The paradox of plastid transit peptides: conservation of function despite divergence in primary structure.

Transit peptides are N-terminal extensions that facilitate the targeting and translocation of cytosolically synthesized precursors into plastids via a post-translational mechanism. With the complete Arabidopsis genome in hand, it is now evident that transit peptides direct more than 3500 different proteins into the plastid during the life of a typical plant. Deciphering a common mechanism for how this multitude of targeting sequences function has been hampered by the realization that at a primary sequence level, transit peptides are highly divergent in length, composition, and organization. This review addresses recent findings on several of the diverse functions that transit peptides must perform, including direct interaction with envelope lipids, association with a cis-acting guidance complex, recognition by envelope receptors, insertion into the Toc/Tic translocon, interaction with molecular motors, and finally, recognition/cleavage by the stromal processing peptidase. In addition to higher plants, transit peptides also direct the import of proteins into complex plastids derived from secondary endosymbiosis. An emerging concept suggests that transit peptides contain multiple domains that provide either distinct or possibly overlapping functions. Although still poorly characterized, evolutionary processes could yield transit peptides with alternative domain organizations.     

Amino-terminal and hydrophobic regions of the Chlamydomonas reinhardtii plastocyanin transit peptide are required for efficient protein accumulation in vivo

Nucleus-encoded chloroplast proteins of vascular plants are synthesized as precursors and targeted to the chloroplast by stroma-targeting domains in N-terminal transit peptides. Transit peptides in Chlamydomonas reinhardtii are considerably shorter than those in vascular plants, and their stroma-targeting domains have similarities to both mitochondrial and chloroplast targeting sequences. To examine Chlamydomonas transit peptide function in vivo, deletions were introduced into the transit peptide coding region of the petE gene, which encodes the thylakoid lumen protein plastocyanin (PC). The mutant petE genes were introduced into a plastocyanin-deficient Chlamydomonas strain, and transformants that accumulated petE mRNA were analyzed for PC accumulation. The most profound defects were observed with deletions at the N-terminus and those that extended into the hydrophobic region in the C-terminal half of the transit peptide. PC precursors were detected among pulse-labeled proteins in transformants with N-terminal deletions, suggesting that these precursors cannot be imported and are degraded in the cytosol. Intermediate PC species were observed in a transformant deleted for part of the hydrophobic region, suggesting that this protein is defective in lumen translocation and/or processing. Thus, despite its shorter length, the bipartite nature of the Chlamydomonas PC transit peptide appears similar to that of lumen-targeted proteins in vascular plants. Analysis of the synthesis, stability, and accumulation of PC species in transformants bearing deletions in the stroma-targeting domain suggests that specific regions probably have distinct roles in vivo. Abbreviations: cyt, cytochrome; ECL, enhanced chemiluminescence; LSU, large subunit; PC, plastocyanin; TP, transit peptide     

Domain structure of mitochondrial and chloroplast targeting peptides

Representative samples of mitochondrial and chloroplast targeting peptides have been analyzed in terms of amino acid composition, positional amino acid preferences and amphiphilic character. No highly conserved 'homology blocks' are found in either class of topogenic sequence. Mitochondrial-matrix-targeting peptides are composed of two domains with different amphiphilic properties. Arginine is frequently found either at position -10 or -2 relative to the cleavage site, suggesting that some targeting peptides may be cleaved twice in succession by two different matrix proteases. In stroma-targeting chloroplast transit peptides three distinct regions are evident: an uncharged amino-terminal domain, a central domain lacking acidic residues and a carboxy-terminal domain with the potential to form an amphiphilic beta-strand. Targeting peptides that route proteins to the mitochondrial intermembrane space or the lumen of chloroplast thylakoids have a mosaic design with an amino-terminal matrix- or stroma-targeting part attached to a carboxy-terminal extension that shares many characteristics with secretory signal peptides.     

A truncated analog of a pre-light-harvesting chlorophyll a/b protein II transit peptide inhibits protein import into chloroplasts

It is unclear how transit peptides target nuclear-encoded precursor proteins to the chloroplast. This study establishes the feasibility of using synthetic peptides as competitive inhibitors of chloroplast protein import and as probes for the function of domains within transit peptides. We show that peptide pL(1-20), MAASTMALSSPAFAGKAVNY, an analog of the NH2 terminus of a pre-light harvesting chlorophyll a/b protein II from Arabidopsis, inhibits the import of several Arabidopsis and pea precursor proteins into pea chloroplasts. Inhibition occurs at a step between the initial binding of precursors to the chloroplast and the first proteolytic cleavage event and is not due to interference with ATP availability or chloroplast integrity. Presumably this reflects specific binding of the peptide to the import machinery in the chloroplast envelope. Our data are consistent with the suggestion (Karlin-Neumann, G. A., and Tobin, E. M. (1986) EMBO J. 5, 9-13) that two conserved blocks of amino acids near the NH2-terminus of transit peptides (spanned by peptide pL(1-20] participate in protein targeting. Computer analysis also shows peptide pL(1-20) lacks the amphiphilic properties characteristic of pre-sequences of many nuclear-encoded mitochondrial proteins. This shows a difference in the mechanisms for targeting proteins to chloroplasts and mitochondria.     

Arabidopsis nuclear-encoded plastid transit peptides contain multiple sequence subgroups with distinctive chloroplast-targeting sequence motifs

The N-terminal transit peptides of nuclear-encoded plastid proteins are necessary and sufficient for their import into plastids, but the information encoded by these transit peptides remains elusive, as they have a high sequence diversity and lack consensus sequences or common sequence motifs. Here, we investigated the sequence information contained in transit peptides. Hierarchical clustering on transit peptides of 208 plastid proteins showed that the transit peptide sequences are grouped to multiple sequence subgroups. We selected representative proteins from seven of these multiple subgroups and confirmed that their transit peptide sequences are highly dissimilar. Protein import experiments revealed that each protein contained transit peptide-specific sequence motifs critical for protein import into chloroplasts. Bioinformatics analysis identified sequence motifs that were conserved among members of the identified subgroups. The sequence motifs identified by the two independent approaches were nearly identical or significantly overlapped. Furthermore, the accuracy of predicting a chloroplast protein was greatly increased by grouping the transit peptides into multiple sequence subgroups. Based on these data, we propose that the transit peptides are composed of multiple sequence subgroups that contain distinctive sequence motifs for chloroplast targeting.     

Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving protein.

Various chimeric precursors and deletions of the 33 kd oxygen-evolving protein (OEE1) were constructed to study the mechanism by which chloroplast proteins are imported and targeted to the thylakoid lumen. The native OEE1 precursor was imported into isolated chloroplasts, processed and localized in the thylakoid lumen. Replacement of the OEE1 transit peptide with the transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase, a stromal protein, resulted in redirection of mature OEE1 into the stromal compartment of the chloroplast. Utilizing chimeric transit peptides and block deletions we demonstrated that the 85 residue OEE1 transit peptide contains separate signal domains for importing and targeting the thylakoid lumen. The importing domain, which mediates translocation across the two membranes of the chloroplast envelope, is present in the N-terminal 58 amino acids. The thylakoid lumen targeting domain, which mediates translocation across the thylakoid membrane, is located within the C-terminal 27 residues of the OEE1 transit peptide. Chimeric precursors were constructed and used in in vitro import experiments to demonstrate that the OEE1 transit peptide is capable of importing and targeting foreign proteins to the thylakoid lumen.     

Synthetic transit peptides inhibit import and processing of mitochondrial precursor proteins

We have demonstrated that a synthetic peptide corresponding to the rat mitochondrial malate dehydrogenase (mMDH) transit peptide (TP-28) inhibits the binding of pre-mMDH to isolated mitochondria. Synthetic peptides derived from chloroplast transit peptide sequences, which have a similar net charge, did not inhibit import. In addition, this peptide (TP-28) inhibits import of ornithine transcarbamylase, another mitochondrial matrix protein, thus suggesting that common import pathways exist for both mMDH and ornithine transcarbamylase. A smaller synthetic peptide corresponding to residues 1-20 of the mMDH transit peptide (TP-20) also inhibits binding. However, several substitutions for leucine-13 in the smaller peptide relieve import inhibition, thus providing evidence that this neutral residue plays a crucial role in transit peptide binding to the mitochondrial surface. Proteolytic processing of pre-mMDH by a mitochondrial matrix fraction to both the mature and intermediate forms of mMDH was also inhibited by TP-28. The ability of synthetic peptides to inhibit distinct steps in the import of mitochondrial precursor proteins corresponds precisely to their ability to interact with the same components used by transit peptides on intact precursors. Furthermore, inhibition at multiple points along the import pathway reflects the functions of several independent structures contained within transit peptides.     

Transit peptides of nuclear-encoded chloroplast proteins share a common amino acid framework.

We have identified three major blocks of amino acid homology shared by the transit peptides of two nuclear-encoded chloroplast proteins, the light-harvesting chlorophyll a/b-protein (LHCP) II of the thylakoid membrane and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of the stroma. These previously unrecognized homology blocks lie at the beginning, middle and end of both transit sequences, and are separated by differing lengths of unshared (interblock) sequence in the two proteins. These interblocks may be dispensible or they might confer a specific property on the individual proteins, such as facilitating proper compartmentalization within the chloroplast. We propose that these three shared sequence elements form a common framework in transit-bearing chloroplast precursors which mediates the common functions performed by each transit peptide. Ferredoxin, the only other such nuclear-encoded protein for which a published transit sequence exists, conforms to the predictions of this hypothesis. These findings stand in contrast to mitochondrial leader sequences and the well-studied signal peptides of secretory and certain integral membrane proteins in which no such framework has been observed.     

Import into chloroplasts of a yeast mitochondrial protein directed by ferredoxin and plastocyanin transit peptides

Many chloroplast proteins are synthesized in the cytoplasm as precursors which contain an amino terminal transit peptide. These precursors are subsequently imported into chloroplast and targeted to one of several organellar locations. This import is mediated by the transit peptide, which is cleaved off during import. We have used the transit peptides of ferredoxin (chloroplast stroma) and plastocyanin (thylakoid lumen) to study chloroplast protein import and intra-organellar routing toward different compartments. Chimeric genes were constructed that encode precursor proteins in which the transit peptides are linked to yeast mitochondrial manganese superoxide dismutase. Chloroplast protein import and localization experiments show that both chimeric proteins are imported into the chloroplast stroma and processed. The plastocyanin transit sequence did not direct superoxide dismutase to the thylakoids; this protein was found in the stroma as an intermediate that still contains part of the plastocyanin transit peptide. The organelle specificity of these chimeric precursors reflected the transit peptide parts of the molecules, because neither the ferredoxin and plastocyanin precursors nor the chimeric proteins were imported into isolated yeast mitochondria.     

Complex protein targeting to dinoflagellate plastids

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

The role of the transit peptide in the routing of precursors toward different chloroplast compartments

The role of the transit peptide in the routing of imported proteins inside the chloroplast was investigated with chimeric proteins in which the transit peptides for the nuclear-encoded ferredoxin and plastocyanin precursors were exchanged. Import and localization experiments with a reconstituted chloroplast system show that the ferredoxin transit peptide directs mature plastocyanin away from its correct location, the thylakoid lumen, to the stroma. With the plastocyanin transit peptide-mature ferredoxin chimera, a processing intermediate is arrested on its way to the lumen. We propose a two domain hypothesis for the plastocyanin transit peptide: the first domain functions in the chloroplast import process, whereas the second is responsible for transport across the thylakoid membrane. Thus, the transit peptide not only targets proteins to the chloroplast, but also is a major determinant in their subsequent localization within the organelle.     

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.     

Silene cDNA clones for a divergent chlorophyll-a/b-binding protein and a small subunit of ribulosebisphosphate carboxylase

Summary We have isolated and analyzed cDNA clones for aSilene pratensis chlorophyll-a/b-binding protein (CAB) and a small subunit (SS) of ribulosebisphosphate carboxylase. These cDNA clones contain the coding information for the complete transit peptides. The CAB clone codes for a divergent CAB protein that differs from most published CAB sequences in both the transit peptide part and in the amino terminal part of the mature protein, a region with an important regulatory function. The SS clone codes for a precursor that is homologous to other published precursor sequences. In the mature part some non-conservative changes are observed.Silene cDNA clones for four chloroplast specific precursor proteins that are directed towards three different chloroplast compartments have been analyzed and the transit peptides compared.     

Chloroplast Import Signals: The Length Requirement for Translocation In Vitro and In Vivo

Protein translocation of cytosolically synthesized proteins requires signals for both targeting of precursor proteins to the surface of the respective compartment and their transfer across its membrane. In contrast to signals for peroxisomal and endoplasmic reticulum translocation, the signals for mitochondrial and chloroplast transport are less well defined with respect to length and amino acid requirements. To study the properties of signals required for translocation into chloroplasts in vitro and in vivo, we used fusion proteins composed of transit peptides and the Ig-like module of the muscle protein titin as passenger. We observed that about 60 amino acids—longer than the transit peptide length of many experimentally confirmed chloroplast proteins—are required for efficient translocation. However, within native chloroplast precursor proteins with transit peptides shorter than 60 amino acids, extension appears to be present as they are efficiently imported into organelles. In addition, the interaction of an unfolded polypeptide stretch of 60 or more amino acids with receptors at the chloroplast surface results in the unidirectionality of protein translocation into chloroplasts even in the presence of a competing C-terminal peroxisomal targeting signal. These findings prove the existing ideas that initial targeting is defined by the N-terminal signal and that the C-terminal signal is sensed only subsequently.     

Plastid-targeting peptides from the chlorarachniophyte Bigelowiella natans

Chlorarachniophytes are marine amoeboflagellate protists that have acquired their plastid (chloroplast) through secondary endosymbiosis with a green alga. Like other algae, most of the proteins necessary for plastid function are encoded in the nuclear genome of the secondary host. These proteins are targeted to the organelle using a bipartite leader sequence consisting of a signal peptide (allowing entry in to the endomembrane system) and a chloroplast transit peptide (for transport across the chloroplast envelope membranes). We have examined the leader sequences from 45 full-length predicted plastid-targeted proteins from the chlorarachniophyte Bigelowiella natans with the goal of understanding important features of these sequences and possible conserved motifs. The chemical characteristics of these sequences were compared with a set of 10 B. natans endomembrane-targeted proteins and 38 cytosolic or nuclear proteins, which show that the signal peptides are similar to those of most other eukaryotes, while the transit peptides differ from those of other algae in some characteristics. Consistent with this, the leader sequence from one B. natans protein was tested for function in the apicomplexan parasite, Toxoplasma gondii, and shown to direct the secretion of the protein.     

Carboxyl-terminal sequences can influence the in vitro import and intraorganellar targeting of chloroplast protein precursors.

The transit peptide of the lumenal 33-kDa oxygen-evolving polypeptide (OEE1) is capable of directing the import and targeting of the foreign protein dihydrofolate reductase (DHFR) to the thylakoid lumen. The import results from the first part of this study indicate that methotrexate cannot block the import or intraorganellar targeting of OEE1-DHFR in chloroplasts in contrast to that reported for the import of cytochrome oxidase subunit IV (COXIV)-DHFR in mitochondria. These results suggest that the fusion of the OEE1 transit sequence to DHFR affected the protein's methotrexate binding properties. We further examined and compared the transport characteristics of a number of carboxyl-terminal truncated native chloroplast precursors to determine whether carboxyl domains contribute to the import and intraorganellar targeting mechanism of these proteins. The plastid precursors chosen for this study are targeted to one of the following chloroplast compartments: the stroma, the thylakoid membrane, and the lumen. In most cases, removal of carboxyl domains had a dramatic effect on one or more stages of the translocation pathway, such as import, processing, and intraorganellar targeting. The effects of carboxyl deletions varied from precursor to precursor and were dependent on the extent of the deletion. These combined results suggest that carboxyl domains in the mature part of the proteins can influence the function of the transit peptide, and as a result play an important role in determining the import and targeting competence of chloroplast precursors.     

Functional characterization of sequence motifs in the transit peptide of Arabidopsis small subunit of rubisco

The transit peptides of nuclear-encoded chloroplast proteins are necessary and sufficient for targeting and import of proteins into chloroplasts. However, the sequence information encoded by transit peptides is not fully understood. In this study, we investigated sequence motifs in the transit peptide of the small subunit of the Rubisco complex by examining the ability of various mutant transit peptides to target green fluorescent protein reporter proteins to chloroplasts in Arabidopsis (Arabidopsis thaliana) leaf protoplasts. We divided the transit peptide into eight blocks (T1 through T8), each consisting of eight or 10 amino acids, and generated mutants that had alanine (Ala) substitutions or deletions, of one or two T blocks in the transit peptide. In addition, we generated mutants that had the original sequence partially restored in single- or double-T-block Ala (A) substitution mutants. Analysis of chloroplast import of these mutants revealed several interesting observations. Single-T-block mutations did not noticeably affect targeting efficiency, except in T1 and T4 mutations. However, double-T mutants, T2A/T4A, T3A/T6A, T3A/T7A, T4A/T6A, and T4A/T7A, caused a 50% to 100% loss in targeting ability. T3A/T6A and T4A/T6A mutants produced only precursor proteins, whereas T2A/T4A and T4A/T7A mutants produced only a 37-kD protein. Detailed analyses revealed that sequence motifs ML in T1, LKSSA in T3, FP and RK in T4, CMQVW in T6, and KKFET in T7 play important roles in chloroplast targeting. In T1, the hydrophobicity of ML is important for targeting. LKSSA in T3 is functionally equivalent to CMQVW in T6 and KKFET in T7. Furthermore, subcellular fractionation revealed that Ala substitution in T1, T3, and T6 produced soluble precursors, whereas Ala substitution in T4 and T7 produced intermediates that were tightly associated with membranes. These results demonstrate that the transit peptide contains multiple motifs and that some of them act in concert or synergistically.     

Protein trafficking to the plastid of Plasmodium falciparum is via the secretory pathway

The plastid of Plasmodium falciparum (or 'apicoplast') is the evolutionary homolog of the plant chloroplast and represents a vestige of a photosynthetic past. Apicoplast indispensability indicates that it still provides essential functions to parasites. Similar to plant chloroplasts, the apicoplast is dependent on many nucleus-encoded genes to provide these functions. The apicoplast is surrounded by four membranes, two more than plant chloroplasts. Thus, protein targeting to the apicoplast must overcome additional membrane barriers. In P.falciparum we have analyzed apicoplast targeting using green fluorescent protein (GFP). We demonstrate that protein targeting is at least a two-step process mediated by bipartite N-terminal pre-sequences that consist of a signal peptide for entry into the secretory pathway and a plant-like transit peptide for subsequent import into the apicoplast. The P.falciparum transit peptide is exceptional compared with other known plastid transit peptides in not requiring serine or threonine residues. The pre-sequence components are removed stepwise during apicoplast targeting. Targeting GFP to the apicoplast has also provided the first opportunity to examine apicoplast morphology in live P. falciparum.