The signal distinguishing between targeting of outer membrane β-barrel protein to plastids and mitochondria in plants

The proteome of the outer membrane of mitochondria and chloroplasts consists of membrane proteins anchored by α-helical or β-sheet elements. While proteins with α-helical transmembrane domains are present in all cellular membranes, proteins with β-barrel structure are specific for these two membranes. The organellar β-barrel proteins are encoded in the nuclear genome and thus, have to be targeted to the outer organellar membrane where they are recognized by surface exposed translocation complexes. In the last years, the signals that ensure proper targeting of these proteins have been investigated as essential base for an understanding of the regulation of cellular protein distribution. However, the organellar β-barrel proteins are unique as most of them do not contain a typical targeting information in form of an N-terminal cleavable targeting signal. Recently, it was discovered that targeting and surface recognition of mitochondrial β-barrel proteins in yeast, humans and plants depends on the hydrophobicity of the last β-hairpin of the β-barrel. However, we demonstrate that the hydrophobicity is not sufficient for the discrimination of targeting to chloroplasts or mitochondria. By domain swapping between mitochondrial and chloroplast targeted β-barrel proteins atVDAC1 and psOEP24 we demonstrate that the presence of a hydrophilic amino acid at the C-terminus of the penultimate β-strand is required for mitochondrial targeting. A mutation of the chloroplast β-barrel protein psOEP24 which mimics such profile is efficiently targeted to mitochondria. Thus, we present the properties of the signal for mitochondrial targeting of β-barrel proteins in plants.     

How can organellar protein N-terminal sequences be dual targeting signals? In silico analysis and mutagenesis approach

Organellar nuclear-encoded proteins can be mitochondrial, chloroplastic or localized in both mitochondria and chloroplasts. Most of the determinants for organellar targeting are localized in the N-terminal part of the proteins, which were therefore analyzed in Arabidopsis thaliana. The mitochondrial, chloroplastic and dual N-terminal sequences have an overall similar composition. However, Arg is rare in the first 20 residues of chloroplastic and dual sequences, and Ala is more frequent at position 2 of these two types of sequence as compared to mitochondrial sequences. According to these observations, mutations were performed in three dual targeted proteins and analyzed by in vitro import into isolated mitochondria and chloroplasts. First, experiments performed with wild-type proteins suggest that the binding of precursor proteins to mitochondria is highly efficient, whereas the import and processing steps are more efficient in chloroplasts. Moreover, different processing sites are recognized by the mitochondrial and chloroplastic processing peptidases. Second, the mutagenesis approach shows the positive role of Arg residues for enhancing mitochondrial import or processing, as expected by the in silico analysis. By contrast, mutations at position 2 have dramatic and unpredicted effects, either enhancing or completely abolishing import. This suggests that the nature of the second amino acid residue of the N-terminal sequence is essential for the import of dual targeted sequences.     

Dual-domain, dual-targeting organellar protein presequences in Arabidopsis can use non-AUG start codons

The processes accompanying endosymbiosis have led to a complex network of interorganellar protein traffic that originates from nuclear genes encoding mitochondrial and plastid proteins. A significant proportion of nucleus-encoded organellar proteins are dual targeted, and the process by which a protein acquires the capacity for both mitochondrial and plastid targeting may involve intergenic DNA exchange coupled with the incorporation of sequences residing upstream of the gene. We evaluated targeting and sequence alignment features of two organellar DNA polymerase genes from Arabidopsis thaliana. Within one of these two loci, protein targeting appeared to be plastidic when the 5' untranslated leader region (UTR) was deleted and translation could only initiate at the annotated ATG start codon but dual targeted when the 5' UTR was included. Introduction of stop codons at various sites within the putative UTR demonstrated that this region is translated and influences protein targeting capacity. However, no ATG start codon was found within this upstream, translated region, suggesting that translation initiates at a non-ATG start. We identified a CTG codon that likely accounts for much of this initiation. Investigation of the 5' region of other nucleus-encoded organellar genes suggests that several genes may incorporate upstream sequences to influence targeting capacity. We postulate that a combination of intergenic recombination and some relaxation of constraints on translation initiation has acted in the evolution of protein targeting specificity for those proteins capable of functioning in both plastids and mitochondria.     

Dual targeting of antioxidant and metabolic enzymes to the mitochondrion and the apicoplast of Toxoplasma gondii

Toxoplasma gondii is an aerobic protozoan parasite that possesses mitochondrial antioxidant enzymes to safely dispose of oxygen radicals generated by cellular respiration and metabolism. As with most Apicomplexans, it also harbors a chloroplast-like organelle, the apicoplast, which hosts various biosynthetic pathways and requires antioxidant protection. Most apicoplast-resident proteins are encoded in the nuclear genome and are targeted to the organelle via a bipartite N-terminal targeting sequence. We show here that two antioxidant enzymes-a superoxide dismutase (TgSOD2) and a thioredoxin-dependent peroxidase (TgTPX1/2)-and an aconitase are dually targeted to both the apicoplast and the mitochondrion of T. gondii. In the case of TgSOD2, our results indicate that a single gene product is bimodally targeted due to an inconspicuous variation within the putative signal peptide of the organellar protein, which significantly alters its subcellular localization. Dual organellar targeting of proteins might occur frequently in Apicomplexans to serve important biological functions such as antioxidant protection and carbon metabolism.     

Direct Targeting of Proteins from the Cytosol to Organelles: The ER versus Endosymbiotic Organelles

In eukaryotic cells consisting of many different types of organelles, targeting of organellar proteins is one of the most fundamental cellular processes. Proteins belonging to the endoplasmic reticulum (ER), chloroplasts and mitochondria are targeted individually from the cytosol to their cognate organelles. As the targeting to these organelles occurs in the cytosol during or after translation, the most crucial aspect is how specific targeting to these three organelles can be achieved without interfering with other targeting pathways. For these organelles, multiple mechanisms are used for targeting proteins, but the exact mechanism used depends on the type of protein and organelle, the location of targeting signals in the protein and the location of the protein in the organelle. In this review, we discuss the various mechanisms involved in protein targeting to the ER, chloroplasts and mitochondria, and how the targeting specificity is determined for these organelles in plant cells .     

Protein targeting of an unusual, evolutionarily conserved adenylate kinase to a eukaryotic flagellum

The eukaryotic flagellum is a large structure into which specific constituent proteins must be targeted, transported and assembled after their synthesis in the cytoplasm. Using Trypanosoma brucei and a proteomic approach, we have identified and characterized a novel set of adenylate kinase proteins that are localized to the flagellum. These proteins represent unique isoforms that are targeted to the flagellum by an N-terminal extension to the protein and are incorporated into an extraaxonemal structure (the paraflagellar rod). We show that the N-terminal extension is both necessary for isoform location in the flagellum and sufficient for targeting of a green fluorescent protein reporter protein to the flagellum. Moreover, these N-terminal extension sequences are conserved in evolution and we find that they allow the identification of novel adenylate kinases in the genomes of humans and worms. Given the existence of specific isoforms of certain central metabolic enzymes, and targeting sequences for these isoforms, we suggest that these isoforms form part of a complex, "solid-phase" metabolic capability that is built into the eukaryotic flagellum.     

Duplication and Quadruplication of Arabidopsis thaliana Cysteinyl- and Asparaginyl-tRNA Synthetase Genes of Organellar Origin

Two cysteinyl-tRNA synthetases (CysRS) and four asparaginyl-tRNA synthetases (AsnRS) from Arabidopsis thaliana were characterized from genome sequence data, EST sequences, and RACE sequences. For one CysRS and one AsnRS, sequence alignments and prediction programs suggested the presence of an N-terminal organellar targeting peptide. Transient expression of these putative targeting sequences joined to jellyfish green fluorescent protein (GFP) demonstrated that both presequences can efficiently dual-target GFP to mitochondria and plastids. The other CysRS and AsnRSs lack targeting sequences and presumably aminoacylate cytosolic tRNAs. Phylogenetic analysis suggests that the four AsnRSs evolved by repeated duplication of a gene transferred from an ancestral plastid and that the CysRSs also arose by duplication of a transferred organelle gene (possibly mitochondrial). These case histories are the best examples to date of capture of organellar aminoacyl-tRNA synthetases by the cytosolic protein synthesis machinery. Received: 8 October 1999 / Accepted: 23 January 2000     

Protein import into hydrogenosomes of Trichomonas vaginalis involves both N-terminal and internal targeting signals: a case study of thioredoxin reductases

The parabasalian flagellate Trichomonas vaginalis harbors mitochondrion-related and H₂-producing organelles of anaerobic ATP synthesis, called hydrogenosomes, which harbor oxygen-sensitive enzymes essential to its pyruvate metabolism. In the human urogenital tract, however, T. vaginalis is regularly exposed to low oxygen concentrations and therefore must possess antioxidant systems protecting the organellar environment against the detrimental effects of molecular oxygen and reactive oxygen species. We have identified two closely related hydrogenosomal thioredoxin reductases (TrxRs), the hitherto-missing component of a thioredoxin-linked hydrogenosomal antioxidant system. One of the two hydrogenosomal TrxR isoforms, TrxRh1, carried an N-terminal extension resembling known hydrogenosomal targeting signals. Expression of hemagglutinin-tagged TrxRh1 in transfected T. vaginalis cells revealed that its N-terminal extension was necessary to import the protein into the organelles. The second hydrogenosomal TrxR isoform, TrxRh2, had no N-terminal targeting signal but was nonetheless efficiently targeted to hydrogenosomes. N-terminal presequences from hydrogenosomal proteins with known processing sites, i.e., the alpha subunit of succinyl coenzyme A synthetase (SCSα) and pyruvate:ferredoxin oxidoreductase A, were investigated for their ability to direct mature TrxRh1 to hydrogenosomes. Neither presequence directed TrxRh1 to hydrogenosomes, indicating that neither extension is, by itself, sufficient for hydrogenosomal targeting. Moreover, SCSα lacking its N-terminal extension was efficiently imported into hydrogenosomes, indicating that this extension is not required for import of this major hydrogenosomal protein. The finding that some hydrogenosomal enzymes require N-terminal signals for import but that in others the N-terminal extension is not necessary for targeting indicates the presence of additional targeting signals within the mature subunits of several hydrogenosome-localized proteins.     

The <Emphasis Type="Italic">Arabidopsis</Emphasis> calmodulin-like proteins AtCML30 and AtCML3 are targeted to mitochondria and peroxisomes,respectively

Calmodulin (CaM) is a ubiquitous sensor/transducer of calcium signals in eukaryotic organisms. While CaM mediated calcium regulation of cytosolic processes is well established, there is growing evidence for the inclusion of organelles such as chloroplasts, mitochondria and peroxisomes into the calcium/calmodulin regulation network. A number of CaM-binding proteins have been identified in these organelles and processes such as protein import into chloroplasts and mitochondria have been shown to be governed by CaM regulation. What have been missing to date are the mediators of this regulation since no CaM or calmodulin-like protein (CML) has been identified in any of these organelles. Here we show that two Arabidopsis CMLs, AtCML3 and AtCML30, are localized in peroxisomes and mitochondria, respectively. AtCML3 is targeted via an unusual C-terminal PTS1-like tripeptide while AtCML30 utilizes an N-terminal, non-cleavable transit peptide. Both proteins possess the typical structure of CaMs, with two pairs of EF-hand motifs separated by a short linker domain. They furthermore display common characteristics, such as calcium-dependent alteration of gel mobility and calcium-dependent exposure of a hydrophobic surface. This indicates that they can function in a similar manner as canonical CaMs. The presence of close homologues to AtCML3 and AtCML30 in other plants further indicates that organellar targeting of these CMLs is not a specific feature of Arabidopsis. The identification of peroxisomal and mitochondrial CMLs is an important step in the understanding how these organelles are integrated into the cellular calcium/calmodulin signaling pathways.     

Presequence Acquisition During Secondary Endocytobiosis and thePossible Role of Introns

Targeting of nucleus-encoded proteins into chloroplasts is mediated by N-terminal presequences. During evolution of plastids from formerly free-living cyanobacteria by endocytobiosis, genes for most plastid proteins have been transferred from the plastid genome to the nucleus and subsequently had to be equipped with such plastid targeting sequences. So far it is unclear how the gene domains coding for presequences and the respective mature proteins may have been assembled. While land plant plastids are supposed to originate from a primary endocytobiosis event (a prokaryotic cyanobacterium was taken up by a eukaryotic cell), organisms with secondary plastids like diatoms experienced a second endocytobiosis step involving a eukaryotic alga taken up by a eukaryotic host cell. In this group of algae, apparently most genes encoding chloroplast proteins have been transferred a second time (from the nucleus of the endosymbiont to the nucleus of the secondary host) and thus must have been equipped with additional targeting signals. We have analyzed cDNAs and the respective genomic DNA fragments of seven plastid preproteins from the diatom Phaeodactylum tricornutum. In all of these genes we found single spliceosomal introns, generally located within the region coding for the N-terminal plastid targeting sequences or shortly downstream of it. The positions of the introns can be related to the putative phylogenetic histories of the respective genes, indicating that the bipartite targeting sequences in these secondary algae might have evolved by recombination events via introns.The nucleotide sequences have been deposited at Genbank under accession numbers AY191862, AY191863, AY191864, AY191865, AY191866, AY191867, and AY191868.     

Fidelity of organellar protein targeting

The majority of cellular proteins are targeted to organelles. Cytosolic ribosomes produce these proteins as precursors with cleavable or non-cleavable targeting sequences that direct them to receptor proteins on the organelle surface. Multiple targeting factors ensure cellular sorting of the precursor proteins. In co-translational protein import, the ribosome-nascent chain complex is transported to the organellar protein translocase to couple protein synthesis and protein import. In post-translational mode, targeting factors like molecular chaperones guide the precursor proteins from ribosomes to the cell organelle. Defects in protein targeting and import cause mistargeting of proteins to different cellular compartments and challenge the balance of cellular proteostasis. Specific dislocases and degradation machineries remove such mislocalized proteins from the membrane to allow retargeting or their proteasomal turnover. In this review, we discuss targeting and quality control factors that ensure fidelity of protein targeting to mitochondria.     

Bimodal targeting of cytochrome P450s to endoplasmic reticulum and mitochondria: the concept of chimeric signals

Targeting signals are critical for proteins to find their specific cellular destination. Signals for protein targeting to the endoplasmic reticulum (ER), mitochondria, peroxisome and nucleus are distinct and the mechanisms of protein translocation across these membrane compartments also vary markedly. Recently, however, a number of proteins have been shown to be present in multiple cellular sites such as mitochondria and ER, cytosol and mitochondria, plasma membrane and mitochondria, and peroxisome and mitochondria suggesting the occurrence of multimodal targeting signals in some cases. Cytochrome P450 monooxygenases (CYPs), which play crucial roles in pharmacokinetics and pharmacodynamics of drugs and toxins, are the prototype of bimodally targeted proteins. Several members of family 1, 2 and 3 CYPs have now been reported to be associated with mitochondria and plasma membrane in addition to the ER. This review highlights the mechanisms of bimodal targeting of CYP1A1, 2B1, 2E1 and 2D6 to mitochondria and ER. The bimodal targeting of these proteins is driven by their N-terminal signals which carry essential elements of both ER targeting and mitochondria targeting signals. These multimodal signals have been termed chimeric signals appropriately to describe their dual targeting property. The cryptic mitochondrial targeting signals of CYP2B1, 2D6, 2E1 require activation by protein kinase A or protein kinase C mediated phosphorylation at sites immediately flanking the targeting signal and/or membrane anchoring regions. The cryptic mitochondria targeting signal of CYP1A1 requires activation by endoproteolytic cleavage by a cytosolic endoprotease, which exposes the mitochondrial signal. This review discusses both mechanisms of bimodal targeting and toxicological consequences of mitochondria targeted CYP proteins.     

Defining the Determinants for Dual Targeting of Amino Acyl-tRNA Synthetases to Mitochondria and Chloroplasts

Most of the organellar amino acyl-tRNA synthetases (aaRSs) are dually targeted to both mitochondria and chloroplasts using dual targeting peptides (dTPs). We have investigated the targeting properties and domain structure of dTPs of seven aaRSs by studying the in vitro and in vivo import of N-terminal deleted constructs of dTPs fused to green fluorescent protein. The deletion constructs were designed based on prediction programs, TargetP and Predotar, as well as LogoPlots derived from organellar proteomes in Arabidopsis thaliana. In vitro import was performed either into a single isolated organelle or as dual import (i.e., into a mixture of isolated mitochondria and chloroplasts followed by reisolation of the organelles). In vivo import was investigated as transient expression of the green fluorescent protein constructs in Nicotiana benthamiana protoplasts. Characterization of recognition determinants showed that the N-terminal portions of TyrRS-, ValRS- and ThrRS-dTPs (27, 22 and 23 amino acids, respectively) are required for targeting into both mitochondria and chloroplasts. Surprisingly, these N-terminal portions contain no or very few arginines (or lysines) but very high number of hydroxylated residues (26-51%). For two aaRSs, a domain structure of the dTP became evident. Removal of 20 residues from the dTP of ProRS abolished chloroplastic import, indicating that the N-terminal region was required for chloroplast targeting, whereas deletion of 16 N-terminal amino acids from AspRS-dTP inhibited the mitochondrial import, showing that in this case, the N-terminal portion was required for the mitochondrial import. Finally, deletion of N-terminal regions of dTPs for IleRS and LysRS did not affect dual targeting. In summary, it can be concluded that there is no general rule for how the determinants for dual targeting are distributed within dTPs; in most cases, the N-terminal portion is essential for import into both organelles, but in a few cases, a domain structure was observed.     

Non-canonical peroxisome targeting signals: identification of novel PTS1 tripeptides and characterization of enhancer elements by computational permutation analysis

ABSTRACT: BACKGROUND: High-accuracy prediction tools are essential in the post-genomic era to define organellar proteomes in their full complexity. We recently applied a discriminative machine learning approach to predict plant proteins carrying peroxisome targeting signals (PTS) type 1 from genome sequences. For Arabidopsis thaliana 392 gene models were predicted to be peroxisome-targeted. The predictions were extensively tested in vivo, resulting in a high experimental verification rate of Arabidopsis proteins previously not known to be peroxisomal. RESULTS: In this study, we experimentally validated the predictions in greater depth by focusing on the most challenging Arabidopsis proteins with unknown non-canonical PTS1 tripeptides and prediction scores close to the threshold. By in vivo subcellular targeting analysis, three novel PTS1 tripeptides (QRL>, SQM>, and SDL>) and two novel tripeptide residues (Q at position -3 and D at pos. -2) were identified. To understand why, among many Arabidopsis proteins carrying the same C-terminal tripeptides, these proteins were specifically predicted as peroxisomal, the residues upstream of the PTS1 tripeptide were computationally permuted and the changes in prediction scores were analyzed. The newly identified Arabidopsis proteins were found to contain four to five amino acid residues of high predicted targeting enhancing properties at position -4 to -12 in front of the non-canonical PTS1 tripeptide. The identity of the predicted targeting enhancing residues was unexpectedly diverse, comprising besides basic residues also proline, hydroxylated (Ser, Thr), hydrophobic (Ala, Val), and even acidic residues. CONCLUSIONS: Our computational and experimental analyses demonstrate that the plant PTS1 tripeptide motif is more diverse than previously thought, including an increasing number of non-canonical sequences and allowed residues. Specific targeting enhancing elements can be predicted for particular sequences of interest and are far more diverse in amino acid composition and positioning than previously assumed. Machine learning methods become indispensable to predict which specific proteins, among numerous candidate proteins carrying the same non-canonical PTS1 tripeptide, contain sufficient enhancer elements in terms of number, positioning and total strength to cause peroxisome targeting.     

Analysis of dinoflagellate mitochondrial protein sorting signals indicates a highly stable protein targeting system across eukaryotic diversity

Protein targeting into mitochondria from the cytoplasm is fundamental to the cell biology of all eukaryotes. Our understanding of this process is heavily biased towards “model” organisms, such as animals and fungi, and it is less clear how conserved this process is throughout diverse eukaryotes. In this study, we have surveyed mitochondrial protein sorting signals from a representative of the dinoflagellate algae. Dinoflagellates are a phylum belonging to the group Alveolata, which also includes apicomplexan parasites and ciliates. We generated 46 mitochondrial gene sequences from the dinoflagellate Karlodinium micrum and analysed these for mitochondrial sorting signals. Most of the sequences contain predicted N-terminal peptide extensions that conform to mitochondrial targeting peptides from animals and fungi in terms of length, amino acid composition, and propensity to form amphipathic α-helices. The remainder lack predicted mitochondrial targeting peptides and represent carrier proteins of the inner mitochondrial membrane that have internal targeting signals in model eukaryotes. We tested for functional conservation of the dinoflagellate mitochondrial sorting signals by expressing K. micrum mitochondrial proteins in the fungus Saccharomyces cerevisiae. Both the N-terminal and internal targeting signals were sufficiently conserved to operate in this distantly related system. This study indicates that the character of mitochondrial sorting signals was well established prior to the radiation of major eukaryotic lineages and has shown remarkable conservation during long periods of evolution.     

Dual targeting of the tRNA nucleotidyltransferase in plants: not just the signal

Enzymes involved in tRNA maturation are essential for cytosolic, mitochondrial, and plastid protein synthesis and are therefore localized to these different compartments of the cell. Interestingly, only one isoform of tRNA nucleotidyltransferase (responsible for adding the 3'-terminal cytidine-cytidine-adenosine to tRNAs) has been identified in plants. The present study therefore explored how signals contained on this enzyme allow it to be distributed among the different cell compartments. It is demonstrated that the N-terminal portion of the protein acts as an organellar targeting signal and that differential use of multiple in-frame start codons alters the localization of the protein. Moreover, it is shown that the mature domain has a major impact on the distribution of the protein within the cell. These data indicate that regulation of dual localization involves not only specific N-terminal signals, but also additional factors within the protein or the cell.     

Genetic toggling of alkaline phosphatase folding reveals signal peptides for all major modes of transport across the inner membrane of bacteria

Prediction of export pathway specificity in prokaryotes is a challenging endeavor due to the similar overall architecture of N-terminal signal peptides for the Sec-, SRP- (signal recognition particle), and Tat (twin arginine translocation)-dependent pathways. Thus, we sought to create a facile experimental strategy for unbiased discovery of pathway specificity conferred by N-terminal signals. Using a limited collection of Escherichia coli strains that allow protein oxidation in the cytoplasm or, conversely, disable protein oxidation in the periplasm, we were able to discriminate the specific mode of export for PhoA (alkaline phosphatase) fusions to signal peptides for all of the major modes of transport across the inner membrane (Sec, SRP, or Tat). Based on these findings, we developed a mini-Tn5 phoA approach to isolate pathway-specific export signals from libraries of random fusions between exported proteins and the phoA gene. Interestingly, we observed that reduced PhoA was exported in a Tat-independent manner when targeted for Tat export in the absence of the essential translocon component TatC. This suggests that initial docking to TatC serves as a key specificity determinant for Tat-specific routing of PhoA, and in its absence, substrates can be rerouted to the Sec pathway, provided they remain compatible with the Sec export mechanism. Finally, the utility of our approach was demonstrated by experimental verification that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signals are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen.     

Identification of novel plant peroxisomal targeting signals by a combination of machine learning methods and in vivo subcellular targeting analyses

In the postgenomic era, accurate prediction tools are essential for identification of the proteomes of cell organelles. Prediction methods have been developed for peroxisome-targeted proteins in animals and fungi but are missing specifically for plants. For development of a predictor for plant proteins carrying peroxisome targeting signals type 1 (PTS1), we assembled more than 2500 homologous plant sequences, mainly from EST databases. We applied a discriminative machine learning approach to derive two different prediction methods, both of which showed high prediction accuracy and recognized specific targeting-enhancing patterns in the regions upstream of the PTS1 tripeptides. Upon application of these methods to the Arabidopsis thaliana genome, 392 gene models were predicted to be peroxisome targeted. These predictions were extensively tested in vivo, resulting in a high experimental verification rate of Arabidopsis proteins previously not known to be peroxisomal. The prediction methods were able to correctly infer novel PTS1 tripeptides, which even included novel residues. Twenty-three newly predicted PTS1 tripeptides were experimentally confirmed, and a high variability of the plant PTS1 motif was discovered. These prediction methods will be instrumental in identifying low-abundance and stress-inducible peroxisomal proteins and defining the entire peroxisomal proteome of Arabidopsis and agronomically important crop plants.