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Targeting heterologous multi-transmembrane domain (TMD) proteins to plant chloroplasts requires sequences in addition to the chloroplast transit peptide (cTP). The N-terminal domain (N-region), located C-terminal to the cTP in chloroplast inner envelope membrane proteins, is an essential region for import. However, it was unclear if the N-region functions solely as a spacer sequence to facilitate cTP access or if it plays an active role in the import process. This study addresses the N-region’s role by using combinations of cTPs and N-regions from Arabidopsis chloroplast inner envelope membrane proteins to direct the cyanobacterial protein SbtA to the chloroplast. We find that the sequence context of the N-region affects the chloroplast import efficiency of SbtA, with particular sequences mis-targeting the protein to different cellular sub-compartments. Additionally, specific cTP and N-region pairs exhibit varying targeting efficiencies for different heterologous proteins. Substituting individual N-region motifs did not significantly alter the chloroplast targeting efficiency of a particular cTP and N-region pair. We conclude that the N-region exhibits contextual functioning and potentially functional redundancy in motifs.Key message: The N-region, a sequence stretch C-terminal to the transit peptide (cTP), is necessary for the chloroplast import of multi-transmembrane domain proteins. The N-region sequence context provided to the cTP influences the chloroplast import efficiency of a heterologous multi-TMD protein. A particular cTP and N-region pair exhibits varying import efficiencies for different cargo proteins but is not dependent on the cargo size. Substituting amino acids in a specific N-region did not significantly alter the protein import efficiency, which suggests functional redundancy in N-region motifs. [ABSTRACT FROM AUTHOR]

Plastoglobules (PGs) are plastidial lipid droplets enclosed by a polar monolayer born from the thylakoid membrane when plants require active lipid metabolism, including carotenogenesis, under the environmental stress and during plastid transition. Despite the fact that many proteins are reported to target PGs, their translocation mechanism has remained largely unexplored. To elucidate this process, we studied the influence of three hydrophobic regions (HR)--HR1 (1-45th aa), HR2 (46-80th aa), and HR3 (229-247th aa)--of rice phytoene synthase 2 (OsPSY2, 398 aa), which has previously shown to target PGs. As results, HR1 includes the crucial sequence (31-45th aa) for chloroplast import and the stromal cleavage occurs at a specific alanine site (64th aa) within HR2, verifying that a Nterminal 64-aa-region works as the transit peptide (Tp). HR2 has a weak PGtargeting signal by showing synchronous and asynchronous localization patterns in both PGs and stroma of chloroplasts. HR3 exhibited a strong PG-targeting role with the required positional specificity to prevent potential issues such as nonaccumulation, aggregation, and folding errors in proteins. Herein, we characterized a Tp and two transmembrane domains in three HRs of OsPSY2 and propose a spontaneous pathway for its PG-translocation with a shape embedded in the PG-monolayer. Given this subplastidial localization, we suggest six sophisticated tactics for plant biotechnology applications, including metabolic engineering and molecular farming. [ABSTRACT FROM AUTHOR]

The spectacular diversity of plastids in non-green organs such as flowers, fruits, roots, tubers, and senescing leaves represents a Universe of metabolic processes in higher plants that remain to be completely characterized. The endosymbiosis of the plastid and the subsequent export of the ancestral cyanobacterial genome to the nuclear genome, and adaptation of the plants to all types of environments has resulted in the emergence of diverse and a highly orchestrated metabolism across the plant kingdom that is entirely reliant on a complex protein import and translocation system. The TOC and TIC translocons, critical for importing nuclear-encoded proteins into the plastid stroma, remain poorly resolved, especially in the case of TIC. From the stroma, three core pathways (cpTat, cpSec, and cpSRP) may localize imported proteins to the thylakoid. Non-canonical routes only utilizing TOC also exist for the insertion of many inner and outer membrane proteins, or in the case of some modified proteins, a vesicular import route. Understanding this complex protein import system is further compounded by the highly heterogeneous nature of transit peptides, and the varying transit peptide specificity of plastids depending on species and the developmental and trophic stage of the plant organs. Computational tools provide an increasingly sophisticated means of predicting protein import into highly diverse non-green plastids across higher plants, which need to be validated using proteomics and metabolic approaches. The myriad plastid functions enable higher plants to interact and respond to all kinds of environments. Unraveling the diversity of non-green plastid functions across the higher plants has the potential to provide knowledge that will help in developing climate resilient crops.

The N-terminal sequence stretch that defines subcellular targeting for most nuclear encoded chloroplast proteins is usually considered identical to the sequence that is cleaved upon import. Yet here this study shows that for eight out of ten tested Chlamydomonas chloroplast transit peptides, significant additional sequence stretches past the cleavage site are required to enable efficient chloroplast import of heterologous cargo proteins. Analysis of Chlamydomonas cTPs with known cleavage sites and replacements of native post-cleavage residues with alternative sequences points to a role for unstructured sequence at mature protein N-termini. [ABSTRACT FROM AUTHOR]

The N-terminal sequence stretch that defines subcellular targeting for most nuclear encoded chloroplast proteins is usually considered identical to the sequence that is cleaved upon import. Yet here this study shows that for eight out of ten tested Chlamydomonas chloroplast transit peptides, significant additional sequence stretches past the cleavage site are required to enable efficient chloroplast import of heterologous cargo proteins. Analysis of Chlamydomonas cTPs with known cleavage sites and replacements of native post-cleavage residues with alternative sequences points to a role for unstructured sequence at mature protein N-termini.

Although the chloroplasts in plants are characterized by an inherent genome, the chloroplast proteome is composed of proteins encoded by not only the chloroplast genome but also the nuclear genome. Nuclear-encoded chloroplast proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the chloroplasts. In the latter process, an N-terminal cleavable transit peptide serves as a targeting signal required for the import of nuclear-encoded chloroplast interior proteins. This import process is mediated via an interaction between the sequence motifs in transit peptides and the components of the TOC/TIC (translocon at the outer/inner envelope of chloroplasts) translocons. Despite a considerable diversity in primary structures, several common features have been identified among transit peptides, including N-terminal moderate hydrophobicity, multiple proline residues dispersed throughout the transit peptide, preferential usage of basic residues over acidic residues, and an absence of N-terminal arginine residues. In this review, we will recapitulate and discuss recent progress in our current understanding of the functional organization of sequence elements commonly present in diverse transit peptides, which are essential for the multi-step import of chloroplast proteins. [ABSTRACT FROM AUTHOR]

Although the chloroplasts in plants are characterized by an inherent genome, the chloroplast proteome is composed of proteins encoded by not only the chloroplast genome but also the nuclear genome. Nuclear-encoded chloroplast proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the chloroplasts. In the latter process, an N-terminal cleavable transit peptide serves as a targeting signal required for the import of nuclear-encoded chloroplast interior proteins. This import process is mediated via an interaction between the sequence motifs in transit peptides and the components of the TOC/TIC (translocon at the outer/inner envelope of chloroplasts) translocons. Despite a considerable diversity in primary structures, several common features have been identified among transit peptides, including N-terminal moderate hydrophobicity, multiple proline residues dispersed throughout the transit peptide, preferential usage of basic residues over acidic residues, and an absence of N-terminal arginine residues. In this review, we will recapitulate and discuss recent progress in our current understanding of the functional organization of sequence elements commonly present in diverse transit peptides, which are essential for the multi-step import of chloroplast proteins.

Summary: Organellogenesis, a key aspect of eukaryotic cell evolution, critically depends on the successful establishment of organellar protein import mechanisms. Phylogenetic analysis revealed that the evolution of the two endosymbiotic organelles, the mitochondrion and the chloroplast, is thought to have occurred at time periods far from each other. Despite this, chloroplasts and mitochondria have highly similar protein import mechanisms. This raises intriguing questions such as what underlies such similarity in the import mechanisms and how these similar mechanisms have evolved. In this review, we summarise the recent findings regarding sorting and specific targeting of these organellar proteins. Based on these findings, we propose possible evolutionary scenarios regarding how the signal sequences of chloroplasts and mitochondrial proteins ended up having such relationship. [ABSTRACT FROM AUTHOR]

Nucleus-encoded plastid proteins are synthesized as precursors with N-terminal targeting signals called transit peptides (TPs), which mediate interactions with the translocon complexes at the outer (TOC) and inner (TIC) plastid membranes. These complexes exist in multiple isoforms in higher plants and show differential specificity and tissue abundance. While some show specificity for photosynthesis-related precursor proteins, others distinctly recognize nonphotosynthetic and housekeeping precursor proteins. Here we used TPs from four Arabidopsis thaliana proteins, three related to photosynthesis (chlorophyll a/b binding protein, Rubisco activase) and photo-protection (tocopherol cyclase) and one involved in the assimilation of ammonium into amino-acids, and whose expression is most abundant in the root (ferredoxin dependent glutamate synthase 2), to determine whether they were able to mediate import of a nuclear-encoded marker protein into plastids of different tissues of a dicot and a monocot species. In A. thaliana, import and processing efficiency was high in all cases, while TP from the rice Rubisco small chain 1, drove very low import in Arabidopsis tissues. Noteworthy, our results show that Arabidopsis photosynthesis TPs also mediate plastid import in rice callus, and in leaf and root tissues with almost a 100% efficiency, providing new biotechnological tools for crop improvement strategies based on recombinant protein accumulation in plastids by the expression of nuclear-encoded transgenes.

Subcellular relocalization of proteins determines an organism’s metabolic repertoire and thereby its survival in unique evolutionary niches. In plants, the plastid and its various morphotypes import a large and varied number of nuclear-encoded proteins to orchestrate vital biochemical reactions in a spatiotemporal context. Recent comparative genomics analysis and high-throughput shotgun proteomics data indicate that there are a large number of plastid-targeted proteins that are either semi-conserved or non-conserved across different lineages. This implies that homologs are differentially targeted across different species, which is feasible only if proteins have gained or lost plastid targeting peptides during evolution. In this study, a broad, multi-genome analysis of 15 phylogenetically diverse genera and in-depth analyses of pangenomes from Arabidopsis and Brachypodium were performed to address the question of how proteins acquire or lose plastid targeting peptides. The analysis revealed that random insertions or deletions were the dominant mechanism by which novel transit peptides are gained by proteins. While gene duplication was not a strict requirement for the acquisition of novel subcellular targeting, 40% of novel plastid-targeted genes were found to be most closely related to a sequence within the same genome, and of these, 30.5% resulted from alternative transcription or translation initiation sites. Interestingly, analysis of the distribution of amino acids in the transit peptides of known and predicted chloroplast-targeted proteins revealed monocot and eudicot-specific preferences in residue distribution.

Plants have two endosymbiotic organelles, chloroplast and mitochondrion. Although they have their own genomes, proteome assembly in these organelles depends on the import of proteins encoded by the nuclear genome. Previously, we elucidated the general design principles of chloroplast and mitochondrial targeting signals, transit peptide, and presequence, respectively, which are highly diverse in primary structure. Both targeting signals are composed of N-terminal specificity domain and C-terminal translocation domain. Especially, the N-terminal specificity domain of mitochondrial presequences contains multiple arginine residues and hydrophobic sequence motif. In this study we investigated whether the design principles of plant mitochondrial presequences can be applied to those in other eukaryotic species. We provide evidence that both presequences and import mechanisms are remarkably conserved throughout the species. In addition, we present evidence that the N-terminal specificity domain of presequence might have evolved from the bacterial TAT (twin-arginine translocation) signal sequence.

Nucleus-encoded plastid proteins are synthesized as precursors with N-terminal targeting signals called transit peptides (TPs), which mediate interactions with the translocon complexes at the outer (TOC) and inner (TIC) plastid membranes. These complexes exist in multiple isoforms in higher plants and show differential specificity and tissue abundance. While some show specificity for photosynthesis-related precursor proteins, others distinctly recognize nonphotosynthetic and housekeeping precursor proteins. Here we used TPs from four Arabidopsis thaliana proteins, three related to photosynthesis (chlorophyll a/b binding protein, Rubisco activase) and photo-protection (tocopherol cyclase) and one involved in the assimilation of ammonium into amino-acids, and whose expression is most abundant in the root (ferredoxin dependent glutamate synthase 2), to determine whether they were able to mediate import of a nuclear-encoded marker protein into plastids of different tissues of a dicot and a monocot species. In A. thaliana , import and processing efficiency was high in all cases, while TP from the rice Rubisco small chain 1, drove very low import in Arabidopsis tissues. Noteworthy, our results show that Arabidopsis photosynthesis TPs also mediate plastid import in rice callus, and in leaf and root tissues with almost a 100% efficiency, providing new biotechnological tools for crop improvement strategies based on recombinant protein accumulation in plastids by the expression of nuclear-encoded transgenes. [ABSTRACT FROM AUTHOR]

Gene losses in plastid genomes (plastomes) are often accompanied by functional transfer to the nucleus or substitution of an alternative nuclear-encoded gene. Despite the highly conserved gene content in plastomes of photosynthetic land plants, recent gene loss events have been documented in several disparate angiosperm clades. Among these lineages, Passiflora lacks several essential ribosomal genes, rps7 , rps16 , rpl20 , rpl22 , and rpl32 , the two largest plastid genes, ycf1 and ycf2 , and has a highly divergent rpoA. Comparative transcriptome analyses were performed to determine the fate of the missing genes in Passiflora. Putative functional transfers of rps7 , rpl22 , and rpl32 to nucleus were detected, with the nuclear transfer of rps7 , representing a novel event in angiosperms. Plastid-encoded rps7 was transferred into the intron of a nuclear-encoded plastid-targeted thioredoxin m-type gene, acquiring its plastid transit peptide (TP). Plastid rpl20 likely experienced a novel substitution by a duplicated, nuclear-encoded mitochondrial-targeted rpl20 that has a similar gene structure. Additionally, among rosids, evidence for a third independent transfer of rpl22 in Passiflora was detected that gained a TP from a nuclear gene containing an organelle RNA recognition motif. Nuclear transcripts representing rpoA , ycf1 , and ycf2 were not detected. Further analyses suggest that the divergent rpoA remains functional and that the gene is under positive or purifying selection in different clades. Comparative analyses indicate that alternative translocon and motor protein complexes may have substituted for the loss of ycf1 and ycf2 in Passiflora. [ABSTRACT FROM AUTHOR]

Subcellular relocalization of proteins determines an organism's metabolic repertoire and thereby its survival in unique evolutionary niches. In plants, the plastid and its various morphotypes import a large and varied number of nuclear-encoded proteins to orchestrate vital biochemical reactions in a spatiotemporal context. Recent comparative genomics analysis and high-throughput shotgun proteomics data indicate that there are a large number of plastid-targeted proteins that are either semi-conserved or non-conserved across different lineages. This implies that homologs are differentially targeted across different species, which is feasible only if proteins have gained or lost plastid targeting peptides during evolution. In this study, a broad, multi-genome analysis of 15 phylogenetically diverse genera and in-depth analyses of pangenomes from Arabidopsis and Brachypodium were performed to address the question of how proteins acquire or lose plastid targeting peptides. The analysis revealed that random insertions or deletions were the dominant mechanism by which novel transit peptides are gained by proteins. While gene duplication was not a strict requirement for the acquisition of novel subcellular targeting, 40% of novel plastid-targeted genes were found to be most closely related to a sequence within the same genome, and of these, 30.5% resulted from alternative transcription or translation initiation sites. Interestingly, analysis of the distribution of amino acids in the transit peptides of known and predicted chloroplast-targeted proteins revealed monocot and eudicot-specific preferences in residue distribution. [ABSTRACT FROM AUTHOR]

Summary: I had the fortune to start my scientific carrier during the early stages of the development of plant transformation in one of the leading laboratories in the field. Here, I describe my personal experience in the laboratory of Marc van Montagu and Jeff Schell, and some important contributions that the group made to the development of the technology to produce transgenic plants. I also briefly summarize the impact of this technology on the development of modern plant biology and in plant molecular improvement. [ABSTRACT FROM AUTHOR]

Plants have two endosymbiotic organelles, chloroplast and mitochondrion. Although they have their own genomes, proteome assembly in these organelles depends on the import of proteins encoded by the nuclear genome. Previously, we elucidated the general design principles of chloroplast and mitochondrial targeting signals, transit peptide, and presequence, respectively, which are highly diverse in primary structure. Both targeting signals are composed of N-terminal specificity domain and C-terminal translocation domain. Especially, the N-terminal specificity domain of mitochondrial presequences contains multiple arginine residues and hydrophobic sequence motif. In this study we investigated whether the design principles of plant mitochondrial presequences can be applied to those in other eukaryotic species. We provide evidence that both presequences and import mechanisms are remarkably conserved throughout the species. In addition, we present evidence that the N-terminal specificity domain of presequence might have evolved from the bacterial TAT (twin-arginine translocation) signal sequence. [ABSTRACT FROM AUTHOR]

The evolution of chloroplasts from the original endosymbiont involved the transfer of thousands of genes from the ancestral bacterial genome to the host nucleus, thereby combining the two genetic systems to facilitate coordination of gene expression and achieve integration of host and organelle functions. A key element of successful endosymbiosis was the evolution of a unique protein import system to selectively and efficiently target nuclear-encoded proteins to their site of function within the chloroplast after synthesis in the cytoplasm. The chloroplast TOC–TIC (translocon at the outer chloroplast envelope–translocon at the inner chloroplast envelope) general protein import system is conserved across the plant kingdom, and is a system of hybrid origin, with core membrane transport components adapted from bacterial protein targeting systems, and additional components adapted from host genes to confer the specificity and directionality of import. In vascular plants, the TOC–TIC system has diversified to mediate the import of specific, functionally related classes of plastid proteins. This functional diversification occurred as the plastid family expanded to fulfill cell- and tissue-specific functions in terrestrial plants. In addition, there is growing evidence that direct regulation of TOC–TIC activities plays an essential role in the dynamic remodeling of the organelle proteome that is required to coordinate plastid biogenesis with developmental and physiological events. [ABSTRACT FROM AUTHOR]

Preprotein import into chloroplasts is mediated by the coordinated actions of translocons at the outer and inner envelopes of chloroplasts (Toc and Tic, respectively). The cleavable N-terminal transit peptide (TP) of preproteins plays an essential role in the import of preproteins into chloroplasts. The Tic40 protein, a component of the Tic complex, is believed to mediate the import of preproteins through the inner envelope. In this study, we aimed to obtain in vivo evidence supporting the role of Tic40 in preprotein import into chloroplasts. Contrary to previous findings, the import of various preproteins with wild-type TPs showed no difference between tic40 and wild-type protoplasts of Arabidopsis thaliana. However, the import of N-terminal mutants of the RbcS protein (RbcS-nt), in which basic amino acid residues (arginine and lysine) in the central region of the TP were substituted with neutral (alanine) or acidic (glutamic acid) amino acid residues, was dependent on Tic40. In addition, in tic40 protoplasts, the inner envelope protein Tic40 tagged with HA (hemagglutinin) showed more intermediate form present in the stroma. Based on these results, we propose that protein can be imported into chloroplast by either Tic40-independent or Tic40-dependent pathways depending on the types of TP. • Transit peptides determine the dependence on Tic40 for chloroplast protein import. • Basic residues in RbcS transit peptide are critical for Tic40-independent import. • Tic40 mediates the reinsertion of inner envelope proteins from the stroma. [ABSTRACT FROM AUTHOR]

Secretory proteins are of particular importance to apicomplexan parasites and comprise over 15% of the genomes of the human pathogens that cause diseases like malaria, toxoplasmosis and babesiosis as well as other diseases of agricultural significance. Here, we developed an approach that allows us to control the trafficking destination of secretory proteins in the human malaria parasite Plasmodium falciparum. Based on the unique structural requirements of apicoplast transit peptides, we designed three conditional localization domains (CLD1, 2 and 3) that can be used to control protein trafficking via the addition of a cell permeant ligand. Studies comparing the trafficking dynamics of each CLD show that CLD2 has the most optimal trafficking efficiency. To validate this system, we tested whether CLD2 could conditionally localize a biotin ligase called holocarboxylase synthetase 1 (HCS1) without interfering with the function of the enzyme. In a parasite line expressing CLD2‐HCS1, we were able to control protein biotinylation in the apicoplast in a ligand‐dependent manner, demonstrating the full functionality of the CLD tool. We have developed and validated a novel molecular tool that may be used in future studies to help elucidate the function of secretory proteins in malaria parasites. [ABSTRACT FROM AUTHOR]

Summary: The mint family (Lamiaceae) is well documented as a rich source of terpene natural products. More than 200 diterpene skeletons have been reported from mints, but biosynthetic pathways are known for just a few of these.We crossreferenced chemotaxonomic data with publicly available transcriptomes to select common selfheal (Prunella vulgaris) and its highly unusual vulgarisin diterpenoids as a case study for exploring the origins of diterpene skeletal diversity in Lamiaceae. Four terpene synthases (TPS) from the TPS‐a subfamily, including two localised to the plastid, were cloned and functionally characterised. Previous examples of TPS‐a enzymes from Lamiaceae were cytosolic and reported to act on the 15‐carbon farnesyl diphosphate. Plastidial TPS‐a enzymes using the 20‐carbon geranylgeranyl diphosphate are known from other plant families, having apparently arisen independently in each family.All four new enzymes were found to be active on multiple prenyl‐diphosphate substrates with different chain lengths and stereochemistries. One of the new enzymes catalysed the cyclisation of geranylgeranyl diphosphate into 11‐hydroxy vulgarisane, the likely biosynthetic precursor of the vulgarisins.We uncovered the pathway to a rare diterpene skeleton. Our results support an emerging paradigm of substrate and compartment switching as important aspects of TPS evolution and diversification. [ABSTRACT FROM AUTHOR]

Plants possess both types of endosymbiotic organelles, chloroplasts and mitochondria. Transit peptides and presequences function as signal sequences for specific import into chloroplasts and mitochondria, respectively. However, how these highly similar signal sequences confer the protein import specificity remains elusive. Here, we show that mitochondrial- or chloroplast-specific import involves two distinct steps, specificity determination and translocation across envelopes, which are mediated by the N-terminal regions and functionally interchangeable C-terminal regions, respectively, of transit peptides and presequences. A domain harboring multiple-arginine and hydrophobic sequence motifs in the N-terminal regions of presequences was identified as the mitochondrial specificity factor. The presence of this domain and the absence of arginine residues in the N-terminal regions of otherwise common targeting signals confers specificity of protein import into mitochondria and chloroplasts, respectively. AtToc159, a chloroplast import receptor, also contributes to determining chloroplast import specificity. We propose that common ancestral sequences were functionalized into mitochondrial- and chloroplast-specific signal sequences by the presence and absence, respectively, of multiple-arginine and hydrophobic sequence motifs in the N-terminal region. Presequences and transit peptides mediate specific protein import into mitochondria and chloroplasts, respectively. The specific protein import into these two organelles involves two distinct steps, specificity determination and translocation across envelopes, which are mediated by the N-terminal regions and functionally interchangeable C-terminal regions, respectively, of transit peptides and presequences. [ABSTRACT FROM AUTHOR]

More than 2500 nuclear encoded preproteins are required for the function of chloroplasts in terrestrial plants. These preproteins are imported into chloroplasts via the concerted action of two multi-subunit translocons of the outer (TOC) and inner (TIC) membranes of the chloroplast envelope. This general import machinery functions to recognize and import proteins with high fidelity and efficiency to ensure that organelle biogenesis is properly coordinated with developmental and physiological events. Two components of the TOC machinery, Toc34 and Toc159, act as the primary receptors for preproteins at the chloroplast surface. They interact with the intrinsic targeting signals (transit peptides) of preproteins to mediate the selectivity of targeting, and they contribute to the quality control of import by constituting a GTP-dependent checkpoint in the import reaction. The TOC receptor family has expanded to regulate the import of distinct classes of preproteins that are required for remodeling of organelle proteomes during plastid-type transitions that accompany developmental changes. As such, the TOC receptors function as central regulators of the fidelity, specificity and selectivity of the general import machinery, thereby contributing to the integration of protein import with plastid biogenesis. [ABSTRACT FROM AUTHOR]

The plastid proteome changes according to developmental stages. Accruing evidence shows that, in addition to transcriptional and translational controls, preprotein import into plastids is also part of the process regulating plastid proteomes. Different preproteins have distinct preferences for plastids of different tissues. Preproteins are also divided into at least three age-selective groups based on their import preference for chloroplasts of different ages. Both tissue and age selectivity are determined by the transit peptide of each preprotein, and a transit-peptide motif for older-chloroplast preference has been identified. Future challenges lie in identifying other motifs for tissue and age selectivity, as well as in identifying the receptor components that decipher these motifs. Developmental regulation also suggests that caution should be exercised when comparing protein import data generated with plastids isolated from different tissues or with chloroplasts isolated from plants of different ages. [ABSTRACT FROM AUTHOR]

Protoplasts have been a useful unicellular system for various molecular biological analyses based on transient expression and single cell analysis using fluorescence-activated cell sorting (FACS), widely used as a powerful method in functional genomics. Despite the versatility of these methods, some limits based on low fluorescence intensity of a flow cytometric analysis (FCA) using protoplasts have been reported. In this study, the chloroplast targeting of fluorescent proteins (FPs) led to an eight-fold increase in fluorescence intensity and a 4.5-fold increase of transfection ratio from 14.7% to 65.7% as compared with their targeting into the cytoplasm. Moreover, the plot data of FCA shows that 83.3% of the K-sGFP population is under the threshold level, regarded as a non-transgenic population with background signals, while 65.7% of the K-sGFP population is spread on overall intervals. To investigate the reason underlying this finding, mRNA/protein levels and transfection efficiency were analyzed, and results suggest that mRNA/protein levels and transfection ratio are not much different between K-sGFP and KR-sGFP. From those results, we hypothesized that the difference of fluorescence intensity is not only derived from cellular events such as molecular level or transfection efficiency. Taken together, we suggest that the translocation of FPs into chloroplasts contributes to the improvement of fluorescence intensity in FCA and, apparently, plays an important role in minimizing the loss of the transfected population. Our study could be usefully applicable for highly sensitive FACS and FCA-investigations of green tissue.

Abstract The stromal processing peptidase (SPP) removes transit peptides as precursor proteins enter the chloroplast and different plastid types. SPP is synthesized as a latent, inactive precursor (preSPP) with an atypically long transit peptide. Determinants in the pea ( Pisum sativum ) SPP transit peptide that regulate mature SPP activation were investigated. Mutational and chemical analyses with protein modifying agents (NEM and APMA) showed a conserved transit peptide Cys-X-Ser/Thr-Cys motif did not inhibit SPP via a “cysteine switch” mechanism through His-X-X-Glu-His site interactions, although cysteines in mature SPP contribute to an active conformation. Significantly, a transit peptide deletion of only the N-terminal 28 amino acids activates SPP located downstream. Short deletions within this region suggest removal of the initial Met plays a pivotal, mechanistic role. Other deletions of ∼30 amino acids along the length of the transit peptide do not individually trigger activity, but larger deletions including Met have an additive effect indicating its removal may be a critical early step during preSPP import. Interestingly, the active preSPP deletion mutants no longer possess predicted Hsp70 binding sites including initial Met, thus Hsp70 interactions may restrict SPP from attaining an active conformation. Highlights • Inactivation of SPP is not through a “cysteine switch” mechanism. • Thiol-modifying reagents do not stimulate preSPP activity but diminish mature SPP activity. • NEM disrupts both substrate binding and peptide bond cleavage by SPP. • Deletion of N terminus of preSPP transit peptide including initial Met triggers processing activity. • The active preSPP deletion mutants no longer possess predicted Hsp70 binding sites. [ABSTRACT FROM AUTHOR]

Chloroplasts are present in organisms belonging to the kingdom Plantae. These organelles are thought to have originated from photosynthetic cyanobacteria through endosymbiosis. During endosymbiosis, most cyanobacterial genes were transferred to the host nucleus. Therefore, most chloroplast proteins became encoded in the nuclear genome and must return to the chloroplast after translation. The N-terminal cleavable transit peptide (TP) is necessary and sufficient for the import of nucleus-encoded interior chloroplast proteins. Over the past decade, extensive research on the TP has revealed many important characteristic features of TPs. These studies have also shed light on the question of how the many diverse TPs could have evolved to target specific proteins to the chloroplast. In this review, we summarize the characteristic features of TPs. We also highlight recent advances in our understanding of TP evolution and provide future perspectives about this important research area. [ABSTRACT FROM AUTHOR]