Multiplicity of different cell- and organ-specific import routes for the NADPH-protochlorophyllide oxidoreductases A and B in plastids of Arabidopsis seedlings

The NADPH-dependent protochlorophyllide (Pchlide) oxidoreductase (POR) is a photoenzyme that requires light for its catalytic activity and uses Pchlide itself as a photoreceptor. In Arabidopsis there are three PORs denoted PORA, PORB and PORC. The PORA and PORB genes are strongly expressed early in seedling development. In contrast to PORB the import of PORA into plastids of cotyledons is substrate-dependent and organ-specific. These differences in the import reactions between PORA and PORB most likely are due to different import mechanisms that are responsible for the uptake of these proteins. The two major core constituents of the translocon of the outer plastid envelope, Toc159 and Toc34, have been implicated in the binding and recognition of precursors of nuclear-encoded plastid proteins. Their involvement in conferring substrate dependency and organ specificity of PORA import was analyzed in intact Arabidopsis seedlings of wild type and the three mutants ppi3, ppi1 and ppi2 that are deficient in atToc34, atToc33, a closely related isoform of atToc34, and atToc159. Whereas none of these three Toc constituents is required for maintaining the organ specificity and substrate dependency of PORA import, atToc33 is indispensable for the import of PORB in cotyledons and true leaves suggesting that in these parts of the plant translocation of PORA and PORB occurs via two distinct import pathways. The analysis of PORA and PORB import into plastids of intact seedlings revealed an unexpected multiplicity of import routes that differed by their substrate, cell, tissue and organ specificities. This versatility of pathways for protein targeting to plastids suggests that in intact seedlings not only the constituents of the core complex of import channels but also other factors are involved in mediating the import of nuclear-encoded plastid proteins.     

Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts

The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.     

AtToc90, a New GTP-Binding Component of the Arabidopsis Chloroplast Protein Import Machinery

AtToc159 is a GTP-binding chloroplast protein import receptor. In vivo, atToc159 is required for massive accumulation of photosynthetic proteins during chloroplast biogenesis. Yet, in mutants lacking atToc159 photosynthetic proteins still accumulate, but at strongly reduced levels whereas non-photosynthetic proteins are imported normally: This suggests a role for the homologues of atToc159 (atToc132, -120 and -90). Here, we show that atToc90 supports accumulation of photosynthetic proteins in plastids, but is not required for import of several constitutive proteins. Part of atToc90 associates with the chloroplast surface in vivo and with the Toc-complex core components (atToc75 and atToc33) in vitro suggesting a function in chloroplast protein import similar to that of atToc159. As both proteins specifically contribute to the accumulation of photosynthetic proteins in chloroplasts they may be components of the same import pathway.     

Methotrexate does not block import of a DHFR fusion protein into chloroplasts

Protein import into chloroplasts requires the movement of a precursor protein across the envelope membranes. The conformation of a precursor as it passes from the aqueous medium across the hydrophobic membranes is not known in detail. To address this problem we examined precursor conformation during translocation using the chimeric precursor PCDHFR, which contains the plastocyanin (PC) transit peptide in front of mouse cytosolic dihydrofolate reductase (DHFR). The chimeric protein is targeted to chloroplasts and is competent for import. The conformation of PCDHFR can be stabilized by complexing with methotrexate, an analogue of the substrate of DHFR. Methotrexate strongly inhibits DHFR import into yeast mitochondria (M. Eilers and G. Schatz, Nature 322 (1986) 228–232), presumably because the precursor must unfold to cross the membrane and it cannot do so when complexed with methotrexate. We show here that methotrexate does not block PCDHFR import into chloroplasts. Methotrexate does slow the rate of import, and protects DHFR from degradation once inside chloroplasts. The processed protein is localized in the stroma, indicating that import into thylakoids is impeded. Protease sensitivity assays indicate that the complex of precursor protein with methotrexate changes in conformation during the translocation across the envelope.     

An outer envelope membrane component of the plastid protein import apparatus plays an essential role in Arabidopsis

T ranslocon at the o uter envelope membrane of c hloroplasts, 34  kDa (Toc34) is a GTP-binding component of the protein import apparatus within the outer envelope membrane of plastids. The Arabidopsis genome encodes two homologues of Toc34, designated atToc33 and atToc34. In this report, we describe the identification and characterization of two atToc34 knockout mutants, plastid protein import 3-1 ( ppi3-1 ) and ppi3-2 . Aerial tissues of the ppi3 mutants appeared similar to the wild type throughout development, and contained structurally normal chloroplasts that were able to efficiently import the Rubisco small subunit precursor (prSS) in vitro . The absence of an obvious ppi3 phenotype in green tissues presumably reflects the ability of atToc33 to substitute for atToc34 in the mutant, and the relatively high level of expression of the atTOC33 gene in these tissues. In the roots, where atTOC33 is expressed at a much lower level, significant growth defects were observed in both mutants: ppi3 roots were approximately 20–30% shorter than wild-type roots. Attempts to identify a double homozygote lacking atToc34 and atToc33 (by crossing the ppi3 mutants with ppi1 , an atToc33 knockout mutant) were unsuccessful, indicating that the function provided by atToc33/atToc34 is essential during early development. Plants that were homozygous for ppi1 and heterozygous for ppi3 displayed a chlorotic phenotype much more severe than that of the ppi1 single mutant. Furthermore, the siliques of these plants contained approximately 25% aborted seeds, indicating that the double homozygous mutation is embryo lethal. The data demonstrate that atToc33/atToc34 performs a central and essential role during plastid protein import, and indicate that the atToc34 isoform is relatively more important for plastid biogenesis in roots.     

Calcium regulation of chloroplast protein import

The majority of chloroplast proteins is nuclear-encoded and therefore synthesized on cytosolic ribosomes. In order to enter the chloroplast, these proteins have to cross the double-membrane surrounding the organelle. This is achieved by means of two hetero-oligomeric protein complexes in the outer and inner envelope, the Toc and Tic translocon. The process of chloroplast import is highly regulated on both sides of the envelope membranes. Our studies indicate the existence of an undescribed mode of control for this process so far, at the same time providing further evidence that the chloroplast is integrated into the calcium-signalling network of the cell. In pea chloroplasts, the calmodulin inhibitor Ophiobolin A as well as the calcium ionophores A23187 and Ionomycin affect the translocation of those chloroplast proteins that are imported with an N-terminal cleavable presequence. Import of these proteins is inhibited in a concentration-dependent manner. Addition of external calmodulin or calcium can counter the effect of these inhibitors. Translocation of chloroplast proteins that do not possess a cleavable transit peptide, that is outer envelope proteins or the inner envelope protein Tic32, is not affected. These results suggest that the import of a certain subset of chloroplast proteins is regulated by calcium. Our studies furthermore indicate that this regulation occurs downstream of the Toc translocon either within the intermembrane space or at the inner envelope translocon. A potential promoter of the calcium regulation is calmodulin, a protein well known as part of the plant's calcium signalling system.     

Nucleotide binding and dimerization at the chloroplast pre‐protein import receptor,atToc33, are not essential in vivo but do increase import efficiency

The atToc33 protein is one of several pre‐protein import receptors in the outer envelope of Arabidopsis chloroplasts. It is a GTPase with motifs characteristic of such proteins, and its loss in the plastid protein import 1 (ppi1) mutant interferes with the import of photosynthesis‐related pre‐proteins, causing a chlorotic phenotype in mutant plants. To assess the significance of GTPase cycling by atToc33, we generated several atToc33 point mutants with predicted effects on GTP binding (K49R, S50N and S50N/S51N), GTP hydrolysis (G45R, G45V, Q68A and N101A), both binding and hydrolysis (G45R/K49N/S50R), and dimerization or the functional interaction between dimeric partners (R125A, R130A and R130K). First, a selection of these mutants was assessed in vitro, or in yeast, to confirm that the mutations have the desired effects: in relation to nucleotide binding and dimerization, the mutants behaved as expected. Then, activities of selected mutants were tested in vivo, by assessing for complementation of ppi1 in transgenic plants. Remarkably, all tested mutants mediated high levels of complementation: complemented plants were similar to the wild type in growth rate, chlorophyll accumulation, photosynthetic performance, and chloroplast ultrastructure. Protein import into mutant chloroplasts was also complemented to >50% of the wild‐type level. Overall, the data indicate that neither nucleotide binding nor dimerization at atToc33 is essential for chloroplast import (in plants that continue to express the other TOC receptors in native form), although both processes do increase import efficiency. Absence of atToc33 GTPase activity might somehow be compensated for by that of the Toc159 receptors. However, overexpression of atToc33 (or its close relative, atToc34) in Toc159‐deficient plants did not mediate complementation, indicating that the receptors do not share functional redundancy in the conventional sense.     

Further in vivo studies on the role of the molecular chaperone, Hsp93, in plastid protein import

In Arabidopsis, Hsp93 is encoded by two genes, atHSP93-V and atHSP93-III. We identified two T-DNA mutants for atHSP93-III: one being a partial 'knockdown' (hsp93-III-1) and the other a complete 'knockout' (hsp93-III-2). Homozygotes for both mutants were indistinguishable from wild type. We crossed each mutant to an atHSP93-V knockout, and identified double mutants with strongly chlorotic phenotypes. This implied redundancy, which was confirmed by the complementation of mildly chlorotic hsp93-V plants by atHSP93-III over-expression. While the hsp93-V hsp93-III-1 mutant was doubly homozygous, the second double mutant was heterozygous for hsp93-III-2 (genotype: hsp93-V/hsp93-V; +/hsp93-III-2). Attempts to identify an hsp93-V hsp93-III-2 double homozygote were unsuccessful, indicating that the Hsp93 pool is essential for viability. Consistently, siliques of the second double mutant contained aborted seeds (because of a block in the zygote-embryo transition) and failed ovules (because of a moderate defect in female gametophytes). Double-mutant plants were chlorophyll-deficient, contained under-developed chloroplasts, and exhibited stunted growth. In import assays using a chimeric pre-protein (plastocyanin transit peptide fused to dihydrofolate reductase; PC-DHFR), a clear defect was observed in hsp93-V hsp93-III-1 chloroplasts. Interestingly, while denaturation or stabilization of the DHFR moiety had a strong effect on import efficiency in the wild type, no such effects were observed with double-mutant (or tic40) chloroplasts. This indicated that pre-protein unfolding is not rate-limiting for import into mutant chloroplasts, and suggested that (unlike the situation in mitochondria) the inner membrane import machinery does not contribute to pre-protein unfolding at the organellar surface.     

Chromoplast development in ripening tomato fruit: identification of cDNAs for chromoplast-targeted proteins and characterization of a cDNA encoding a plastid-localized low-molecular-weight heat shock protein

During tomato fruit ripening, photosynthetically competent thylakoid membranes are broken down and replaced by membranous deposits of carotenoids. Few of the proteins involved in this transition have been identified. We have used chloroplast protein import assays as a means to identify two cDNAs that encode proteins destined for the developing chromoplast. One of the cDNAs had unexpected properties and its biological function has not been determined. However, the other cDNA encodes a plastid-localized low-MW heat shock protein (hsp). The steady-state level of RNA corresponding to this cDNA increased several-fold during tomato ripening, and the amount of RNA induced by heat stress increased dramatically during this process. These observations suggest a new role for this stress protein in protecting the plastid during the dismantling of the thylakoid membranes or during the buildup of carotenoids.     

Diatom plastids: Secondary endocytobiosis, plastid genome and protein import

Plastids of diatoms and other chromophytic algae have four surrounding membranes. In contrast to plastids of green algae, higher plants and red algae chromophytic cells are thought to have evolved by secondary endocytobiosis, i.e. by uptake of a eukaryotic photosynthetic organism by a eukaryotic host cell. This review gives a brief summary of the current views about the origin of diatom plastids and discusses possible mechanisms the cells might employ to transport nucleus-encoded plastid proteins into these organelles.     

Structural diversity of signal recognition particle RNAs in plastids

One of the pathways for protein targeting to the plasma membrane in bacteria utilizes the co-translationally acting signal recognition particle (SRP), a universally conserved ribonucleoprotein complex consisting of a 54 kDa protein and a functional RNA. An interesting exception is the higher plant chloroplast SRP, which lacks the otherwise essential RNA component. Furthermore, green plant chloroplasts have an additional post-translational SRP-dependent transport system in which the chloroplast-specific cpSRP43 protein binds to imported substrate proteins and to the conserved 54 kDa SRP subunit (cpSRP54). While homologs to the bacterial SRP protein and RNA component previously have been identified in genome sequences of red algae and diatoms, a recent study investigated the evolution of the green plant SRP system.¹ Analysis of hundreds of plastid and nuclear genomes showed a surprising pattern of multiple losses of the plastid SRP RNA during evolution and a widespread presence in all non-spermatophyte plants and green algae. Contrary to expectations, all green organisms that have an identified cpSRP RNA also contain a cpSRP43. Notably, the structure of the plastid SRP RNAs is much more diverse than that of bacterial SRP RNAs. The apical GNRA tetraloop is only conserved in organisms of the red lineage and basal organisms of the green lineage, whereas further chloroplast SRP RNAs are characterized by atypical, mostly enlarged apical loops.     

Deletion of core components of the plastid protein import machinery causes differential arrest of embryo development in Arabidopsis thaliana

Among the genes that have recently been pinpointed to be essential for plant embryo development a large number encodes plastid proteins suggesting that embryogenesis is linked to plastid localized processes. However, nuclear encoded plastid proteins are synthesized as precursors in the cytosol and subsequently have to be transported across the plastid envelopes by a complex import machinery. We supposed that deletion of components of this machinery should allow a more general assessment of the role of plastids in embryogenesis since it will not only affect single proteins but instead inhibit the accumulation of most plastid proteins. Here we have characterized three Arabidopsis thaliana mutants lacking core components of the Toc complex, the protein translocase in the outer plastid envelope membrane, which indeed show embryo lethal phenotypes. Remarkably, embryo development in the atToc75-III mutant, lacking the pore forming component of the translocase, was arrested extremely early at the two-cell stage. In contrast, despite the complete or almost complete lack of the import receptors Toc34 and Toc159, embryo development in the a tToc33/34 and atToc132/159 mutants proceeded slowly and was arrested later at the transition to the globular and the heart stage, respectively. These data demonstrate a strict dependence of cell division and embryo development on functional plastids as well as specific functions of plastids at different stages of embryogenesis. In addition, our analysis suggest that not all components of the translocase are equally essential for plastid protein import in vivo.     

DCL is a plant-specific protein required for plastid ribosomal RNA processing and embryo development

The defective chloroplast and leaf-mutable (dcl-m) mutation of tomato blocks chloroplast differentiation in leaf mesophyll cells and a signaling system that appears to be required for morphogenesis of palisade cells during leaf growth. To dissect the function of DCL, mutants with stable dcl alleles (dcl-s) were generated and examined for their phenotype. DCL/dcl-s plant produce dcl-s/dcl-s seeds with embryos arrested at the globular stage of development. The levels of several chloroplast- and nuclear-encoded proteins are strongly reduced in dcl-m mutant leaf sectors without significant changes in their corresponding mRNAs. The 4.5S rRNA fails to be processed efficiently, however, suggesting that DCL has a direct or indirect function in rRNA processing or correct ribosome assembly. Accordingly, chloroplasts in dcl-m sectors are impaired in polysome assembly, which can explain the reduced accumulation of chloroplast-encoded proteins. These results suggest that DCL is required for chloroplast rRNA processing, and emphasize the importance of plastid function during embryogenesis.     

Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic -helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F₁ subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.Abbreviations cTP chloroplast transit peptide - mTP mitochondrial targeting peptide - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - pF₁(1,25) a synthetic peptide derived from the first 25 residues of the Nicotiana plumbaginifolia mitochondrial ATP synthase F₁ subunit - PsaF(2–30) and PsaF(2–61) mutant proteins lacking regions corresponding to residues 2–30 and 2–61 in the PsaF precursor protein, respectively     

Targeting of an abundant cytosolic form of the protein import receptor at Toc159 to the outer chloroplast membrane

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.     

A role of Toc33 in the protochlorophyllide-dependent plastid import pathway of NADPH:protochlorophyllide oxidoreductase (POR) A

NADPH:protochlorophyllide oxidoreductase (POR) A is a key enzyme of chlorophyll biosynthesis in angiosperms. It is nucleus-encoded, synthesized as a larger precursor in the cytosol and imported into the plastids in a substrate-dependent manner. Plastid envelope membrane proteins, called protochlorophyllide-dependent translocon proteins, Ptcs, have been identified that interact with pPORA during import. Among them are a 16-kDa ortholog of the previously characterized outer envelope protein Oep16 (named Ptc16) and a 33-kDa protein (Ptc33) related to the GTP-binding proteins Toc33 and Toc34 of Arabidopsis. In the present work, we studied the interactions and roles of Ptc16 and Ptc33 during pPORA import. Radiolabeled Ptc16/Oep16 was synthesized from a corresponding cDNA and imported into isolated Arabidopsis plastids. Crosslinking experiments revealed that import of 35S-Oep16/Ptc16 is stimulated by GTP. 35S-Oep16/Ptc16 forms larger complexes with Toc33 but not Toc34. Plastids of the ppi1 mutant of Arabidopsis lacking Toc33, were unable to import pPORA in darkness but imported the small subunit precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSSU), precursor ferredoxin (pFd) as well as pPORB which is a close relative of pPORA. In white light, partial suppressions of pSSU, pFd and pPORB import were observed. Our results unveil a hitherto unrecognized role of Toc33 in pPORA import and suggest photooxidative membrane damage, induced by excess Pchlide accumulating in ppi1 chloroplasts because of the lack of pPORA import, to be the cause of the general drop of protein import.     

Structure of a TOC-TIC supercomplex spanning two chloroplast envelope membranes

1. Download : Download high-res image (389KB)
2. Download : Download full-size image

     

PTS2 protein import into mammalian peroxisomes

Peroxisome targeting signal (PTS)2 directs proteins from their site of synthesis in the cytosol to the lumen of the peroxisome. Unlike PTS1 which is present in the great majority of peroxisomal matrix proteins and whose import mechanics have been dissected in considerable detail, PTS2 is a relatively rare topogenic signal whose import mechanisms are far less well understood. However, as is the case for PTS1 proteins, an inability to import PTS2 proteins leads to human disease. In this report, we describe the biochemical characterization of mammalian PTS2 protein import using a semi-permeabilized cell system. We show that a PTS2-containing reporter molecule is taken up by peroxisomes in a reaction that is time-, temperature-, ATP-, and cytosol-dependent. Furthermore, the import process is specific, saturable, and requires action of the chaperone Hsc70, the cochaperone Hsp40, and the peroxins Pex5p and Pex14p. We also demonstrate peroxisomal translocation of PTS2 reporter/antibody complexes confirming the import competence of higher order structures. Importantly, cultured fibroblasts from patients with the rhizomelic form of chondrodysplasia punctata (RCDP) which are deficient for the PTS2 receptor protein, Pex7p, are unable to import the PTS2 reporter in this assay. The ability to monitor PTS2 import in vitro will permit, for the first time, a detailed comparison of the biochemical properties of PTS1 and PTS2 protein import.     

The mitochondrial intermembrane space: the most constricted mitochondrial sub-compartment with the largest variety of protein import pathways

The mitochondrial intermembrane space (IMS) is the most constricted sub-mitochondrial compartment, housing only about 5% of the mitochondrial proteome, and yet is endowed with the largest variability of protein import mechanisms. In this review, we summarize our current knowledge of the major IMS import pathway based on the oxidative protein folding pathway and discuss the stunning variability of other IMS protein import pathways. As IMS-localized proteins only have to cross the outer mitochondrial membrane, they do not require energy sources like ATP hydrolysis in the mitochondrial matrix or the inner membrane electrochemical potential which are critical for import into the matrix or insertion into the inner membrane. We also explore several atypical IMS import pathways that are still not very well understood and are guided by poorly defined or completely unknown targeting peptides. Importantly, many of the IMS proteins are linked to several human diseases, and it is therefore crucial to understand how they reach their normal site of function in the IMS. In the final part of this review, we discuss current understanding of how such IMS protein underpin a large spectrum of human disorders.