Chloroplast-targeting Peptide Research Articles

Identification of a highly efficient chloroplast-targeting peptide for plastid engineering.

Plastids are pivotal target organelles for comprehensively enhancing photosynthetic and metabolic traits in plants via plastid engineering. Plastidial proteins predominantly originate in the nucleus and must traverse membrane-bound multiprotein translocons to access these organelles. This import process is meticulously regulated by chloroplast-targeting peptides (cTPs). Whereas many cTPs have been employed to guide recombinantly expressed functional proteins to chloroplasts, there is a critical need for more efficient cTPs. Here, we performed a comprehensive exploration and comparative assessment of an advanced suite of cTPs exhibiting superior targeting capabilities. We employed a multifaceted approach encompassing computational prediction, in planta expression, fluorescence tracking, and in vitro chloroplast import studies to identify and analyze 88 cTPs associated with Arabidopsis thaliana mutants with phenotypes linked to chloroplast function. These polypeptides exhibited distinct abilities to transport green fluorescent protein (GFP) to various compartments within leaf cells, particularly chloroplasts. A highly efficient cTP derived from Arabidopsis plastid ribosomal protein L35 (At2g24090) displayed remarkable effectiveness in chloroplast localization. This cTP facilitated the activities of chloroplast-targeted RNA-processing proteins and metabolic enzymes within plastids. This cTP could serve as an ideal transit peptide for precisely targeting biomolecules to plastids, leading to advancements in plastid engineering.

Arabidopsis transcription factor ANAC102 predominantly expresses a nuclear protein and acts as a negative regulator of methyl viologen-induced oxidative stress responses.

Plants, being sessile organisms, constantly need to respond to environmental stresses, often leading to the accumulation of reactive oxygen species (ROS). While ROS can be harmful, they also act as second messengers guiding plant growth and stress responses. Because chloroplasts are sensitive to environmental changes and are both a source and a target of ROS during stress conditions, they are important in conveying environmental changes to the nucleus, where acclimation responses are coordinated to maintain organellar and overall cellular homeostasis. ANAC102 has previously been established as a regulator of β-cyclocitral-mediated chloroplast-to-nucleus signaling, protecting plants against photooxidative stress. However, debates persist about where ANAC102 is located-in chloroplasts or in the nucleus. Our study, utilizing the genomic ANAC102 sequence driven by its native promoter, establishes ANAC102 primarily as a nuclear protein, lacking a complete N-terminal chloroplast-targeting peptide. Moreover, our research reveals the sensitivity of plants overexpressing ANAC102 to severe superoxide-induced chloroplast oxidative stress. Transcriptome analysis unraveled a dual role of ANAC102 in negatively and positively regulating genome-wide transcriptional responses to chloroplast oxidative stress. Through the integration of published data and our own study, we constructed a comprehensive transcriptional network, which suggests that ANAC102 exerts direct and indirect control over transcriptional responses through downstream transcription factor networks, providing deeper insights into the ANAC102-mediated regulatory landscape during oxidative stress.

The journey of preproteins across the chloroplast membrane systems.

The photosynthetic capacity of chloroplasts is vital for autotrophic growth in algae and plants. The origin of the chloroplast has been explained by the endosymbiotic theory that proposes the engulfment of a cyanobacterium by an ancestral eukaryotic cell followed by the transfer of many cyanobacterial genes to the host nucleus. As a result of the gene transfer, the now nuclear-encoded proteins acquired chloroplast targeting peptides (known as transit peptides; transit peptide) and are translated as preproteins in the cytosol. Transit peptides contain specific motifs and domains initially recognized by cytosolic factors followed by the chloroplast import components at the outer and inner envelope of the chloroplast membrane. Once the preprotein emerges on the stromal side of the chloroplast protein import machinery, the transit peptide is cleaved by stromal processing peptidase. In the case of thylakoid-localized proteins, cleavage of the transit peptides may expose a second targeting signal guiding the protein to the thylakoid lumen or allow insertion into the thylakoid membrane by internal sequence information. This review summarizes the common features of targeting sequences and describes their role in routing preproteins to and across the chloroplast envelope as well as the thylakoid membrane and lumen.

Arabidopsis ANAC102, Chloroplastic or Nucleocytosolic Localization?

ANAC102 is a transcription factor involved in stress response and brassinosteroids signaling, with circadian regulation controlled by phytochromes. ANAC102 has been proposed to have a role in downregulating chloroplast transcription, which may be very useful in reducing photosynthesis and chloroplast energy demand under stress conditions. However, its localization in the chloroplast has mainly been demonstrated by using constitutive promoters. In this work, we recapitulate the literature, clarify which are ANAC102 isoforms in Arabidopsis and analyze their expressions under control conditions and in response to stress. Based on our results, the most highly expressed ANAC102 isoform encodes for a nucleocytoplasmic protein and the N-terminal chloroplast-targeting peptide appears to be present only in Brassicaceae, and not involved in stress response.

Converting antimicrobial into targeting peptides reveals key features governing protein import into mitochondria and chloroplasts

We asked what peptide features govern targeting to the mitochondria versus the chloroplast, using antimicrobial peptides as a starting point. This approach was inspired by the endosymbiotic hypothesis that organelle-targeting peptides derive from antimicrobial amphipathic peptides delivered by the host cell, to which organelle progenitors became resistant. To explore the molecular changes required to convert antimicrobial into targeting peptides, we expressed a set of 13 antimicrobial peptides in Chlamydomonas reinhardtii. Peptides were systematically modified to test distinctive features of mitochondrion- and chloroplast-targeting peptides, and we assessed their targeting potential by following the intracellular localization and maturation of a Venus fluorescent reporter used as a cargo protein. Mitochondrial targeting can be achieved by some unmodified antimicrobial peptide sequences. Targeting to both organelles is improved by replacing lysines with arginines. Chloroplast targeting is enabled by the presence of flanking unstructured sequences, additional constraints consistent with chloroplast endosymbiosis having occurred in a cell that already contained mitochondria. If indeed targeting peptides evolved from antimicrobial peptides, then required modifications imply a temporal evolutionary scenario with an early exchange of cationic residues and a late acquisition of chloroplast-specific motifs.

Cell-penetrating peptide for targeted macromolecule delivery into plant chloroplasts.

Reports on chloroplast-targeted protein delivery using cell-penetrating peptides are scarce. In this study, a novel peptide-based macromolecule delivery strategy targeting chloroplasts was successfully developed in wheat mesophyll protoplasts. A peptide derived from the signal sequence of the chloroplast-targeted protein of ferredoxin-thioredoxin reductase catalytic chain of Spinacia oleracea with UniProtKB Id-P41348 exhibits properties of cellular internalization. DNase I was efficiently delivered into the chloroplast using 10 μM cTP with an efficiency of more than 90%. This cell-penetrating peptide-mediated approach offers various advantages over the existing chloroplast targeting methods, such as non-invasiveness, biocompatibility, low-toxicity, and target-specific delivery. The present study shows that peptide-based strategies hold tremendous potential in the field of chloroplast biotechnology. KEY POINTS: • Screening of database of chloroplast targeting peptides in order to develop an efficient cell-penetrating peptide termed as cTP. • cTP efficiently crosses the cell barrier and demonstrated chloroplast-localization. • cTP can be incorporated as a promising strategy for delivering macromolecules for crop improvement.

Targeted Gene Delivery into Various Plastids Mediated by Clustered Cell-Penetrating and Chloroplast-Targeting Peptides.

The plastid is an organelle that functions as a cell factory to supply food and oxygen to the plant cell and is therefore a potential target for genetic engineering to acquire plants with novel photosynthetic traits or the ability to produce valuable biomolecules. Conventional plastid genome engineering technologies are laborious for the preparation of plant material, require expensive experimental instruments, and are time consuming for obtaining a transplastomic plant line that produces significant levels of the biomolecule of interest. Herein, a transient plastid transformation technique is presented using a peptide‐based gene carrier. By formulating peptide/plasmid DNA complexes that combine the functions of both a cell‐penetrating peptide and a chloroplast‐targeting peptide, DNA molecules are translocated across the plant cell membrane and delivered to the plastid efficiently via vesicle formation and intracellular vesicle trafficking. A simple infiltration method enables the introduction of a complex solution into intact plants, and plastid‐localized transgene expression is expeditiously observed in various types of plastids in differentiated cell types of several plants. The gene delivery technology thus provides a useful tool to rapidly engineer plastids in crop species.

Plastoglobule-Targeting Competence of a Putative Transit Peptide Sequence from Rice Phytoene Synthase 2 in Plastids.

Plastoglobules (PGs) are thylakoid membrane microdomains within plastids that are known as specialized locations of carotenogenesis. Three rice phytoene synthase proteins (OsPSYs) involved in carotenoid biosynthesis have been identified. Here, the N-terminal 80-amino-acid portion of OsPSY2 (PTp) was demonstrated to be a chloroplast-targeting peptide by displaying cytosolic localization of OsPSY2(ΔPTp):mCherry in rice protoplast, in contrast to chloroplast localization of OsPSY2:mCherry in a punctate pattern. The peptide sequence of a PTp was predicted to harbor two transmembrane domains eligible for a putative PG-targeting signal. To assess and enhance the PG-targeting ability of PTp, the original PTp DNA sequence (PTp) was modified to a synthetic DNA sequence (stPTp), which had 84.4% similarity to the original sequence. The motivation of this modification was to reduce the GC ratio from 75% to 65% and to disentangle the hairpin loop structures of PTp. These two DNA sequences were fused to the sequence of the synthetic green fluorescent protein (sGFP) and drove GFP expression with different efficiencies. In particular, the RNA and protein levels of stPTp-sGFP were slightly improved to 1.4-fold and 1.3-fold more than those of sGFP, respectively. The green fluorescent signals of their mature proteins were all observed as speckle-like patterns with slightly blurred stromal signals in chloroplasts. These discrete green speckles of PTp-sGFP and stPTp-sGFP corresponded exactly to the red fluorescent signal displayed by OsPSY2:mCherry in both etiolated and greening protoplasts and it is presumed to correspond to distinct PGs. In conclusion, we identified PTp as a transit peptide sequence facilitating preferential translocation of foreign proteins to PGs, and developed an improved PTp sequence, a stPTp, which is expected to be very useful for applications in plant biotechnologies requiring precise micro-compartmental localization in plastids.

Enantioselective microbial synthesis of the indigenous natural product (-)-α-bisabolol by a sesquiterpene synthase from chamomile (Matricaria recutita).

(-)-α-Bisabolol, a sesquiterpene alcohol, is a major ingredient in the essential oil of chamomile (Matricaria recutita) and is used in many health products. The current supply of (-)-α-bisabolol is mainly dependent on the Brazilian candeia tree (Eremanthus erythropappus) by distillation or by chemical synthesis. However, the distillation method using the candeia tree is not sustainable, and chemical synthesis suffers from impurities arising from undesirable α-bisabolol isomers. Therefore enzymatic synthesis of (-)-α-bisabolol is a viable alternative. In the present study, a cDNA encoding (-)-α-bisabolol synthase (MrBBS) was identified from chamomile and used for enantioselective (-)-α-bisabolol synthesis in yeast. Chamomile MrBBS was identified by Illumina and 454 sequencing, followed by activity screening in yeast. When MrBBS was expressed in yeast, 8mg of α-bisabolol was synthesized de novo per litre of culture. The structure of purified α-bisabolol was elucidated as (S,S)-α-bisabolol [or (-)-α-bisabolol]. Although MrBBS possesses a putative chloroplast-targeting peptide, it was localized in the cytosol, and a deletion of its N-terminal 23 amino acids significantly reduced its stability and activity. Recombinant MrBBS showed kinetic properties comparable with those of other sesquiterpene synthases. These data provide compelling evidence that chamomile MrBBS synthesizes enantiopure (-)-α-bisabolol as a single sesquiterpene product, opening a biotechnological opportunity to produce (-)-α-bisabolol.

Molecular cloning and characterization of SoNCED, a novel gene encoding 9-cis-epoxycarotenoid dioxygenase from sugarcane (Saccharum officinarum L.)

Abscisic acid (ABA) plays important roles in adaptive responses to various environmental stresses. The rate-limiting step in ABA biosynthesis is the oxidative cleavage of cis-epoxycarotenoids, which is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). In this experiment, a full-length cDNA encoding NCED gene was cloned by RT-PCR and RACE from sugarcane (Saccharum officinarum L.). The full-length of SoNCED is 2,521 bp with 1,827 bp open reading frame, encoding a peptide of 608 amino acids. The calculated molecular weight of protein was 65.9 kDa with isoelectric point of 6.04. Conserved domains prediction indicated a chloroplast-targeting peptide located at N-terminus of SoNCED. Phylogenetic tree, constructed by Neighbor-Joining method indicated that SoNCED shared high identity with the NCEDs reported from other plant species. Sequence alignment revealed that the basic secondary structure including α-helices, β-strands, β-propeller and His residues coordinating catalytic sites of SoNCED were highly conserved as in the NCEDs from other plants. Tissue specific expression analysis using quantitative real-time PCR showed a significant increase in SoNCED mRNA level and its correlation with O2 – production rate and ABA accumulation in leaves and roots of sugarcane variety GT21 when exposed to water stress. Further, the stimulation of SoNCED mRNA level, O2 – production rate and ABA content after exogenous application of ABA (100 μMol l−1) proved its involvement in pathways providing tolerance to drought stress.

Novel Shuttle Markers for Nuclear Transformation of the Green Alga Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii today is a premier model organism for the study of green algae and plants. Yet the efficient engineering of its nuclear genome requires development of new antibiotic resistance markers. We have recoded, based on codon usage in the nuclear genome, the AadA marker that has been used previously for chloroplast transformation. The recoded AadA gene, placed under the control of the HSP70A-RBCS2 hybrid promoter and preceded by the RbcS2 chloroplast-targeting peptide, can be integrated into the nuclear genome by electroporation, conferring resistance to spectinomycin and streptomycin. Transformation efficiency is markedly increased when vector sequences are completely eliminated from the transforming DNA. Antibiotic resistance is stable for several months in the absence of selection pressure. Shuttle markers allowing selection in both Chlamydomonas and Escherichia coli would also be a useful asset. By placing an artificial bacterial promoter and Shine-Dalgarno sequence in frame within the AadA coding sequence, we generated such a shuttle marker. To our surprise, we found that the classical AphVIII construct already functions as a shuttle marker. Finally, we developed a method to introduce the AadA and AphVIII markers into the vector part of the bacterial artificial chromosomes (BACs) of the Chlamydomonas genomic DNA library. Our aim was to facilitate complementation studies whenever the test gene cannot be selected for directly. After transformation of a petC mutant with a modified BAC carrying the AphVIII marker along with the PETC gene in the insert, almost half of the paromomycin-resistant transformants obtained showed restoration of phototrophy, indicating successful integration of the unselected test gene. With AadA, cotransformation was also observed, but with a lower efficiency.

Targeting and localization of wound-inducible leucine aminopeptidase A in tomato leaves

The constitutive and wound-inducible leucine aminopeptidases (LAP-N and LAP-A, respectively) of tomato encode 60-kDa proteins with 5-kDa presequences that resemble chloroplast-targeting peptides. Cell fractionation studies and immunoblot analyses of chloroplast and total proteins have suggested a dual location of the mature LAP-A proteins in the cytosol and the plastids. In this study, the subcellular localization of tomato LAPs was further investigated using in vitro chloroplast-targeting assays and immunocytochemical techniques at the light and TEM levels. In vitro-translated LAP-A1 and LAP-N preproteins were readily transported into pea chloroplasts and processed into mature proteins of 55 kDa indicating the presence of a functional chloroplast-targeting signal in the LAP-A1 and LAP-N protein precursors. In addition, a LAP polyclonal and a LAP-A-specific antisera were used to immunolocalize LAP proteins in leaves from healthy, wounded and methyl jasmonate (MeJA)-treated plants. Low levels of LAPs and/or LAP-like proteins were detected in leaves from unwounded plants. The LAP polyclonal antiserum, which detected LAP-A, LAP-N and LAP-like proteins, and the LAP-A specific antibodies, which detected only LAP-A, showed that LAP levels increased in leaf sections after wounding and MeJA treatments. LAP-A proteins were primarily detected within the chloroplasts of spongy and palisade mesophyll cells. The localization of LAP-A was distinct from the location of early wound-response proteins that are important in the biosynthesis of jasmonic acid or systemin and more similar to the late wound-response proteins with primary roles in defense. The importance of these findings relative to the potential roles of LAP-A in defense is discussed.

The role of the N-terminal domain of chloroplast targeting peptides in organellar protein import and miss-sorting

We have analysed 385 mitochondrial and 567 chloroplastic signal sequences of proteins found in the organellar proteomes of Arabidopsis thaliana. Despite overall similarities, the first 16 residues of transit peptides differ remarkably. To test the hypothesis that the N-terminally truncated transit peptides would redirect chloroplastic precursor proteins to mitochondria, we studied import of the N-terminal deletion mutants of ELIP, PetC and Lhcb2.1. The results show that the deletion mutants were neither imported into chloroplasts nor miss-targeted to mitochondria in vitro and in vivo, showing that the entire transit peptide is necessary for correct targeting as well as miss-sorting.

Catalysis, Subcellular Localization, Expression and Evolution of the Targeting Peptides Degrading Protease, AtPreP2

We have previously identified a zinc metalloprotease involved in the degradation of mitochondrial and chloroplast targeting peptides, the presequence protease (PreP). In the Arabidopsis thaliana genomic database, there are two genes that correspond to the protease, the zinc metalloprotease (AAL90904) and the putative zinc metalloprotease (AAG13049). We have named the corresponding proteins AtPreP1 and AtPreP2, respectively. AtPreP1 and AtPreP2 show significant differences in their targeting peptides and the proteins are predicted to be localized in different compartments. AtPreP1 was shown to degrade both mitochondrial and chloroplast targeting peptides and to be dual targeted to both organelles using an ambiguous targeting peptide. Here, we have overexpressed, purified and characterized proteolytic and targeting properties of AtPreP2. AtPreP2 exhibits different proteolytic subsite specificity from AtPreP1 when used for degradation of organellar targeting peptides and their mutants. Interestingly, AtPreP2 precursor protein was also found to be dual targeted to both mitochondria and chloroplasts in a single and dual in vitro import system. Furthermore, targeting peptide of the AtPreP2 dually targeted green fluorescent protein (GFP) to both mitochondria and chloroplasts in tobacco protoplasts and leaves using an in vivo transient expression system. The targeting of both AtPreP1 and AtPreP2 proteases to chloroplasts in A. thaliana in vivo was confirmed via a shotgun mass spectrometric analysis of highly purified chloroplasts. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that AtPreP1 and AtPreP2 are differentially expressed in mature A. thaliana plants. Phylogenetic evidence indicated that AtPreP1 and AtPreP2 are recent gene duplicates that may have diverged through subfunctionalization.

Molecular cloning and characterization of a dehydration-inducible cDNA encoding a putative 9-cis-epoxycarotenoid dioxygenase inArachis hygogaeaL.

A rate-limiting step in abscisic acid (ABA) biosynthesis in plants is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). Here we present the cloning, characterization of a cDNA from dehydrated peanut (Arachis hygogaea L.) leaves that encodes a putative NCED. The 2486-bp full-length cDNA (designated as AhNCED1), obtained by rapid amplification of cDNA ends (RACE), has an open reading frame of 601 amino acid residues and encodes a protein with a calculated molecular weight of 66.86 kDa and an isoelectric point of 8.39. Sequence analysis shows that the deduced amino acid sequence of AhNCED1 shares high identity with the reported NCED protein sequences. There is a 30-amino-acid chloroplast-targeting peptide at the N-terminus of the AhNCED1 protein predicted by iPSORT algorithm. Semi-quantification by duplex RT-PCR reveals that the expression of AhNCED1 is up-regulated by dehydration and that rehydration represses its expression. The organ specific expression pattern of AhNCED1 has been examined, which indicates its dominant expression in leaves and stems. Molecular analysis of the drought-inducible gene of peanut may be useful to investigate the response of agricultural crops to drought stress.

Folate synthesis in plants: the last step of the p‐aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyase

In plants, the last step in the synthesis of p-aminobenzoate (PABA) moiety of folate remains to be elucidated. In Escherichia coli, this step is catalyzed by the PabC protein, a beta-lyase that converts 4-amino-4-deoxychorismate (ADC)--the reaction product of the PabA and PabB enzymes--to PABA and pyruvate. So far, the only known plant enzyme involved in PABA synthesis is ADC synthase, which has fused domains homologous to E. coli PabA and PabB and is located in plastids. ADC synthase has no lyase activity, implying that plants have a separate ADC lyase. No such lyase is known in any eukaryote. Genomic and phylogenetic approaches identified Arabidopsis and tomato cDNAs encoding PabC homologs with putative chloroplast-targeting peptides. These cDNAs were shown to encode functional enzymes by complementation of an E. coli pabC mutant, and by demonstrating that the partially purified recombinant proteins convert ADC to PABA. Plant ADC lyase is active as dimer and is not feedback inhibited by physiologic concentrations of PABA, its glucose ester, or folates. The full-length Arabidopsis ADC lyase polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to Arabidopsis chloroplasts in transient expression experiments. These data indicate that ADC lyase, like ADC synthase, is present in plastids. As shown previously for the ADC synthase transcript, the level of ADC lyase mRNA in the pericarp of tomato fruit falls sharply as ripening advances, suggesting that the expression of these two enzymes is coregulated.

Identification of Three Previously Unknown in Vivo Protein Phosphorylation Sites in Thylakoid Membranes of Arabidopsis thaliana

The proteins in plant photosynthetic thylakoid membranes undergo light-induced phosphorylation, but only a few phosphoproteins have been characterized. To access the unknown sites of in vivo protein phosphorylation the thylakoid membranes were isolated from Arabidopsis thaliana grown in normal light, and the surface-exposed peptides were cleaved from the membranes by trypsin. The peptides were methylated and subjected to immobilized metal affinity chromatography, and the enriched phosphopeptides were sequenced using tandem nanospray quadrupole time-of-flight mass spectrometry. Three new phosphopeptides were revealed in addition to the five known phosphorylation sites in photosystem II proteins. All phosphopeptides are found phosphorylated at threonine residues implementing a strict threonine specificity of the thylakoid kinases. For the first time protein phosphorylation is found in photosystem I. The phosphorylation site is localized to the first threonine in the N terminus of PsaD protein that assists in the electron transfer from photosystem I to ferredoxin. A new phosphorylation site is also revealed in the acetylated N terminus of the minor chlorophyll a-binding protein CP29. The third novel phosphopeptide, composed of 25 amino acids, belongs to a nuclear encoded protein annotated as "expressed protein" in the Arabidopsis database. The protein precursor has a chloroplast-targeting peptide followed by the mature protein with two transmembrane helices and a molecular mass of 14 kDa. This previously uncharacterized protein is named thylakoid membrane phosphoprotein of 14 kDa (TMP14). The finding of the novel phosphoproteins extends involvement of the redox-regulated protein phosphorylation in photosynthetic membranes beyond the photosystem II and its light-harvesting antennae.

The Thylakoid ΔpH-dependent Pathway Machinery Facilitates RR-independent N-Tail Protein Integration

The thylakoidal DeltapH-dependent and bacterial twin arginine transport systems are structurally and functionally related protein export machineries. These recently discovered systems have been shown to transport folded proteins but are not known to assemble integral membrane proteins. We determined the translocation pathway of a thylakoidal FtsH homologue, plastid fusion/protein translocation factor, which is synthesized with a chloroplast-targeting peptide, a hydrophobic signal peptide, and a hydrophobic membrane anchor. The twin arginine motif in its signal peptide and its sole integration requirement of a DeltapH suggested that plastid fusion/protein translocation factor employs the DeltapH pathway. Surprisingly, changing the twin arginine to twin lysine or deleting the signal peptide did not abrogate integration capability or characteristics. Nevertheless, three criteria argue that all three forms require the DeltapH pathway for integration. First, integration was competed by an authentic DeltapH pathway precursor. Second, antibodies to DeltapH pathway component Hcf106 specifically inhibited integration. Finally, chloroplasts from the hcf106 null mutant were unable to integrate Pftf into their thylakoids. Thus, DeltapH pathway machinery facilitates both signal peptide-directed and N-tail-mediated membrane integration and does not strictly require the twin arginine motif.

Characterization of Subcellular Localization Signals Using HYPOTHESISCREATOR

Proteins are first synthesized in the cytosol and then carried to specified locations of the cell. The information which determines where the proteins are carried is believed to be encoded in the amino acid sequence. Mitochondrial targeting peptides (mTP), chloroplast targeting peptides (cTP), signal peptide, are some of the known targeting signals. Our approach differs from previous methods [5, 1, 2] which try to characterize these signals in that we try to extract a simple rule or knowledge which may be easily understood. In our experiments, we use an alphabet indexing combined with string pattern matching, similar to [6]. Concerning mitochondrial targeting peptides, we have succeeded in discovering a simple rule which seems to capture important biological knowledge about the signals.

Expression, isolation, and characterization of a chloroplast targeting peptide.

For the first time a method is described in which an N-terminal targeting peptide is isolated from Escherichia coli. After overexpression, purification, and cleavage of a fusion protein the protease-sensitive transit peptide from the chloroplast precursor protein preferredoxin could be isolated by HPLC. It was characterized by N-terminal amino acid sequencing and electrospray mass spectrometry. Its functionality was suggested by in vitro import competition experiments with isolated pea chloroplasts, in which the isolated peptide inhibited the import of radioactively labeled preferredoxin. Results from import competition experiments performed with a transit peptide deletion mutant suggested that the four extreme C-terminal amino acids lack information to interact with the chloroplast import machinery.