Chloroplast protein translocon components atToc159 and atToc33 are not essential for chloroplast biogenesis in guard cells and root cells

Protein import into chloroplasts is mediated by a protein import apparatus located in the chloroplast envelope. Previous results indicate that there may be multiple import complexes in Arabidopsis. To gain further insight into the nature of this multiplicity, we analyzed the Arabidopsis ppi1 and ppi2 mutants, which are null mutants of the atToc33 and atToc159 translocon proteins, respectively. In the ppi2 mutant, in contrast to the extremely defective plastids in mesophyll cells, chloroplasts in guard cells still contained starch granules and thylakoid membranes. The morphology of root plastids in both mutants was similar to that in wild type. After prolonged light treatments, root plastids of both mutants and the wild type differentiated into chloroplasts. Enzymatic assays indicated that the activity of a plastid enzyme was reduced only in leaves but not in roots. These results indicated that both the ppi1 and ppi2 mutants had functional root and guard cell plastids. Therefore, we propose that import complexes are cell type specific rather than substrate or plastid specific.     

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.     

Carotenoid-deficient young wheat etioplasts are able to bind precursor proteins on the plastid surface but are impaired in their translocation ability

Young carotenoid-deficient etioplasts, isolated from Norflurazon (NF)-treated wheat seedlings, were used to study the role of coloured carotenoids in the binding and import reactions of different nuclear-encoded plastid proteins. Plastids from control seedlings exhibited significantly higher import efficiencies than did plastids from NF-treated plants. Etioplasts containing normal levels of carotenoids imported approximately 2000 and 800 molecules per plastid of the precursors of the small Rubisco subunit (pSS) and the Rieske FeS protein (pFeS), respectively. Plastids from NF-treated plants imported approximately 100 and 70 pSS and pFeS molecules per plastid, respectively. In addition, a maximum binding capacity of NF-treated plastids of 1200 protein molecules per plastid was observed for both pSS and pFeS when assayed at 25°C: and a maximum binding capacity of approximately 1300 molecules per plastid was noted at 4°C. For control plastids, a similar amount of binding, or approximately 1400 protein molecules per plastid, could only be observed if import was inhibited by low ATP concentrations at 4°C. When these plastids were washed and transferred to conditions promoting import at 25°C and 10 mM Mg-ATP, close to 60% of the envelope-associated precursor protein molecules were imported. These results indicate that control and NF-treated young etioplasts contain similar amounts of binding sites for precursor proteins. However, only in the case of control plastids the binding was productive and lead to import and processing in the stroma upon transfer to conditions promoting import. Plastids isolated from wheat seedlings grown in weak red light and containing different amounts of carotenoids, were assayed for their ability to bind and import a protein with unusual import characteristics, the Chlamydomonas reinhardtii PsaF precursor of PSI (pPsaF) and transit peptide deletion constructs. The PsaF protein was imported in a transit peptide-dependent manner into control etioplasts, whereas import of pPsaF into young wheat etioplasts isolated from NF-treated plants was inhibited at low levels of plastid carotenoids.     

Is Protein Import into Plastids with Four‐Membrane Envelopes Dependent on Two Toc Systems Operating in Tandem?

Abstract: Plastids with four‐membrane envelopes have evolved by several independent endosymbioses involving a eukaryotic alga as the endosymbiont and a protozoan predator as the host. It is assumed that their outermost membrane is derived from the phagosomal membrane of the host and that protein targeting to and across this membrane proceeds co‐translationally, including ER and the Golgi apparatus (e.g., chlorarachniophytes) or only ER (e.g., heterokonts). Since the two inner membranes (or the plastid envelope) of such a complex plastid are derived from the endosymbiont plastid, they are probably provided with Toc and Tic systems, enabling post‐translational passage of the imported proteins into the stroma. The third envelope membrane, or the periplastid one, originates from the endosymbiont plasmalemma, but what import apparatus operates in it remains enigmatic. Recently, Cavalier‐Smith (1999^[5]) has proposed that the Toc system, pre‐existing in the endosymbiont plastid, has been relocated to the periplastid membrane, and that plastids having four envelope membranes contain two Toc systems operating in tandem and requiring the same targeting sequence, i.e., the transit peptide. Although this model is parsimonious, it encounters several serious obstacles, the most serious one resulting from the complex biogenesis of Toc75 which forms a translocation pore. In contrast to most proteins targeted to the outer membrane of the plastid envelope, this protein carries a complex transit peptide, indicating that a successful integration of the Toc system into the periplastid membrane would have to be accompanied by relocation of the stromal processing peptidase to the endosymbiont cytosol. However, such a relocation would be catastrophic because this enzyme would cleave the transit peptide off all plastid‐destined proteins, thus disabling biogenesis of the plastid compartment. Considering these difficulties, I suggest that in periplastid membranes two Toc‐independent translocation apparatuses have evolved: a porin‐like channel in chlorarachniophytes and cryptophytes, and a vesicular pathway in heterokonts and haptophytes. Since simultaneous evolution of a new transport system in the periplastid membrane and in the phagosomal one would be complicated, it is argued that plastids with four‐membrane envelopes have evolved by replacement of plastids with three‐membrane envelopes. I suggest that during the first round of secondary endosymbioses (resulting in plastids surrounded by three membranes), myzocytotically‐engulfed eukaryotic alga developed a Golgi‐mediated targeting pathway which was added to the Toc/Tic‐based apparatus of the endosymbiont plastid. During the second round of secondary endosymbioses (resulting in plastids surrounded by four membranes), phagocytotically‐engulfed eukaryotic alga exploited the Golgi pathway of the original plastid, and a new translocation system had to originate only in the periplastid membrane, although its emergence probably resulted in modification of the import machinery pre‐existing in the endosymbiont plastid.     

Transport of proteins into cryptomonads complex plastids

Complex plastids, found in many alga groups, are surrounded by three or four membranes. Therefore, proteins of the complex plastids, which are encoded in the cell nucleus, must cross three or four membranes during transport to the plastid. To study this process we have developed a method for isolating transport-competent two membrane-bound plastids derived from the complex plastids of the cryptophyte Guillardia theta. This in vitro protein import system provides the first non-heterologous system for studying the import of proteins into four-membrane complex plastids. We use our import system as well as canine microsomes to demonstrate in the case of cryptomonads how nuclear proteins pass the first nucleomorph-encoded proteins the third and fourth membrane and discuss the potential mechanisms for protein transport across the second membrane.     

In vivo analysis of the role of atTic20 in protein import into chloroplasts

The import of nucleus-encoded preproteins into plastids requires the coordinated activities of membrane protein complexes that facilitate the translocation of polypeptides across the envelope double membrane. Tic20 was identified previously as a component of the import machinery of the inner envelope membrane by covalent cross-linking studies with trapped preprotein import intermediates. To investigate the role of Tic20 in preprotein import, we altered the expression of the Arabidopsis Tic20 ortholog (atTic20) by antisense expression. Several antisense lines exhibited pronounced chloroplast defects exemplified by pale leaves, reduced accumulation of plastid proteins, and significant growth defects. The severity of the phenotypes correlated directly with the reduction in levels of atTic20 expression. In vitro import studies with plastids isolated from control and antisense plants indicated that the antisense plastids are defective specifically in protein translocation across the inner envelope membrane. These data suggest that Tic20 functions as a component of the protein-conducting channel at the inner envelope membrane.     

Assembly of the barley light-harvesting chlorophyll a/b proteins in barley etiochloroplasts involves processing of the precursor on thylakoids

A barley gene encoding the major light-harvesting chlorophyll a/b-binding protein (LHCP) has been sequenced and then expressed in vitro to produce a labelled LHCP precursor (pLHCP). When barley etiochloroplasts are incubated with this pLHCP, both labelled pLHCP and LHCP are found as integral thylakoid membrane proteins, incorporated into the major pigment-protein complex of the thylakoids. The presence of pLHCP in thylakoids and its proportion with respect to labelled LHCP depends on the developmental stage of the plastids used to study the import of pLHCP. The reduced amounts of chlorophyll in a chlorophyll b-less mutant of barley does not affect the proportion of pLHCP to LHCP found in the thylakoids when import of pLHCP into plastids isolated from the mutant plants is examined. Therefore, insufficient chlorophyll during early stages of plastid development does not seem to be responsible for their relative inefficiency in assembling pLHCP. A chase of labelled pLHCP that has been incorporated into the thylakoids of intact plastids, by further incubation of the plastids with unlabelled pLHCP, reveals that the pLHCP incorporated into the thylakoids can be processed to its mature size. Our observations strongly support the hypothesis that after import into plastids, pLHCP is inserted into thylakoids and then processed to its mature size under in vivo conditions.     

Internal plastid-targeting signal found in a RubisCO small subunit protein of a chlorarachniophyte alga

In all plants and algae, most plastid proteins are encoded by the nuclear genome and, consequently, need to be transported into plastids across multiple membranes. In organisms with secondary plastids, which evolved by secondary endosymbioses, and are surrounded by three or four envelope membranes, precursors of nuclear-encoded plastid proteins generally have an N-terminal bipartite targeting sequence that consists of an endoplasmic reticulum (ER)-targeting signal peptide (SP) and a transit peptide-like (TPL) sequence. The bipartite targeting sequences have been demonstrated to be necessary and sufficient for targeting proteins into the plastids of many algal groups, including chlorarachniophytes. Here, we report a new type of targeting signal that is required for delivering a RubisCO small subunit (RbcS) protein into the secondary plastids of chlorarachniophyte algae. In this study, we analyzed the plastid-targeting ability of an RbcS pre-protein, using green fluorescent protein (GFP) as a reporter molecule in chlorarachniophyte cells. We demonstrate that the N-terminal bipartite targeting sequence of the RbcS pre-protein is not sufficient, and that a part of the mature protein is also necessary for plastid targeting. By deletion analyses of amino acids, we determined the approximate location of an internal plastid-targeting signal within the mature protein, which is involved in targeting the protein from the ER into the chlorarachniophyte plastids.     

Genome-based reconstruction of the protein import machinery in the secondary plastid of a chlorarachniophyte alga

Most plastid proteins are encoded by their nuclear genomes and need to be targeted across multiple envelope membranes. In vascular plants, the translocons at the outer and inner envelope membranes of chloroplasts (TOC and TIC, respectively) facilitate transport across the two plastid membranes. In contrast, several algal groups harbor more complex plastids, the so-called secondary plastids, which are surrounded by three or four membranes, but the plastid protein import machinery (in particular, how proteins cross the membrane corresponding to the secondary endosymbiont plasma membrane) remains unexplored in many of these algae. To reconstruct the putative protein import machinery of a secondary plastid, we used the chlorarachniophyte alga Bigelowiella natans, whose plastid is bounded by four membranes and still possesses a relict nucleus of a green algal endosymbiont (the nucleomorph) in the intermembrane space. We identified nine homologs of plant-like TOC/TIC components in the recently sequenced B. natans nuclear genome, adding to the two that remain in the nucleomorph genome (B. natans TOC75 [BnTOC75] and BnTIC20). All of these proteins were predicted to be localized to the plastid and might function in the inner two membranes. We also show that the homologs of a protein, Der1, that is known to mediate transport across the second membrane in the several lineages with secondary plastids of red algal origin is not associated with plastid protein targeting in B. natans. How plastid proteins cross this membrane remains a mystery, but it is clear that the protein transport machinery of chlorarachniophyte plastids differs from that of red algal secondary plastids.     

Mechanism of Protein Targeting to the Chlorarachniophyte Plastids and the Evolution of Complex Plastids with Four Membranes — A Hypothesis

Chlorarachniophyta are phototrophic amoeboflagellates, with plastids surrounded by four membranes. Contrary to other plastids of this type which occur in chromists, their outermost membrane bears no ribosomes. It is argued that the nuclear-encoded chlorarachniophyte plastid proteins are first transported into the ER, then to the Colgi apparatus, and finally to the plastids. The same import mechanism could be originally present in the chromist ancestor, prior to the fusion of their plastids with the RER membranes. According to the most recent concept, the complex plastids of Chromista and Chlorarachniophyta have evolved through replacement of the cyanobacterial plastids. The assumption that these plastids had an envelope composed not of two, but of three membranes makes it possible to avoid the erlier discerned difficulties with conversion of a eukaryotic alga into a complex plastid. My scenario provides an additional support to the hypothesis on polyphy-letic origin of four-membraned plastids.     

Early steps in plastid evolution: current ideas and controversies

Some nuclear‐encoded proteins are imported into higher plant plastids via the endomembrane (EM) system. Compared with multi‐protein Toc and Tic translocons required for most plastid protein import, the relatively uncomplicated nature of EM trafficking led to suggestions that it was the original transport mechanism for nuclear‐encoded endosymbiont proteins, and critical for the early stages of plastid evolution. Its apparent simplicity disappears, however, when EM transport is considered in light of selective constraints likely encountered during the conversion of stable endosymbionts into fully integrated organelles. From this perspective it is more parsimonious to presume the early evolution of post‐translational protein import via simpler, ancestral forms of modern Toc and Tic plastid translocons, with EM trafficking arising later to accommodate glycosylation and/or protein targeting to multiple cellular locations. This hypothesis is supported by both empirical and comparative data, and is consistent with the relative paucity of EM‐based transport to modern primary plastids.     

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.     

The potato granule bound starch synthase chloroplast transit peptide directs recombinant proteins to plastids

The chloroplast targeting transit sequence from potato granule bound starch synthase (gbss) was used to direct the accumulation of recombinant proteins to the plastid stroma. The potato gbss transit sequence was fused to the N-terminus of the green fluorescent protein (GFP) and the Catharanthus roseus strictosidine synthase (Str1) enzyme. Fluorescence microscopy confirmed that the recombinant gbss-GFP fusion protein was exclusively targeted to the plastid stroma in tobacco suspension cells, demonstrating that the transit sequence was functional in vivo. The Str1 fusion protein accumulated to high levels in plastids isolated from transgenic plants. We conclude that the potato gbss transit sequence is functional and directs import of recombinant proteins into the chloroplast stroma.     

The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles

Background  

Today it is widely accepted that plastids are of cyanobacterial origin. During their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. This process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. Such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. At present, most information on protein translocation is obtained by the analysis of land plants. However, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. Here, bacterial outer membrane proteins of the Omp85 family have recently been discussed as evolutionary seeds for the development of translocation systems.     

THE ROLE OF CARBONIC ANHYDRASE IN THE FORMATION OF MOUGEOTIA (CHAROPHYCEAE) BLOOMS IN ACIDIC ENVIRONMENTS

Diatoms and related algae have plastids that are surrounded by four membranes. The outer two membranes are continuous with the endoplasmic reticulum and the inner two membranes are analogous to the plastid envelope membranes of higher plants and green algae. Thus the plastids are completely compartmentalized within the ER membranes. The targeting presequences for nuclear-encoded plastid proteins have two recognizable domains. The first domain is a classic signal sequence, which presumably targets the proteins to the endoplasmic reticulum. The second domain has characteristics of a transit peptide, which targets proteins to the plastids of higher plants. To characterize these targeting domains, the presequence from the nuclear-encoded plastid protein AtpC was utilized. A series of deletions of this presequence were fused to Green Fluorescent Protein (GFP) and transformed into cells of the diatom, Phaeodactylum tricornutum. The intracelluar localization of GFP was visualized by fluorescence microscopy. This work demonstrates that the first domain of the presequence is responsible for targeting proteins to the ER lumen and is the essential first step in the plastid protein import process. The second domain is responsible to directing proteins from the ER and through the plastid envelope and only a short portion of the transit peptide-like domain is necessary to complete this second processing step. In vivo data generated from this study in a fully homologous transformation system has confirmed Gibbs' hypothesis regarding a multistep import process for plastid proteins in chromophytic algae.     

Members of the Toc159 import receptor family represent distinct pathways for protein targeting to plastids

Plastids represent a diverse group of organelles that perform essential metabolic and signaling functions within all plant cells. The differentiation of specific plastid types relies on the import of selective sets of proteins from among the approximately 2500 nucleus-encoded plastid proteins. The Toc159 family of GTPases mediates the initial targeting of proteins to plastids. In Arabidopsis thaliana, the Toc159 family consists of four genes: atTOC159, atTOC132, atTOC120, and atTOC90. In vivo analysis of atToc159 function indicates that it is required specifically for the import of proteins necessary for chloroplast biogenesis. In this report, we demonstrate that atToc120 and atToc132 represent a structurally and functionally unique subclass of protein import receptors. Unlike atToc159, mutants lacking both atToc120 and atToc132 are inviable. Furthermore, atToc120 and atToc132 exhibit preprotein binding properties that are distinct from atToc159. These data indicate that the different members of the Toc159 family represent distinct pathways for protein targeting to plastids and are consistent with the hypothesis that separate pathways have evolved to ensure balanced import of essential proteins during plastid development.     

Dual intracellular localization and targeting of aminoimidazole ribonucleotide synthetase in cowpea

De novo purine biosynthesis is localized to both mitochondria and plastids isolated from Bradyrhizobium sp.-infected cells of cowpea (Vigna unguiculata L. Walp) nodules, but several of the pathway enzymes, including aminoimidazole ribonucleotide synthetase (AIRS [EC 6.3.3.1], encoded by Vupur5), are encoded by single genes. Immunolocalization confirmed the presence of AIRS protein in both organelles. Enzymatically active AIRS was purified separately from nodule mitochondria and plastids. N-terminal sequencing showed that these two isoforms matched the Vupur5 cDNA sequence but were processed at different sites following import; the mitochondrial isoform was five amino acids longer than the plastid isoform. Electrospray tandem mass spectrometry of a trypsin digest of mitochondrial AIRS identified two internal peptides identical with the amino acid sequence deduced from Vupur5 cDNA. Western blots of proteins from mitochondria and plastids isolated from root tips showed a single AIRS protein present at low levels in both organelles. (35)S-AIRS protein translated from a Vupur5 cDNA was imported into isolated pea (Pisum sativum) leaf chloroplasts in vitro by an ATP-dependent process but not into import-competent mitochondria from several plant and non-plant sources. Components of the mature protein are likely to be important for import because the N-terminal targeting sequence was unable to target green fluorescent protein to either chloroplasts or mitochondria in Arabidopsis leaves. The data confirm localization of the protein translated from the AIRS gene in cowpea to both plastids and mitochondria and that it is cotargeted to both organelles, but the mechanism underlying import into mitochondria has features that are yet to be identified.