cyanobacterial protein translocation, the cyanelles of the glaucocystophyte alga Cyanophora paradoxa for which in vitro systems for protein import and intraorganellar sorting were elaborated can serve as a model: the cyanelles are surrounded by a peptidoglycan wall, their thylakoids are covered with phycobilisomes and the composition of their oxygen-evolving complex is another feature shared with cyanobacteria. We demonstrate the operation of the Sec and Tat pathways in cyanelles and show for the first time in vitro protein import across cyanobacteria-like thylakoid membranes and protease protection of the mature protein.     

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.     

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.     

Chloroplast protein translocon components atToc159 and atToc33 are not essential for chloroplast biogenesis in guard cells and root cells

Protein import into chloroplasts is mediated by a protein import apparatus located in the chloroplast envelope. Previous results indicate that there may be multiple import complexes in Arabidopsis. To gain further insight into the nature of this multiplicity, we analyzed the Arabidopsis ppi1 and ppi2 mutants, which are null mutants of the atToc33 and atToc159 translocon proteins, respectively. In the ppi2 mutant, in contrast to the extremely defective plastids in mesophyll cells, chloroplasts in guard cells still contained starch granules and thylakoid membranes. The morphology of root plastids in both mutants was similar to that in wild type. After prolonged light treatments, root plastids of both mutants and the wild type differentiated into chloroplasts. Enzymatic assays indicated that the activity of a plastid enzyme was reduced only in leaves but not in roots. These results indicated that both the ppi1 and ppi2 mutants had functional root and guard cell plastids. Therefore, we propose that import complexes are cell type specific rather than substrate or plastid specific.     

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.     

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.