Publications by authors named "Lilly Maneta-Peyret"

20 Publications

  • Page 1 of 1

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation.

J Biol Chem 2021 Jan-Jun;296:100602. Epub 2021 Mar 27.

Laboratoire de Biogènese Membranaire, UMR 5200, CNRS, Université de Bordeaux, Villenave d'Ornon Cedex, France. Electronic address:

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph-mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.
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http://dx.doi.org/10.1016/j.jbc.2021.100602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099651PMC
March 2021

Doxorubicin Inhibits Phosphatidylserine Decarboxylase and Modifies Mitochondrial Membrane Composition in HeLa Cells.

Int J Mol Sci 2020 Feb 15;21(4). Epub 2020 Feb 15.

INSERM U1211, Rare Diseases: Genetic and Metabolism, F-33076 Bordeaux, France.

Doxorubicin (DXR) is a drug widely used in chemotherapy. Its mode of action is based on its intercalation properties, involving the inhibition of topoisomerase II. However, few studies have reported the mitochondrial effects of DXR while investigating cardiac toxicity induced by the treatment, mostly in pediatric cases. Here, we demonstrate that DXR alters the mitochondrial membrane composition associated with bioenergetic impairment and cell death in human cancer cells. The remodeling of the mitochondrial membrane was explained by phosphatidylserine decarboxylase (PSD) inhibition by DXR. PSD catalyzes phosphatidylethanolamine (PE) synthesis from phosphatidylserine (PS), and DXR altered the PS/PE ratio in the mitochondrial membrane. Moreover, we observed that DXR localized to the mitochondrial compartment and drug uptake was rapid. Evaluation of other topoisomerase II inhibitors did not show any impact on the mitochondrial membrane composition, indicating that the DXR effect was specific. Therefore, our findings revealed a side molecular target for DXR and PSD, potentially involved in DXR anti-cancer properties and the associated toxicity.
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http://dx.doi.org/10.3390/ijms21041317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072979PMC
February 2020

Developmental control of plant Rho GTPase nano-organization by the lipid phosphatidylserine.

Science 2019 04;364(6435):57-62

Laboratoire Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRA, F-69342 Lyon, France.

Rho guanosine triphosphatases (GTPases) are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. We found that the phospholipid phosphatidylserine acts as a developmentally controlled lipid rheostat that tunes Rho GTPase signaling in Live superresolution single-molecule imaging revealed that the protein Rho of Plants 6 (ROP6) is stabilized by phosphatidylserine into plasma membrane nanodomains, which are required for auxin signaling. Our experiments also revealed that the plasma membrane phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work shows that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.
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http://dx.doi.org/10.1126/science.aav9959DOI Listing
April 2019

The multistress-induced Translocator protein (TSPO) differentially modulates storage lipids metabolism in seeds and seedlings.

Plant J 2018 10 16;96(2):274-286. Epub 2018 Aug 16.

Louvain Institute of Biomolecular Science and Technology (LIBST), UCLouvain, Croix du Sud 4-5, L7.07.14, 1348, Louvain-la-Neuve, Belgium.

Translocator proteins (TSPO) are conserved membrane proteins extensively studied in mammals, but their function is still unclear. Angiosperm TSPO are transiently induced by abiotic stresses in vegetative tissues. We showed previously that constitutive expression of the Arabidopsis TSPO (AtTSPO) could be detrimental to the cell. Degradation of AtTSPO requires an active autophagy pathway. We show here that genetic modifications of TSPO expression in plant and yeast cells reduce the levels of cytoplasmic lipid droplets (LD). Transgenic Arabidopsis seedlings overexpressing AtTSPO contain less LD as compared with wild type (WT). LD levels were increased in Arabidopsis AtTSPO knockout (KO) seedlings. Deletion of the Schizosaccharomyces pombe TSPO resulted in an increase in LD level in the cell. As compared with the WT, the mutant strain was more sensitive to cerulenin, an inhibitor of fatty acids and sterol biosynthesis. We found that in contrast with seedlings, overexpression of AtTSPO (OE) resulted in an up to 50% increase in seeds fatty acids as compared with WT. A time course experiment revealed that after 4 days of seed imbibition, the levels of triacylglycerol (TAG) was still higher in the OE seeds as compared with WT or KO seeds. However, the de novo synthesis of phospholipids and TAG after 24 h of imbibition was substantially reduced in OE seeds as compared with WT or KO seeds. Our findings support a plant TSPO role in energy homeostasis in a tissue-specific manner, enhancing fatty acids and LD accumulation in mature seeds and limiting LD levels in seedlings.
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http://dx.doi.org/10.1111/tpj.14028DOI Listing
October 2018

Constraint-Based Modeling Highlights Cell Energy, Redox Status and α-Ketoglutarate Availability as Metabolic Drivers for Anthocyanin Accumulation in Grape Cells Under Nitrogen Limitation.

Front Plant Sci 2018 17;9:421. Epub 2018 May 17.

UMR 1287 Ecophysiologie et Génomique Fonctionnelle de la Vigne, Université de Bordeaux, Institut des Sciences de la Vigne et du Vin, Bordeaux, France.

Anthocyanin biosynthesis is regulated by environmental factors (such as light, temperature, and water availability) and nutrient status (such as carbon, nitrogen, and phosphate nutrition). Previous reports show that low nitrogen availability strongly enhances anthocyanin accumulation in non carbon-limited plant organs or cell suspensions. It has been hypothesized that high carbon-to-nitrogen ratio would lead to an energy excess in plant cells, and that an increase in flavonoid pathway metabolic fluxes would act as an "energy escape valve," helping plant cells to cope with energy and carbon excess. However, this hypothesis has never been tested directly. To this end, we used the grapevine L. cultivar Gamay Teinturier (syn. Gamay Freaux or Freaux Tintorier, VIVC #4382) cell suspension line as a model system to study the regulation of anthocyanin accumulation in response to nitrogen supply. The cells were sub-cultured in the presence of either control (25 mM) or low (5 mM) nitrate concentration. Targeted metabolomics and enzyme activity determinations were used to parametrize a constraint-based model describing both the central carbon and nitrogen metabolisms and the flavonoid (phenylpropanoid) pathway connected by the energy (ATP) and reducing power equivalents (NADPH and NADH) cofactors. The flux analysis (2 flux maps generated, for control and low nitrogen in culture medium) clearly showed that in low nitrogen-fed cells all the metabolic fluxes of central metabolism were decreased, whereas fluxes that consume energy and reducing power, were either increased (upper part of glycolysis, shikimate, and flavonoid pathway) or maintained (pentose phosphate pathway). Also, fluxes of flavanone 3β-hydroxylase, flavonol synthase, and anthocyanidin synthase were strongly increased, advocating for a regulation of the flavonoid pathway by alpha-ketoglutarate levels. These results strongly support the hypothesis of anthocyanin biosynthesis acting as an energy escape valve in plant cells, and they open new possibilities to manipulate flavonoid production in plant cells. They do not, however, support a role of anthocyanins as an effective mechanism for coping with carbon excess in high carbon to nitrogen ratio situations in grape cells. Instead, constraint-based modeling output and biomass analysis indicate that carbon excess is dealt with by vacuolar storage of soluble sugars.
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http://dx.doi.org/10.3389/fpls.2018.00421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5966944PMC
May 2018

A Combinatorial Lipid Code Shapes the Electrostatic Landscape of Plant Endomembranes.

Dev Cell 2018 05 10;45(4):465-480.e11. Epub 2018 May 10.

Laboratoire Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRA, Lyon 69342, France. Electronic address:

Membrane surface charge is critical for the transient, yet specific recruitment of proteins with polybasic regions to certain organelles. In eukaryotes, the plasma membrane (PM) is the most electronegative compartment of the cell, which specifies its identity. As such, membrane electrostatics is a central parameter in signaling, intracellular trafficking, and polarity. Here, we explore which are the lipids that control membrane electrostatics using plants as a model. We show that phosphatidylinositol-4-phosphate (PI4P), phosphatidic acidic (PA), and phosphatidylserine (PS) are separately required to generate the electrostatic signature of the plant PM. In addition, we reveal the existence of an electrostatic territory that is organized as a gradient along the endocytic pathway and is controlled by PS/PI4P combination. Altogether, we propose that combinatorial lipid composition of the cytosolic leaflet of organelles not only defines the electrostatic territory but also distinguishes different functional compartments within this territory by specifying their varying surface charges.
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http://dx.doi.org/10.1016/j.devcel.2018.04.011DOI Listing
May 2018

The odd one out: Arabidopsis reticulon 20 does not bend ER membranes but has a role in lipid regulation.

Sci Rep 2018 02 2;8(1):2310. Epub 2018 Feb 2.

Plant Cell Biology, Biological and Medical Sciences, Oxford Brookes University, Oxford, OX3 0BP, United Kingdom.

Reticulons are integral ER membrane proteins characterised by a reticulon homology domain comprising four transmembrane domains which results in the proteins sitting in the membrane in a W-topology. Here we report on a novel subgroup of reticulons with an extended N-terminal domain and in particular on arabidopsis reticulon 20. Using high resolution confocal microscopy we show that reticulon 20 is located in a unique punctate pattern on the ER membrane. Its closest homologue reticulon 19 labels the whole ER. Other than demonstrated for the other members of the reticulon protein family RTN20 and 19 do not display ER constriction phenotypes on over expression. We show that mutants in RTN20 or RTN19, respectively, display a significant change in sterol composition in roots indicating a role in lipid regulation. A third homologue in this family -3BETAHSD/D1- is unexpectedly localised to ER exit sites resulting in an intriguing location difference for the three proteins.
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http://dx.doi.org/10.1038/s41598-018-20840-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797236PMC
February 2018

ER Membrane Lipid Composition and Metabolism: Lipidomic Analysis.

Methods Mol Biol 2018 ;1691:125-137

CNRS-University of Bordeaux, UMR 5200 Membrane Biogenesis Laboratory, INRA Bordeaux Aquitaine, 33140, Villenave d'Ornon, France.

Plant ER membranes are the major site of biosynthesis of several lipid families (phospholipids, sphingolipids, neutral lipids such as sterols and triacylglycerols). The structural diversity of lipids presents considerable challenges to comprehensive lipid analysis. This chapter will briefly review the various biosynthetic pathways and will detail several aspects of the lipid analysis: lipid extraction, handling, separation, detection, identification, and data presentation. The different tools/approaches used for lipid analysis will also be discussed in relation to the studies to be carried out on lipid metabolism and function.
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http://dx.doi.org/10.1007/978-1-4939-7389-7_10DOI Listing
June 2018

The Qb-SNARE Memb11 interacts specifically with Arf1 in the Golgi apparatus of Arabidopsis thaliana.

J Exp Bot 2015 Nov 24;66(21):6665-78. Epub 2015 Jul 24.

CNRS-University of Bordeaux, UMR 5200 Membrane Biogenesis Laboratory, INRA Bordeaux Aquitaine, 33140 Villenave d'Ornon, France Bordeaux Imaging Center, UMS 3420 CNRS, US4 INSERM, University of Bordeaux, 33000 Bordeaux, France

The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are critical for the function of the secretory pathway. The SNARE Memb11 is involved in membrane trafficking at the ER-Golgi interface. The aim of the work was to decipher molecular mechanisms acting in Memb11-mediated ER-Golgi traffic. In mammalian cells, the orthologue of Memb11 (membrin) is potentially involved in the recruitment of the GTPase Arf1 at the Golgi membrane. However molecular mechanisms associated to Memb11 remain unknown in plants. Memb11 was detected mainly at the cis-Golgi and co-immunoprecipitated with Arf1, suggesting that Arf1 may interact with Memb11. This interaction of Memb11 with Arf1 at the Golgi was confirmed by in vivo BiFC (Bimolecular Fluorescence Complementation) experiments. This interaction was found to be specific to Memb11 as compared to either Memb12 or Sec22. Using a structural bioinformatic approach, several sequences in the N-ter part of Memb11 were hypothesized to be critical for this interaction and were tested by BiFC on corresponding mutants. Finally, by using both in vitro and in vivo approaches, we determined that only the GDP-bound form of Arf1 interacts with Memb11. Together, our results indicate that Memb11 interacts with the GDP-bound form of Arf1 in the Golgi apparatus.
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http://dx.doi.org/10.1093/jxb/erv373DOI Listing
November 2015

Phospholipid biosynthesis increases in RHD3-defective mutants.

Plant Signal Behav 2014 ;9(9):e29657

a Laboratoire de Biogenèse Membranaire ; UMR 5200 CNRS-University of Bordeaux, INRA Bordeaux Aquitaine ; Villenave d'Ornon, France.

RHD3, a member of the ER-shaping dynamin-like GTPases, is required in the transition from a cisternal to a tubular ER architecture during cell growth. The aberrant ER morphology in rhd3 mutants may be correlated with alterations of the ER lipid bilayer. We analyzed the lipid fraction of rhd3 mutants at qualitative and quantitative levels. We observed an increase of the amount of phospholipids but also of proteins in the mutants, indicating an overall increase of ER membranes. This increase may indicate that phospholipid biosynthesis is deregulated in rhd3 mutants. It was shown that overexpression of PIS1 and PIS2 (involved in phosphatidylinositol biosynthesis) induces the synthesis of phosphatidylinositol (PI) but also of phosphatidic acid and that overexpression of PIS1 also induces the synthesis of phosphatidylethanolamine and diacylglycerol. (1) We wondered whether PIS1 or PIS2 could be linked to the increase of the amount of phospholipids in rhd3 mutants. To answer, we measured the phospholipid composition in the double mutants rhd3-7/pis1 and rhd3-7/pis2. The phospholipid increase in the rhd3 mutant was compensated in rhd3-7/pis1 but not rhd3-7/pis2. Our results suggest a possible deregulation of PIS1 in the rhd3 mutant.
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http://dx.doi.org/10.4161/psb.29657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203640PMC
November 2015

Characterisation of detergent-insoluble membranes in pollen tubes of Nicotiana tabacum (L.).

Biol Open 2015 Feb 20;4(3):378-99. Epub 2015 Feb 20.

Laboratoire de Biogenèse Membranaire, Université Bordeaux Segalen, 71 Avenue Edouard Bourlaux, 33883 Villenave d'Ornon, France.

Pollen tubes are the vehicle for sperm cell delivery to the embryo sac during fertilisation of Angiosperms. They provide an intriguing model for unravelling mechanisms of growing to extremes. The asymmetric distribution of lipids and proteins in the pollen tube plasma membrane modulates ion fluxes and actin dynamics and is maintained by a delicate equilibrium between exocytosis and endocytosis. The structural constraints regulating polarised secretion and asymmetric protein distribution on the plasma membrane are mostly unknown. To address this problem, we investigated whether ordered membrane microdomains, namely membrane rafts, might contribute to sperm cell delivery. Detergent insoluble membranes, rich in sterols and sphingolipids, were isolated from tobacco pollen tubes. MALDI TOF/MS analysis revealed that actin, prohibitins and proteins involved in methylation reactions and in phosphoinositide pattern regulation are specifically present in pollen tube detergent insoluble membranes. Tubulins, voltage-dependent anion channels and proteins involved in membrane trafficking and signalling were also present. This paper reports the first evidence of membrane rafts in Angiosperm pollen tubes, opening new perspectives on the coordination of signal transduction, cytoskeleton dynamics and polarised secretion.
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http://dx.doi.org/10.1242/bio.201410249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4359744PMC
February 2015

The importance of cardiolipin synthase for mitochondrial ultrastructure, respiratory function, plant development, and stress responses in Arabidopsis.

Plant Cell 2013 Oct 22;25(10):4195-208. Epub 2013 Oct 22.

Institut de Biologie des Plantes, Saclay Plant Science, Université de Paris-Sud XI, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8618, 91405 Orsay cedex, France.

Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane. In animals and yeast (Saccharomyces cerevisiae), CL depletion affects the stability of respiratory supercomplexes and is thus crucial to the energy metabolism of obligate aerobes. In eukaryotes, the last step of CL synthesis is catalyzed by CARDIOLIPIN SYNTHASE (CLS), encoded by a single-copy gene. Here, we characterize a cls mutant in Arabidopsis thaliana, which is devoid of CL. In contrast to yeast cls, where development is little affected, Arabidopsis cls seedlings are slow developing under short-day conditions in vitro and die if they are transferred to long-day (LD) conditions. However, when transferred to soil under LD conditions under low light, cls plants can reach the flowering stage, but they are not fertile. The cls mitochondria display abnormal ultrastructure and reduced content of respiratory complex I/complex III supercomplexes. The marked accumulation of tricarboxylic acid cycle derivatives and amino acids demonstrates mitochondrial dysfunction. Mitochondrial and chloroplastic antioxidant transcripts are overexpressed in cls leaves, and cls protoplasts are more sensitive to programmed cell death effectors, UV light, and heat shock. Our results show that CLS is crucial for correct mitochondrial function and development in Arabidopsis under both optimal and stress conditions.
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http://dx.doi.org/10.1105/tpc.113.118018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3877823PMC
October 2013

Cell wall constrains lateral diffusion of plant plasma-membrane proteins.

Proc Natl Acad Sci U S A 2012 Jul 11;109(31):12805-10. Epub 2012 Jun 11.

Department of Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom.

A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in animal cells. This anomalous diffusion results from a combination of structuring factors including protein-protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction.
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http://dx.doi.org/10.1073/pnas.1202040109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3411962PMC
July 2012

Glucosylceramide biosynthesis is involved in Golgi morphology and protein secretion in plant cells.

Traffic 2010 Apr 17;11(4):479-90. Epub 2009 Dec 17.

Université V. Segalen Bordeaux 2, Laboratoire de Biogenèse Membranaire, CNRS UMR 5200, 146, rue Léo Saignat, 33076 Bordeaux Cedex, France.

Lipids have an established role as structural components of membranes or as signalling molecules, but their role as molecular actors in protein secretion is less clear. The complex sphingolipid glucosylceramide (GlcCer) is enriched in the plasma membrane and lipid microdomains of plant cells, but compared to animal and yeast cells, little is known about the role of GlcCer in plant physiology. We have investigated the influence of GlcCer biosynthesis by glucosylceramide synthase (GCS) on the efficiency of protein transport through the plant secretory pathway and on the maintenance of normal Golgi structure. We determined that GlcCer is synthesized at the beginning of the plant secretory pathway [mainly endoplasmic reticulum (ER)] and that D,L-threo-1-phenyl-2-decanoyl amino-3-morpholino-propanol (PDMP) is a potent inhibitor of plant GCS activity in vitro and in vivo. By an in vivo confocal microscopy approach in tobacco leaves infiltrated with PDMP, we showed that the decrease in GlcCer biosynthesis disturbed the transport of soluble and membrane secretory proteins to the cell surface, as these proteins were partly retained intracellularly in the ER and/or Golgi. Electron microscopic observations of Arabidopsis thaliana root cells after high-pressure freezing and freeze substitution evidenced strong morphological changes in the Golgi bodies, pointing to a link between decreased protein secretion and perturbations of Golgi structure following inhibition of GlcCer biosynthesis in plant cells.
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http://dx.doi.org/10.1111/j.1600-0854.2009.01030.xDOI Listing
April 2010

A novel di-acidic motif facilitates ER export of the syntaxin SYP31.

J Exp Bot 2009 10;60(11):3157-65. Epub 2009 Jun 10.

University of Bordeaux 2, Membrane Biogenesis Laboratory, CNRS UMR 5200, 146, rue Léo Saignat, 33076 Bordeaux Cedex, France.

It is generally accepted that ER protein export is largely influenced by the transmembrane domain (TMD). The situation is unclear for membrane-anchored proteins such as SNAREs, which are anchored to the membrane by their TMD at the C-terminus. For example, in plants, Sec22 and SYP31 (a yeast Sed5 homologue) have a 17 aa TMD but different locations (ER/Golgi and Golgi), indicating that TMD length alone is not sufficient to explain their targeting. To establish the identity of factors that influence SNARE targeting, mutagenesis and live cell imaging experiments were performed on SYP31. It was found that deletion of the entire N-terminus domain of SYP31 blocked the protein in the ER. Several deletion mutants of different parts of this N-terminus domain indicated that a region between the SNARE helices Hb and Hc is required for Golgi targeting. In this region, replacement of the aa sequence MELAD by GAGAG or MALAG retained the protein in the ER, suggesting that MELAD may function as a di-acidic ER export motif EXXD. This suggestion was further verified by replacing the established di-acidic ER export motif DLE of a type II Golgi protein AtCASP and a membrane-anchored type I chimaera, TMcCCASP, by MELAD or GAGAG. The MELAD motif allowed the proteins to reach the Golgi, whereas the motif GAGAG was found to be insufficient to facilitate ER protein export. Our analyses indicate that we have identified a novel and transplantable di-acidic motif that facilitates ER export of SYP31 and may function for type I and type II proteins in plants.
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http://dx.doi.org/10.1093/jxb/erp155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2718219PMC
October 2009

Immunolocalization and high affinity interactions of acyl-CoAs with proteins: an original study with anti-acyl-CoA antibodies.

Biochim Biophys Acta 2006 Jan 26;1761(1):91-9. Epub 2006 Jan 26.

Laboratoire de Biogenèse Membranaire, CNRS-UMR 5200, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.

Anti-acyl-Coenzyme A (acyl-CoA) antibodies were used to detect fatty acyl-CoAs in cultured rat hippocampal neurons, in which important lipid metabolism and transport occur. Hippocampus was chosen because of his involvement in many cerebral functions and diseases. Immunofluorescence experiments showed an intense labelling within neurites and cell bodies. Labelling seems to be associated with vesicles and membrane domains. We have shown by immunoblot experiments that the labelling corresponded to acyl-CoAs which were in strong interaction with proteins, without being covalently bound to them. Immunoprecipitation experiments, followed by proteomic analysis, showed that anti-acyl-CoA antibodies were also able to immunoprecipitate multiprotein complexes, principally related to vesicle trafficking and/or to membrane rafts.
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http://dx.doi.org/10.1016/j.bbalip.2005.12.012DOI Listing
January 2006

Chromatographic purification of an insoluble histidine tag recombinant Ykt6p SNARE from Arabidopsis thaliana over-expressed in E. coli.

J Chromatogr B Analyt Technol Biomed Life Sci 2004 Aug;808(1):83-9

Laboratoire de Biogenèse Membranaire, CNRS-UMR 5544, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.

In order to undertake in plant cell the study of the endoplasmic reticulum (ER)-Golgi apparatus (GA) protein and/or lipid vesicular transport pathway, expressed sequence tag (EST) coding for a homologue to the yeast soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Ykt6p has been cloned in Arabidopsis thaliana by reverse transcription polymerase chain reaction (RT-PCR). The corresponding protein was over-expressed as a recombinant histidine-tag (his-tag) protein in E. coli. Starting from one litter of culture, an ultrasonic homogenization was performed for cell disruption and after centrifugation the Arabidopsis Ykt6p SNARE present in inclusion bodies in the pellet was solubilized. After centrifugation, the clarified feedstock obtained was injected onto an immobilized metal affinity chromatography (IMAC) in presence of 6 M guanidine and on-column refolding was performed. Folded and subsequently purified (94% purity) recombinant protein was obtained with 82% of recovery.
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http://dx.doi.org/10.1016/j.jchromb.2004.03.028DOI Listing
August 2004

Lipids are co-eluted with immunoglobulins G during purification by recombinant streptococcal protein G affinity chromatography.

J Immunol Methods 2002 Dec;271(1-2):107-11

Laboratoire de Biogenèse Membranaire CNRS-UMR 5544, Université Victor Segalen, Bordeaux 2, France.

The efficiency of recombinant streptococcal protein G (rec-spG) affinity column chromatography in purifying immunoglobulins G (IgG) from lipids has been studied, with particular reference to IgG fractions from bronchoalveolar lavage (BAL) fluid and serum samples from different sources. It was found that the IgG fractions purified by rec-spG affinity column chromatography also contained cholesterol and phospholipids.
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http://dx.doi.org/10.1016/s0022-1759(02)00345-9DOI Listing
December 2002

Anti-phospholipid antibodies in diabetes mellitus.

Clin Chem Lab Med 2002 Jun;40(6):604-8

Laboratoire de Biogenèse Membranaire, CNRS-UMR 5544, Université Victor Segalen Bordeaux, France.

The presence of autoantibodies to phospholipids may be associated with various pathological disorders; diabetes could be one of them because of the changes occurring in lipid metabolism but there are only few reports examining this question, and they are not always leading to the same conclusions because of the differences in the procedures or in the phospholipids tested. We carried out a systematic comparative study of diabetic serum antibody binding to all phospholipids, anionic and zwitterionic, by a quantitative ELISA. The implication of the hydrophobic moiety of the lipids was also studied: the presence of autoantibodies to the fatty acyl chains was investigated. Our results show the presence of anti-phospholipid antibodies in diabetic sera, particularly anti-phosphatidylinositol and anti-phosphatidylcholine which have never been tested before, and appear to be associated with macroangiopathic complications. The antigenic epitopes are mainly the polar heads as no antibody binding to the hydrophobic moiety was observed. We discuss the relation of those antibodies to the angiopathic complications and to the direct effects of hyperglycemia on lipid antigenicity.
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http://dx.doi.org/10.1515/CCLM.2002.104DOI Listing
June 2002

Immunolocalization of long-chain acyl-CoAs in plant cells.

Biochim Biophys Acta 2002 Jun;1583(1):85-90

Laboratoire de Biogenèse Membranaire, Université Victor Segalen Bordeaux 2, CNRS-UMR 5544, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.

Long chain acyl-Coenzyme A esters (acyl-CoAs) are key substrates in many enzymic reactions of lipid metabolism. Due to their amphiphilic nature, the membrane localization of these molecules cannot be established by subcellular membrane fractionation and usual biochemical studies. We have developed another approach based on ultrastructural immunogold cytochemistry. To preserve the acyl-CoA membrane content, the plant material was freeze substituted and cryoembedded after short aldehyde fixation followed by quick freezing. Using Arabidopsis thaliana root cells and specific antibodies raised against acyl-CoAs, we show that acyl-CoAs are mainly localized in endoplasmic reticulum membranes. Our results demonstrate the value of cryo-methods for the accurate localization of labile metabolites in plant cells.
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http://dx.doi.org/10.1016/s1388-1981(02)00187-7DOI Listing
June 2002