Altération du système membranaire de protoplastes de feuilles de piment (Capsicum annuum) sous l'action du capsidiol

During infection with Phytophthora capsici, we demonstrated that susceptible induced pepper leaves produced apposition walls that protect host cells, but modifications perturbed the photosynthetic apparatus and crystal-like structures appeared in chloroplasts. We propose that these alterations may be due to the action of capsidiol synthesized by host cells. To confirm this hypothesis, we tested the action (1 h) of capsidiol (400 μg.mL⁻¹) on isolated leaf protoplasts and we observed the formation of crystal-like structures on the site of clustered thylakoïds. The destructuration is initiated by the deposition of a proteic osmiophilic substance within the lumen of each thylakoïd, then the process was extended to the other thylakoïds in the same group. Thus, a transversal arrangement progressively occurred and served as substitute for the initial vertical disposition of the membranes. Plasma membrane and cytoplasmic organelles were also altered and we observed that the endoplasmic reticulum was in cluse association with the plasma membrane, probably to play a detoxicating role.     

Characterization of a chloroplast inner envelope K+ channel.

A K(+)-conducting protein of the chloroplast inner envelope was characterized as a K+ channel. Studies of this transport protein in the native membrane documented its sensitivity to K+ channel blockers. Further studies of native membranes demonstrated a sensitivity of K+ conductance to divalent cations such as Mg2+, which modulate ion conduction through interaction with negative surface charges on the inner-envelope membrane. Purified chloroplast inner-envelope vesicles were fused into an artificial planar lipid bilayer to facilitate recording of single-channel K+ currents. These single-channel K+ currents had a slope conductance of 160 picosiemens. Antibodies generated against the conserved amino acid sequence that serves as a selectivity filter in the pore of K+ channels immunoreacted with a 62-kD polypeptide derived from the chloroplast inner envelope. This polypeptide was fractionated using density gradient centrifugation. Comigration of this immunoreactive polypeptide and K+ channel activity in sucrose density gradients further suggested that this polypeptide is the protein facilitating K+ conductance across the chloroplast inner envelope.     

Specific labelling of the active site of the phosphate translocator in spinach chloroplasts by 2,4,6-trinitrobenzene sulfonate

Phosphate transport across the chloroplast envelope is rapidly inactivated by the amino-group reagent 2,4,6-trinitrobenzene sulfonate. Subsequent exposure to [³H]NaBH₄ leads to an incorporation of the trinitrophenyl moiety into envelope membrane preparations. From the membrane proteins only a polypeptide with 29000 dalton molecular weight is labelled. The inactivation of phosphate transport and the incorporation of radioactivity are both specifically reduced by the presence of substrates.The results lead to the conclusion that a polypeptide with a molecular weight of 29000 dalton and containing a lysyl residue at the substrate binding site is involved in the phosphate translocator function.     

Isolation and characterization of a cDNA clone encoding a cognate 70-kDa heat shock protein of the chloroplast envelope.

The translocation of proteins into the endoplasmic reticulum, the mitochondrion, and the chloroplast has recently been shown to involve homologues of the highly conserved 70-kDa heat shock protein (HSP70) family. In this study, we have isolated and sequenced a full-length cDNA clone encoding a cognate 70-kDa heat shock protein of the spinach chloroplast envelope (SCE70). The cDNA insert is 2,535 base pairs long and codes for 653 amino acid residues of a protein with a predicted molecular mass of 71,731 daltons. The deduced amino acid sequence shows a high degree of homology with HSP70 proteins from other organisms. Southern genomic and RNA analyses reveal different hybridization patterns than that observed for a heat-inducible 70-kDa protein gene. The protein synthesized from the SCE70 cDNA insert co-migrates with a 70-kDa polypeptide of the chloroplast envelope following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis and import studies indicate that SCE70 is associated with the chloroplast outer envelope. The import data suggest that SCE70 is targeted to the envelope membrane via a pathway different from other plastidic precursors but similar to that recently reported for outer envelope proteins SOE1 and OM14.     

Polypeptides of the chloroplast envelope membranes as visualized by immunochemical techniques

The polypeptides of relative molecular masses (Mr) 22,000, 29,000, and 36,000 represent three major constituents of the chloroplast envelope of spinach (Spinacia oleracea L.) leaves. The Mr 22,000 polypeptide has been localized in the outer membrane, whereas the two other peptides have been attributed to the inner envelope membrane (Joyard et al., 1983). The Mr 29,000 polypeptide has been identified as the "phosphate translocator" (Flügge and Heldt, 1979). In this investigation, we studied the three envelope polypeptides by means of immunocytochemistry. Using indirect immunofluorescence, all three polypeptides were visualized in cryostat sections of formaldehyde-fixed leaf tissue. They were found in both palisade and spongy parenchyma cells and in guard cells, as indicated by a strong fluorescence in the chloroplast periphery. In contrast, fluorescein isothiocyanate or protein A-gold labeling of isolated fixed chloroplasts resulted only in visualization of the Mr 22,000 polypeptide, a constituent of the outer membrane. We further studied the morphological distribution and frequency of this peptide by electron microscopic evaluation of platinum-carbon replicas after freeze-etching or label-fracture and of ultra-thin sections. By use of these three methods, the polypeptide was found to be randomly distributed in the outer envelope membrane and easily accessible to the immunomarker. Average marker density, as obtained by freeze-etching and label-fracture, was approximately 130 gold particles per square micron.     

Characterization of a chloroplast inner envelope P-ATPase proton pump

P-ATPases such as the plasma membrane proton pump are known to generate a phosphorylated intermediate as a step in their reaction mechanism; phosphoenzyme formation is a basis for classification of an ATPase as a member of this subfamily of ion pumps. The chloroplast inner envelope is known to contain a H+-ATPase which acts to maintain an alkaline stroma and, thus, optimal photosynthesis. Our characterization of this chloroplast envelope proton pump described in this report focused on determining whether purified chloroplast inner envelope membrane protein preparations containing this ATPase form a phosphorylated intermediate. Incubation of envelope membranes with [-³²P]ATP documented the formation of P-type ATPase phosphoenzyme intermediates by these membrane protein preparations. Our work cannot discount the possibility that more than one chloroplast inner envelope ATPase contributes to this phosphoenzyme formation. However, the kinetics of this phosphoenzyme formation, along with the sensitivity of phosphoenzyme formation to inhibitors and other assay conditions suggested that one of the envelope membrane proteins which is covalently radiolabeled by [-³²P]ATP is a P-type H+-ATPase. Autoradiography of chloroplast envelope membrane proteins size fractionated on lithium dodecyl sulfate-PAGE indicated that the phosphoenzyme intermediate corresponds to a 103 kDa polypeptide. P-type proton pumps are known to be comprised of a single type of 100 kDa subunit. Experimental evidence presented in this report is consistent with the classification of a chloroplast inner envelope H+-ATPase as a P-type proton pump.     

Analysis of suborganellar fractions from spinach and pea chloroplasts for calmodulin-binding proteins

Purified chloroplasts from spinach and pea leaves were subfractionated into envelope, thylakoid, and stroma fractions and were analyzed for calmodulin-binding proteins using a 125I-calmodulin gel overlay assay. Calmodulin binding was primarily associated with a major polypeptide (Mr 33,000) in the envelope membrane fraction. In contrast, major calmodulin-binding proteins were not detected in the thylakoid or stroma fractions. Our results provide the first evidence of calmodulin- binding proteins in the chloroplast envelope, and raise the possibility that calmodulin may contribute to the regulation of chloroplast function through its interaction with calmodulin-binding proteins in the chloroplast envelope. In addition, our results combined with those of other investigators support the proposal that subcellular organelles may be a primary site of calmodulin action.     

Effects of osmotic preconditioning on nuclear replication activity in seeds of pepper (Capsicum annuum)

Routing of cytosolically synthesized precursor proteins into chloroplasts is a specific process which involves a multitude of soluble and membrane components. In this review we wil1 focus on early events of the translocation pathway of nuclear coded plastidic precursor proteins and compare import routes for polypeptide of the outer chloroplast envelope to that of internal chloroplast compartments. A number of proteins housed in the chloroplast envelopes have been implied to be involved in the translocation process, but so far a certain function has not been assigned to any of these proteins. The only exception could be an envelope localized hsc 70 homologue which could retain the import competence of a precursor protein in transit into the organelle.     

The chloroplast protein import receptors Toc34 and Toc159 are phosphorylated by distinct protein kinases

The molecular composition of chloroplast outer and inner envelope translocons is fairly well established, but little is known about mechanisms and elements involved in import regulation. After synthesis in the cytosol, chloroplast targeted precursor proteins are recognized by outer envelope receptors Toc34 and Toc159. Phosphorylation plays an important role in regulation of Toc34 activity and preprotein binding. Using kinase renaturation assays, we have identified an ATP-dependent 98-kDa outer envelope kinase which is able to selectively phosphorylate Toc34 at a specific site. A 70-kDa outer envelope polypeptide phosphorylating Toc159 was identified by the same strategy. Antiserum against the 98-kDa kinase inhibits phosphorylation of Toc34, whereas labeling of Toc159 remains unaffected. Both kinases do not autophosphorylate in vitro and are unable to utilize myelin basic protein as substrate. We propose that distinct kinases are involved in regulation of chloroplast import via desensitization of preprotein receptors.     

Envelope membranes from mature spinach chloroplasts contain a NADPH:protochlorophyllide reductase on the cytosolic side of the outer membrane

Using fluorescence spectroscopy, we have demonstrated that isolated envelope membranes from mature spinach chloroplasts catalyze the phototransformation of endogenous protochlorophyllide into chlorophyllide in presence of NADPH, but not in presence of NADH. Protochlorophyllide reductase was characterized further using monospecific antibodies (anti-protochlorophyllide reductase) raised against the purified enzyme from oat. In mature spinach chloroplasts, protochlorophyllide reductase is present only in envelope membranes. We have demonstrated that the envelope protochlorophyllide reductase, a 37,000-dalton polypeptide, is only a minor envelope component and is present on the outer surface of the outer envelope membrane. This conclusion is supported by several lines of evidence: (a) the envelope polypeptide that was immunodecorated with anti-protochlorophyllide reductase can be distinguished from the major 37,000-dalton envelope polypeptide E37 (which was identified by monospecific antibodies) only after two-dimensional polyacrylamide gel electrophoresis; (b) the envelope protochlorophyllide reductase was hydrolyzed when isolated intact chloroplasts were incubated in presence of thermolysin; and (c) isolated intact chloroplasts strongly agglutinate when incubated in presence of antibodies raised against protochlorophyllide reductase. These results demonstrate that major differences exist between chloroplasts and etioplasts with respect to protochlorophyllide reductase levels and localization. The presence on the chloroplast envelope membrane of both the substrate (protochlorophyllide) and the enzyme (protochlorophyllide reductase) necessary for chlorophyllide synthesis could have major implications for the understanding of chlorophyll biosynthesis in mature chloroplasts.     

Subchloroplast localization of polypeptides synthesized by chloroplasts during senescence

To study the localization of polypeptides synthesized by isolated senescent chloroplasts we have fractionated the chloroplasts into stroma, envelope and thylakoid components. The validity of the fractionation procedure was tested by assaying both chlorophyll and enzyme markers, as well as the polypeptide composition of each fraction. Plastids in the transition of etioplast to chloroplast, senescent chloroplasts and kinetin-treated chloroplasts produced acceptable fractions, although their polypeptide compositions varied considerably during the ontogeny, particularly those of the envelope. Most of the polypeptides synthesized by isolated senescent chloroplasts were incorporated into the thylakoids except for a 58 kDa polypeptide localized in the stroma and some minor polypeptides present in both stroma and envelope. Although most of the polypeptides synthesized by isolated chloroplasts from kinetin-treated leaves were incorporated into the thylakoid membrane, several polypeptides were found in the stroma (90, 80, 65 and 54 kDa) and in the envelope (100, 75, 48 and 28–30 kDa). The results indicate that early in senescence, the polypeptides of the envelope change but, that probably, most of the new polypeptides are synthesized in the cytoplasm.     

Partial purification of a nucleoside triphosphatase from the inner membrane of the chloroplast envelope of pea

A Mg2+-NTPase has been partially purified from the inner membrane of the pea chloroplast envelope. Isolated envelope membranes were solubilized with Triton X-100 and fractionated by DEAE-Sephadex chromatography, followed by ultrafiltration and sucrose density gradient centrifugation. An approximate 35-fold increase in the specific activity of the vanadate and sodium fluoride sensitive NTPase was obtained. Analysis of the partially purified NTPase by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single 37-kDa polypeptide that appeared to be associated with the activity. In support of this identification, it is demonstrated that the 37-kDa polypeptide can be photolabeled with 8-azido-ATP.     

Functional analysis of the 37 kDa inner envelope membrane polypeptide in chloroplast biogenesis using a Ds-tagged Arabidopsis pale-green mutant

To study the functions of the nuclear genes involved in chloroplast development, we systematically analyzed albino and pale-green Arabidopsis thaliana mutants by using a two-component transposon system based on the Ac/Ds element of maize as a mutagen. One of the pale-green mutants, albino or pale green mutant 1 (designated as apg1), did not survive beyond the seedling stage, when germinated on soil. The chloroplasts of the apg1 plants contained decreased numbers of lamellae with reduced levels of chlorophyll. A gene encoding a 37 kDa polypeptide precursor of the chloroplast inner envelope membrane was disrupted by insertion of the Ds transposon in apg1. The 37 kDa protein had partial sequence similarity to the S-adenosylmethionine-dependent methyltransferase. The apg1 plants lacked plastoquinone (PQ), suggesting that the APG1 protein is involved in the methylation step of PQ biosynthesis, which is localized at the envelope membrane. Our results demonstrate the importance of the 37 kDa protein of the chloroplast inner envelope membrane for chloroplast development in Arabidopsis.     

Is E37, a major polypeptide of the inner membrane from plastid envelope,an S-adenosyl methionine-dependent methyltransferase?

Using antibodies raised against E37, one of the major polypeptides of the inner membrane from the chloroplast envelope, it has been demonstrated that a single immunologically related polypeptide was present in total protein extracts from various higher plants (monocots and dicots), in photosynthetic and non-photosynthetic tissues from young spinach plantlets, as well as in the cytoplasmic membrane from the cyanobacteria Synechococcus . This ubiquitous distribution of E37 strongly suggests that this protein plays an envelope-specific function common to all types of plastids. Comparison of tobacco and spinach E37 amino acid sequences deduced from the corresponding cDNA demonstrates that consensus motifs for S-adenosyl methionine-dependent methyltransferases are located in both sequences. This hypothesis was confirmed using a biochemical approach. It was demonstrated that E37, together with two minor spinach chloroplast envelope polypeptides of 32 and 39 kDa, can be specifically photolabeled with [³H]-S-adenosyl methionine upon UV-irradiation. Identification of E37 as a photolabeled polypeptide was established by immunoprecipitation. Furthermore, photolabeling of the three envelope polypeptides was specifically inhibited by very low concentration of S-adenosyl homocysteine, thus providing evidence for the presence within these proteins of S-adenosyl methionine- and S-adenosyl homocysteine-binding sites that were closely associated. Taken as a whole these results strongly suggest that E37 is an ubiquitous plastid envelope protein that probably has an S-adenosyl methionine-dependent methyltransferase activity. The 32 and 39 kDa envelope polypeptides probably have a similar methyltransferase activity.     

A constituent of the chloroplast import complex represents a new type of GTP-binding protein

The 34 kDa polypeptide of the outer envelope membranes from pea chloroplasts (OEP 34) is a major constituent of this membrane. OEP 34 is detected on polyacrylamide gels under non-reducing condition in association with OEP 75, the putative protein translocation pore. An antiserum against OEP 34 is able to co-immunoprecipitate the precursor of Rubisco small subunit from a partially purified import complex of chloroplast outer envelope membranes. A full-length cDNA clone coding for pea OEP 34 has been isolated. Analysis of the deduced amino acid sequence revealed typical and conserved sequence motifs found in GTP-binding proteins, making it a new and unique member of this superfamily. OEP 34 behaves as an integral constituent of the outer chloroplast envelope, which is anchored by its C-terminus into the membrane, while the majority of the protein projects into the cytoplasm. OEP 34 does not possess a cleavable N-terminal transit sequence but it is targeted to the chloroplasts and integrated into the outer membranes by internal sequence information which seems to be present in the C-terminal membrane anchor region. Productive integration of OEP 34 into the outer envelope requires, in contrast to other OEPs, protease-sensitive chloroplast surface components and is stimulated by ATR. The GTP binding specificity of OEP 34 is demonstrated by photo-affinity labelling in the presence of [α-³²P]GTP. Overexpressed and purified OEP 34 possesses endogenous GTPase activity. These results indicate a possible regulatory function of OEP 34 in protein translocation into chloroplasts.     

In situ incorporation of Fatty acids into lipids of the outer and inner envelope membranes of pea chloroplasts

When incubated with [1-¹⁴C]acetate and cofactors (ATP, Coenzyme A, sn-glycerol-3-phosphate, UDPgalactose, and NADH), intact chloroplasts synthesized fatty acids that were subsequently incorporated into most of the lipid classes. To study lipid synthesis at the chloroplast envelope membrane level, ¹⁴C-labeled pea (Pisum sativum) chloroplasts were subfractionated using a single flotation gradient. The different envelope membrane fractions were characterized by their density, lipid and polypeptide composition, and the localization of enzymic activities (UDPgalactose-1,2 diacylglycerol galactosyltransferase, Mg²⁺-dependent ATPase). They were identified as very pure outer membranes (light fraction) and strongly enriched inner membranes (heavy fraction). A fraction of intermediate density, which probably contained double membranes, was also isolated. Labeled glycerolipids recovered in the inner envelope membrane were phosphatidic acid, phosphatidyl-glycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol. Their ¹⁴C-fatty acid composition indicated that a biosynthetic pathway similar to the prokaryotic pathway present in cyanobacteria occurred in the inner membrane. In the outer membrane, phosphatidylcholine was the most labeled glycerolipid. Phosphatidic acid, phosphatidylglycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol were also labeled. The ¹⁴C-fatty acid composition of these lipids showed a higher proportion of oleate than palmitate. This labeling, different from that of the inner membrane, could result either from transacylation activities or from a biosynthetic pathway not yet described in pea and occurring partly in the outer chloroplast envelope membrane. This metabolism would work on an oleate-rich pool of fatty acids, possibly due to the export of oleate from chloroplast toward the extrachloroplastic medium. The respective roles of each membrane for chloroplast lipid synthesis are emphasized.     

From nuclear genes to chloroplast localized proteins

There is broad evidence that an endosymbiotic uptake of a cyanobacterial-type organism was the point of origin for the evolution of chloroplasts. During organelle evolution extensive gene transfer from the symbiont to the host genome occurred, which raises the question of how these gene products, namely proteins, which are still functional in chloroplasts, find their way back ‘home’. Nuclear-encoded proteins enter plastids via a complex import machinery that requires the coordinate interplay of a variety of soluble and membrane-bound factors on the cytosolic site as well as on the stromal side of the chloroplast envelope membranes. We define that the process called ‘import of chloroplast precursor proteins’ begins with the release of the polypeptide from the ribosomes and binding to cytosolic factors, such as a guidance complex, which accompanies (chaperones) proteins to chloroplasts. The translocation across the envelope membranes engages distinct translocation machineries at the outer and the inner envelope membranes. Additionally subsequent sorting events to different subcompartments within the plastids are operated by a number of distinct pathways, all of which seem to involve multiple subunits, which are largely of bacterial (symbiotic) origin. The evolutionary history of proteins mediating the import of chloroplast constituents across the envelope membranes seems more diverse. Since cyanobacteria lack a protein import pathway, it is not surprising that only a few subunits of the chloroplast translocon seem to be of symbiotic origin while others seem to be eukaryotic additions.     

Preparation and characterization of envelope membranes from nongreen plastids

We have developed a reliable procedure for the purification of envelope membranes from cauliflower (Brassica oleracea L.) bud plastids and sycamore (Acer pseudoplatanus L.) cell amyloplasts. After disruption of purified intact plastids, separation of envelope membranes was achieved by centrifugation on a linear sucrose gradient. A membrane fraction, having a density of 1.122 grams per cubic centimeter and containing carotenoids, was identified as the plastid envelope by the presence of monogalactosyldiacylglycerol synthase. Using antibodies raised against spinach chloroplast envelope polypeptides E24 and E30, we have demonstrated that both the outer and the inner envelope membranes were present in this envelope fraction. The major polypeptide in the envelope fractions from sycamore and cauliflower plastids was identified immunologically as the phosphate translocator. In the envelope membranes from cauliflower and sycamore plastids, the major glycerolipids were monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phosphatidylcholine. Purified envelope membranes from cauliflower bud plastids and sycamore amyloplasts also contained a galactolipid:galactolipid galactosyltransferase, enzymes for phosphatidic acid and diacylglycerol biosynthesis, acyl-coenzyme A thioesterase, and acyl-coenzyme A synthetase. These results demonstrate that envelope membranes from nongreen plastids present a high level of homology with chloroplasts envelope membranes.     

The Low CO2-Inducible 36-Kilodalton Protein Is Localized to the Chloroplast Envelope of Chlamydomonas reinhardtii

The localization of the 36-kD polypeptide of Chlamydomonas reinhardtii induced by photoautotrophic growth on low CO2 concentrations (0.03% in air [v/v], low CO2-grown cells) has been investigated. This polypeptide was specifically localized to the chloroplast envelope membranes isolated from low CO2-grown cells and was not present in the chloroplast envelopes isolated from high (5% CO2 in air [v/v]) CO2-grown cells. The 36-kD protein does not show carbonic anhydrase activity and was not present on the plasma membranes isolated from low CO2-grown cells. This protein may, in part, account for the different inorganic carbon uptake characteristics observed in chloroplasts isolated from high and low CO2-grown cells of C. reinhardtii.     

NUCLEAR ENVELOPE-CHLOROPLAST RELATIONSHIPS IN ALGAE

In Ochromonas danica and two related species (Chrysophyceae) and in Rhodomonas lens and Cryptomonas sp. (Cryptophyceae), the chloroplast is surrounded by an outer double-membraned envelope which lies outside the usual double-membraned chloroplast envelope. At the borders of the area where the chloroplast lies adjacent to the nucleus, this outer envelope is continuous with the outer membrane of the nuclear envelope as a double-membraned outfolding, so that the entire chloroplast in these species lies within a double-membraned sac, one wall of which is the nuclear envelope. In Olisthodiscus sp. (Chrysophyceae ?), each of the small peripheral chloroplasts is surrounded by a similar double-membraned outer envelope, but in this species no connections with the nuclear envelope were observed. In the Ochromonadaceae, a characteristic array of tubules is present within the sac in the narrow space which separates the chloroplast from the nucleus. In the other species studied, tubules are present at places between the chloroplast envelope and the outer envelope. In the Cryptophyceae, the starch grains lie outside the chloroplast envelope, but within the outer double-membraned sac. A double-membraned outer envelope appears to be present outside the chloroplasts of the Phaeophyta and Euglenophyta, but seems to be absent in the other groups of algae.