RESUMO
Plant cell chloroplasts are bounded by a two-membrane envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are distinct from the chloroplast envelope, and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three compartments. A protocol is detailed for leaves of spinach, Arabidopsis or pea.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Magnoliopsida , Tilacoides/metabolismo , Cloroplastos/metabolismo , Arabidopsis/metabolismo , Folhas de Planta , Proteínas de Arabidopsis/metabolismoRESUMO
The outer and the inner membranes of the chloroplast envelope, also called OEM and IEM, have distinct lipid and protein compositions. They host molecular systems involved in the biogenesis of the organelle, its cellular function, and its communication with other compartments. Here we describe a method for the isolation of these two membranes starting from intact chloroplast preparations, with two alternative procedures based on the starting material. One was developed from spinach leaves, the other from pea leaves. The two procedures differ in the method used to isolate and rupture chloroplasts and separate each membrane.
Assuntos
Membranas Intracelulares , Magnoliopsida , Membranas Intracelulares/metabolismo , Magnoliopsida/metabolismo , Cloroplastos/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Biogenesis of photosynthetic membranes depends on galactolipid synthesis, which relies on several cell compartments, notably the endoplasmic reticulum (ER) and the chloroplast envelope. Galactolipid synthesis involves lipid trafficking between both membrane compartments. In Arabidopsis, ALA10, a phospholipid flippase of the P4 type-ATPase family, counteracts the limitation of monogalactosyldiacylglycerol (MGDG) production and has a positive effect on leaf development. ALA10 locates in distinct domains of the ER depending on the ALIS (ALA interacting subunit) subunit it interacts with: close to the plasma membrane with ALIS1, or next to chloroplasts with ALIS5. It interacts with FAD2 (Fatty acid desaturase 2) and prevents accumulation of linolenic (18:3) containing phosphatidylcholine (PC) stimulating an increase of MGDG synthesis. Here we report that ALA10 interacts with PUB11 (plant U-box type 11), an E3 protein ubiquitin ligase, in vitro and in vivo. ALA10 is however ubiquitinated and degraded by the 26S proteasome in a PUB11-independent process. In pub11 null mutant, the proteasome-dependent degradation of ALA10 is retained and ALA10 is still subject to ubiquitination although its ubiquitination profile appears different. In the absence of PUB11, ALA10 is constrained to the ER close to chloroplasts, which is the usual location when ALA10 is overexpressed. Additionally, in this condition, the decrease of 18:3 containing PC is no longer observed. Taken together these results suggest, that ALA10 contributes in chloroplast-distal ER interacting domains, to reduce the 18:3 desaturation of PC and that PUB11 is involved in reconditioning of ALA10 from chloroplast-proximal to chloroplast-distal ER interacting domains.
RESUMO
Mono- and digalactosyldiacylglycerol are essential galactolipids for the biogenesis of plastids and functioning of the photosynthetic machinery. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by monogalactosyldiacylglycerol synthase 1 (MGD1), a monotopic protein located in the inner envelope membrane of chloroplasts, which transfers a galactose residue from UDP-galactose to diacylglycerol (DAG). MGD1 needs anionic lipids such as phosphatidylglycerol (PG) to be active, but the mechanism by which PG activates MGD1 is still unknown. Recent studies shed light on the catalytic mechanism of MGD1 and on the possible PG binding site. Particularly, Pro189 was identified as a potential residue implicated in PG binding and His155 as the putative catalytic residue. In the present study, using a multifaceted approach (Langmuir membrane models, atomic force microscopy, molecular dynamics; MD), we investigated the membrane binding properties of native MGD1 and mutants (P189A and H115A). We demonstrated that both residues are involved in PG binding, thus suggesting the existence of a PG-His catalytic dyad that should facilitate deprotonation of the nucleophile hydroxyl group of DAG acceptor. Interestingly, MD simulations showed that MGD1 induces a reorganization of lipids by attracting DAG molecules to create an optimal platform for binding.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Galactosiltransferases/metabolismo , Fosfatidilgliceróis/metabolismo , Adsorção , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Galactosiltransferases/química , Galactosiltransferases/genética , Lipídeos/química , MutaçãoRESUMO
Jasmonic acid (JA) biosynthesis and signaling are activated in Arabidopsis cultivated in phosphate (Pi) deprived conditions. This activation occurs mainly in photosynthetic tissues and is less important in roots. In leaves, the enhanced biosynthesis of JA coincides with membrane glycerolipid remodeling triggered by the lack of Pi. We addressed the possible role of JA on the dynamics and magnitude of glycerolipid remodeling in response to Pi deprivation and resupply. Based on combined analyses of gene expression, JA biosynthesis and glycerolipid remodeling in wild-type Arabidopsis and in the coi1-16 mutant, JA signaling seems important in the determination of the basal levels of phosphatidylcholine, phosphatidic acid (PA), monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol. JA impact on MGDG steady state level and fluctuations seem contradictory. In the coi1-16 mutant, the steady state level of MGDG is higher, possibly due to a higher level of PA in the mutant, activating MGD1, and to an increased expression of MGD3. These results support a possible impact of JA in limiting the overall content of this lipid. Concerning lipid variations, upon Pi deprivation, JA seems rather associated with a specific MGDG increase. Following Pi resupply, whereas the expression of glycerolipid remodeling genes returns to basal level, JA biosynthesis and signaling genes are still upregulated, likely due to a JA-induced positive feedback remaining active. Distinct impacts on enzymes synthesizing MGDG, that is, downregulating MGD3, possibly activating MGD1 expression and limiting the activation of MGD1 via PA, might allow JA playing a role in a sophisticated fine tuning of galactolipid variations.
Assuntos
Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Glicolipídeos/metabolismo , Oxilipinas/metabolismo , Fosfatos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Homeostase , Transdução de SinaisRESUMO
Chloroplasts are specific organelles of plant cells dedicated to photosynthesis and delimited by a two-membrane chloroplast envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are clearly different from the chloroplast envelope and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three main compartments of the chloroplast: the stroma, the thylakoids, and the chloroplast envelope. Three different protocols are provided, adapted for starting leaves of spinach, Arabidopsis, and pea.
Assuntos
Fracionamento Celular , Cloroplastos/fisiologia , Arabidopsis , Fracionamento Celular/métodos , Centrifugação , Pisum sativum , Células Vegetais/fisiologia , Spinacia oleracea , Tilacoides/fisiologiaRESUMO
The outer membrane and the inner membrane of the chloroplast envelope, also called OEM and IEM, have distinct functions connected with chloroplast biogenesis and chloroplast communication with the rest of the cell. Here we describe a method for the isolation of these membranes starting from intact chloroplast preparations, with two alternative procedures based on the starting material. One was developed from spinach leaves, the other one from pea leaves. The two procedures differ by the means that are used to isolate and rupture chloroplasts and to liberate each membrane.
Assuntos
Fracionamento Celular , Cloroplastos , Membranas Intracelulares , Pisum sativum , Spinacia oleracea , Fracionamento Celular/métodos , Centrifugação , Folhas de PlantaRESUMO
Chloroplasts contain about 3000 proteins, with approximately 400 of them located in the chloroplast envelope membranes, 1300 in the soluble stroma, and 1300 in the thylakoid membranes. Most of them are encoded by nuclear genes and translated as precursor proteins in the cytosol before their transport into chloroplasts. As a tool to control and characterize their import into plastids, we here describe an assay for in vitro protein import with isolated pea chloroplasts.
Assuntos
Cloroplastos/metabolismo , Transporte Proteico , Fracionamento Celular/métodos , Cloroplastos/genética , Técnicas In Vitro , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
A unique feature of chloroplasts is their high content of the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), which constitute up to 80% of their lipids. These galactolipids are synthesized in the chloroplast envelope membrane through the concerted action of galactosyltransferases, the so-called 'MGDG synthases (MGDs)' and 'DGDG synthases (DGDs),' which use uridine diphosphate (UDP)-galactose as donor. In Arabidopsis leaves, under standard conditions, the enzymes MGD1 and DGD1 provide the bulk of galactolipids, necessary for the massive expansion of thylakoid membranes. Under phosphate limited conditions, plants activate another pathway involving MGD2/MGD3 and DGD2 to provide additional DGDG that is exported to extraplastidial membranes where they partly replace phospholipids, a phosphate-saving mechanism in plants. A third enzyme system, which relies on the UDP-Gal-independent GGGT (also called SFR2 for SENSITIVE TO FREEZING 2), can be activated in response to a freezing stress. The biosynthesis of galactolipids by these multiple enzyme sets must be tightly regulated to meet the cellular demand in response to changing environmental conditions. The cooperation between MGD and DGD enzymes with a possible substrate channeling from diacylglycerol to MGDG and DGDG is supported by biochemical and biophysical studies and mutant analyses reviewed herein. The fine-tuning of MGDG to DGDG ratio, which allows the reversible transition from the hexagonal II to lamellar α phase of the lipid bilayer, could be a key factor in thylakoid biogenesis.
RESUMO
In plant cells, phosphatidylcholine (PC) is a major glycerolipid of most membranes but practically lacking from the plastid internal membranes. In chloroplasts, PC is absent from the thylakoids and the inner envelope membrane. It is however the main component of the outer envelope membrane, where it exclusively distributes in the outer monolayer. This unique distribution is likely related with operational compartmentalization of plant lipid metabolism. In this review, we summarize the different mechanisms involved in homeostasis of PC in plant cells. The specific origin of chloroplast PC is examined and the involvement of the P4-ATPase family of phospholipid flippases (ALA) is considered with a special attention to the recently reported effect of the endoplasmic reticulum-localized ALA10 on modification of chloroplast PC desaturation. The different possible roles of chloroplast PC are then discussed and analyzed in consideration of plant physiology.
Assuntos
Cloroplastos/metabolismo , Fosfatidilcolinas/metabolismo , Plantas/metabolismo , Homeostase , Propriedades de SuperfícieRESUMO
Photosynthetic membranes, or thylakoids, are the most extensive membrane system found in the biosphere. They form flattened membrane cisternae in the cytosol of cyanobacteria and in the stroma of chloroplasts. The efficiency of light energy capture and conversion, critical for primary production in ecosystems, relies on the rapid expansion of thylakoids and their versatile reorganization in response to light changes. Thylakoid biogenesis results from the assembly of a lipid matrix combined with the incorporation of protein components. Four lipid classes are conserved from cyanobacteria to chloroplasts: mono- and digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, and phosphatidyldiacylglycerol. This review focuses on the production and biophysical properties of galactolipids, making them determinant factors for the nonvesicular/nonlamellar biogenesis and for the three-dimensional architecture of nascent thylakoids. The regulation of MGD1, the committing enzyme of galactolipid biosynthesis in Arabidopsis, via feedback regulatory loops and control of protein binding to membranes, is also detailed.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fotossíntese/fisiologia , Células Vegetais/metabolismo , Tilacoides/metabolismoRESUMO
Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major lipid components of photosynthetic membranes, and hence the most abundant lipids in the biosphere. They are essential for assembly and function of the photosynthetic apparatus. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by MGDG synthase 1 (MGD1), which transfers a galactosyl residue from UDP-galactose to diacylglycerol (DAG). MGD1 is a monotopic protein that is embedded in the inner envelope membrane of chloroplasts. Once produced, MGDG is transferred to the outer envelope membrane, where DGDG synthesis occurs, and to thylakoids. Here we present two crystal structures of MGD1: one unliganded and one complexed with UDP. MGD1 has a long and flexible region (approximately 50 amino acids) that is required for DAG binding. The structures reveal critical features of the MGD1 catalytic mechanism and its membrane binding mode, tested on biomimetic Langmuir monolayers, giving insights into chloroplast membrane biogenesis. The structural plasticity of MGD1, ensuring very rapid capture and utilization of DAG, and its interaction with anionic lipids, possibly driving the construction of lipoproteic clusters, are consistent with the role of this enzyme, not only in expansion of the inner envelope membrane, but also in supplying MGDG to the outer envelope and nascent thylakoid membranes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Galactolipídeos/biossíntese , Galactosiltransferases/metabolismo , Tilacoides/metabolismo , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Biocatálise , Vias Biossintéticas/genética , Domínio Catalítico , Cristalografia por Raios X , Diglicerídeos/química , Diglicerídeos/metabolismo , Eletroforese em Gel de Poliacrilamida , Galactose/química , Galactose/metabolismo , Galactosiltransferases/química , Galactosiltransferases/genética , Membranas Intracelulares/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Difosfato de Uridina/química , Difosfato de Uridina/metabolismo , Difração de Raios XRESUMO
The mitochondrion is an organelle originating from an endosymbiotic event and playing a role in several fundamental processes such as energy production, metabolite syntheses, and programmed cell death. This organelle is delineated by two membranes whose synthesis requires an extensive exchange of phospholipids with other cellular organelles such as endoplasmic reticulum (ER) and vacuolar membranes in yeast. These transfers of phospholipids are thought to occur by a non-vesicular pathway at contact sites between two closely apposed membranes. In plants, little is known about the biogenesis of mitochondrial membranes. Contact sites between ER and mitochondria are suspected to play a similar role in phospholipid trafficking as in yeast, but this has never been demonstrated. In contrast, it has been shown that plastids are able to transfer lipids to mitochondria during phosphate starvation. However, the proteins involved in such transfer are still unknown. Here, we identified in Arabidopsis thaliana a large lipid-enriched complex called the mitochondrial transmembrane lipoprotein (MTL) complex. The MTL complex contains proteins located in the two mitochondrial membranes and conserved in all eukaryotic cells, such as the TOM complex and AtMic60, a component of the MICOS complex. We demonstrate that AtMic60 contributes to the export of phosphatidylethanolamine from mitochondria and the import of galactoglycerolipids from plastids during phosphate starvation. Furthermore, AtMic60 promotes lipid desorption from membranes, likely as an initial step for lipid transfer, and binds to Tom40, suggesting that AtMic60 could regulate the tethering between the inner and outer membranes of mitochondria.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Transporte Proteico , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismoRESUMO
The biogenesis of photosynthetic membranes relies on galactoglycerolipids, which are synthesized via pathways that are dispatched over several cell compartments. This membrane biogenesis requires both trafficking of lipid intermediates and a tight homeostatic regulation. In this work, we address the role of ALA10 (for aminophospholipid ATPase), a P4-type ATPase, in a process counteracting the monogalactosyldiacylglycerol (MGDG) shortage in Arabidopsis (Arabidopsis thaliana) leaves. ALA10 can interact with protein partners, ALIS1 (for ALA-interacting subunit1) or ALIS5, leading to differential endomembrane localizations of the interacting proteins, close to the plasma membrane with ALIS1 or to chloroplasts with ALIS5. ALA10 interacts also with FATTY ACID DESATURASE2 (FAD2), and modification of ALA10 expression affects phosphatidylcholine (PC) fatty acyl desaturation by disturbing the balance between FAD2 and FAD3 activities. Modulation of ALA10 expression downstream impacts the fatty acyl composition of chloroplast PC. ALA10 expression also enhances leaf growth and improves the MGDG-PC ratio, possibly through MGDG SYNTHASE1 (MGD1) activation by phosphatidic acid. The positive effect of ALA10 on leaf development is significant in conditions such as upon treatment of plants with Galvestine-1, an inhibitor of MGDG synthases, or when plants are grown at chilling temperature.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Fosfatidilcolinas/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Cloroplastos/metabolismo , Retículo Endoplasmático/metabolismo , Galactolipídeos/metabolismo , Perfilação da Expressão Gênica , Metabolismo dos Lipídeos , Folhas de Planta/metabolismo , Plantas Geneticamente ModificadasRESUMO
In higher plants, fatty acids (FAs) with 18 carbons (18C) represent about 70% of total FAs, the most abundant species being 18:2 and 18:3. These two polyunsaturated FAs (PUFAs) represent about 55% of total FAs in Arabidopsis cell suspension cultures, whereas 18:1 represents about 10%. The level of PUFAs may vary, depending on ill-defined factors. Here, we compared various sets of plant cell cultures and noticed a correlation between the growth rate of a cell population and the level of unsaturation of 18C FAs. These observations suggest that the final level of PUFAs might depend in part on the rate of cell division, and that FAD2 and FAD3 desaturases, which are respectively responsible for the formation of 18:2 and 18:3 on phospholipids, have limiting activities in fast-growing cultures. In plant cell culture, phosphate (Pi) deprivation is known to impair cell division and to trigger lipid remodeling. We observed that Pi starvation had no effect on the expression of FAD genes, and that the level of PUFAs in this situation was also correlated with the growth rate. Thus, the level of PUFAs appears as a hallmark in determining cell maturity and aging.
Assuntos
Arabidopsis/citologia , Ácidos Graxos Insaturados/farmacologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Divisão Celular/efeitos dos fármacos , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Fosfatos/deficiência , Fosfatos/metabolismo , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Glycerolipid synthesis in plant cells is characterized by an intense trafficking of lipids between the endoplasmic reticulum (ER) and chloroplasts. Initially, fatty acids are synthesized within chloroplasts and are exported to the ER where they are used to build up phospholipids and triacylglycerol. Ultimately, derivatives of these phospholipids return to chloroplasts to form galactolipids, monogalactosyldiacylglycerol and digalactosyldiacylglycerol, the main and essential lipids of photosynthetic membranes. Lipid trafficking was proposed to transit through membrane contact sites (MCSs) connecting both organelles. Here, we review recent insights into ER-chloroplast MCSs and lipid trafficking between chloroplasts and the ER.
Assuntos
Cloroplastos/metabolismo , Retículo Endoplasmático/metabolismo , Metabolismo dos Lipídeos , Animais , Transporte Biológico , LipídeosRESUMO
Diatoms constitute a major phylum of phytoplankton biodiversity in ocean water and freshwater ecosystems. They are known to respond to some chemical variations of the environment by the accumulation of triacylglycerol, but the relative changes occurring in membrane glycerolipids have not yet been studied. Our goal was first to define a reference for the glycerolipidome of the marine model diatom Phaeodactylum tricornutum, a necessary prerequisite to characterize and dissect the lipid metabolic routes that are orchestrated and regulated to build up each subcellular membrane compartment. By combining multiple analytical techniques, we determined the glycerolipid profile of P. tricornutum grown with various levels of nitrogen or phosphorus supplies. In different P. tricornutum accessions collected worldwide, a deprivation of either nutrient triggered an accumulation of triacylglycerol, but with different time scales and magnitudes. We investigated in depth the effect of nutrient starvation on the Pt1 strain (Culture Collection of Algae and Protozoa no. 1055/3). Nitrogen deprivation was the more severe stress, triggering thylakoid senescence and growth arrest. By contrast, phosphorus deprivation induced a stepwise adaptive response. The time scale of the glycerolipidome changes and the comparison with large-scale transcriptome studies were consistent with an exhaustion of unknown primary phosphorus-storage molecules (possibly polyphosphate) and a transcriptional control of some genes coding for specific lipid synthesis enzymes. We propose that phospholipids are secondary phosphorus-storage molecules broken down upon phosphorus deprivation, while nonphosphorus lipids are synthesized consistently with a phosphatidylglycerol-to-sulfolipid and a phosphatidycholine-to-betaine lipid replacement followed by a late accumulation of triacylglycerol.
Assuntos
Diatomáceas/fisiologia , Lipídeos de Membrana/metabolismo , Nitrogênio/metabolismo , Fósforo/metabolismo , Adaptação Fisiológica/fisiologia , Diatomáceas/metabolismo , Perfilação da Expressão Gênica , Lipídeos de Membrana/fisiologia , Tilacoides/metabolismo , Tilacoides/fisiologia , Triglicerídeos/metabolismo , Triglicerídeos/fisiologiaRESUMO
Inorganic phosphate (Pi) is present in most soils at suboptimal concentrations, strongly limiting plant development. Plants have the ability to sense and adapt to the surrounding ionic environment, and several genes involved in the response to Pi starvation have been identified. However, a global understanding of the regulatory mechanisms involved in this process is still elusive. Here, we have initiated a chemical genetics approach and isolated compounds that inhibit the response to Pi starvation in Arabidopsis (Arabidopsis thaliana). Molecules were screened for their ability to inhibit the expression of a Pi starvation marker gene (the high-affinity Pi transporter PHT1;4). A drug family named Phosphatin (PTN; Pi starvation inhibitor), whose members act as partial suppressors of Pi starvation responses, was thus identified. PTN addition also reduced various traits of Pi starvation, such as phospholipid/glycolipid conversion, and the accumulation of starch and anthocyanins. A transcriptomic assay revealed a broad impact of PTN on the expression of many genes regulated by low Pi availability. Despite the reduced amount of Pi transporters and resulting reduced Pi uptake capacity, no reduction of Pi content was observed. In addition, PTN improved plant growth; this reveals that the developmental restrictions induced by Pi starvation are not a consequence of metabolic limitation but a result of genetic regulation. This highlights the existence of signal transduction pathway(s) that limit plant development under the Pi starvation condition.
Assuntos
Arabidopsis/efeitos dos fármacos , Arabidopsis/fisiologia , Fosfatos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Concentração Inibidora 50 , Ferro/metabolismo , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Amido/metabolismo , Relação Estrutura-Atividade , Compostos de Sulfidrila/químicaRESUMO
The understanding of chloroplast function requires the precise localization of proteins in each of its sub-compartments. High-sensitivity mass spectrometry has allowed the inventory of proteins in thylakoid, stroma, and envelope fractions. Concerning membrane association, proteins can be either integral or peripheral or even soluble proteins bound transiently to a membrane complex. We sought a method providing information at the surface of the outer envelope membrane (OEM), based on specific tagging with biotin or proteolysis using thermolysin, a non-membrane permeable protease. To evaluate this method, envelope, thylakoid, and stroma proteins were separated by two-dimensional electrophoresis and analyzed by immunostaining and mass spectrometry. A short selection of proteins associated to the chloroplast envelope fraction was checked after superficial treatments of intact chloroplasts. We showed that this method could allow the characterization of OEM embedded proteins facing the cytosol, as well as peripheral and soluble proteins associated via tight or lose interactions. Some stromal proteins were associated with biotinylated spots and analyzes are still needed to determine whether polypeptides were tagged prior import or if they co-migrated with OEM proteins. This method also suggests that some proteins associated with the inner envelope membrane (IEM) might need the integrity of a trans-envelope (IEM-OEM) protein complex (e.g., division ring-forming components) or at least an intact OEM partner. Following this evaluation, proteomic analyzes should be refined and the putative role of inter-membrane space components stabilizing trans-envelope complexes demonstrated. For future comprehensive studies, perspectives include the dynamic analyses of OEM proteins and IEM-OEM complexes in various physiological contexts and using virtually any other purified membrane organelle.
RESUMO
Mono- and digalactosyldiacylglycerol (MGDG and DGDG) are the most abundant lipids of photosynthetic membranes (thylakoids). In Arabidopsis green tissues, MGD1 is the main enzyme synthesizing MGDG. This monotopic enzyme is embedded in the inner envelope membrane of chloroplasts. DGDG synthesis occurs in the outer envelope membrane. Although the suborganellar localization of MGD1 has been determined, it is still not known how the lipid/glycolipid composition influences its binding to the membrane. The existence of a topological relationship between MGD1 and "embryonic" thylakoids is also unknown. To investigate MGD1 membrane binding, we used a Langmuir membrane model allowing the tuning of both lipid composition and packing. Surprisingly, MGD1 presents a high affinity to MGDG, its product, which maintains the enzyme bound to the membrane. This positive feedback is consistent with the low level of diacylglycerol, the substrate of MGD1, in chloroplast membranes. By contrast, MGD1 is excluded from membranes highly enriched in, or made of, pure DGDG. DGDG therefore exerts a retrocontrol, which is effective on the overall synthesis of galactolipids. Previously identified activators, phosphatidic acid and phosphatidylglycerol, also play a role on MGD1 membrane binding via electrostatic interactions, compensating the exclusion triggered by DGDG. The opposite effects of MGDG and DGDG suggest a role of these lipids on the localization of MGD1 in specific domains. Consistently, MGDG induces the self-organization of MGD1 into elongated and reticulated nanostructures scaffolding the chloroplast membrane.-Sarkis, J., Rocha, J., Maniti, O., Jouhet, J., Vié, V., Block, M. A., Breton, C., Maréchal, E., Girard-Egrot, A. The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts.