Lilly Maneta-Peyret

Cell-free transfer of phospholipids between the endoplasmic reticulum and the Golgi apparatus of leek seedlings

The transfer of lipids between the endoplasmic reticulum and the Golgi apparatus was investigated in vitro using a cell-free system from leek seedlings. Lipids of the donor membranes (endoplasmic reticulum) were radiolabeled either by incubating leek seedlings with [1-14C]acetate or [3H]acetate. Acceptor membranes (Golgi apparatus) were unlabeled and immobilized on nitrocellulose strips. The assay measured the lipid transfer resulting from both an ATP-independent process and an ATP- and cytosol-dependent process. A significant ATP- and cytosol-dependent lipid transfer was observed only in the case of the endoplasmic reticulum as donor and the Golgi apparatus as acceptor. Lipids transferred in an ATP-dependent manner were chiefly phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. The stimulation of lipid transfer by ATP as compared to the ATP-independent process was +79% (PC), +123% (PS) and +69% (PE). On the other hand, PI was not transferred in an ATP-dependent manner (the stimulation by ATP was only 20%). This supports the theory that a sorting of phospholipids takes place in the donor membrane. Moreover, a formation of lysoPC was observed only in the presence of ATP (+330%). The ATP-dependent lipid transfer was inhibited by N-ethylmaleimide, indicating the involvement of cytosolic (but no phospholipid transfer proteins) or membrane proteins in the transfer process. The ATP-dependent transfer of lipids was also diminished at 12 degrees C showing the sensitivity to low temperatures of the transfer of lipids between the endoplasmic reticulum and the Golgi apparatus.

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

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.

Synthesis of a monoclonal antibody-indium-111-porphyrin conjugate

Antibodies were labelled with indium-111 with a view to their use in the radio-immunodetection of cancers. The covalent coupling between indium-111 porphyrin and monoclonal antibodies (IgG and F(ab)2 fragment) was achieved using the ester activated method [N-hydroxy-succinimide/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide]. After purification, this provided conjugated with specific activities of 6 muCi/micrograms Mab (9.3 molecules per Mab) or 1 muCi/microgram (F(ab)2 fragment (1.5 molecule per F(ab)2). ELISA procedures suggested the full retention of immunoreactivity by the radiolabelled antibodies.

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

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.

Phospholipid biosynthesis increases in RHD3-defective mutants

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.

Immunogold Labelling of Fatty Acyl Chains

For the first time, antibodies against a hydrophobic hapten have been used for immunogold labelling of a lipid antigen (BSA-C18:1 conjugate) coated on polystyrene. The labelling was visualised either directly in transmission electron microscopy or in light microscopy after silver enhancement. Good recognition of the fatty acyl chain was obtained even after treatment of the antigen coat with various cross-linking fixatives used for electron microscopy, i.e. formaldehyde, glutaraldehyde and osmium tetroxide.

A Combinatorial Lipid Code Shapes the Electrostatic Landscape of Plant Endomembranes

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 thexa0electrostatic 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.

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

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.

Lipid Traffic to the Plasma Membrane of Leek Cells. Sorting Based on Fatty Acyl Chain Length

The mechanisms by which lipids are sorted and transferred to the plasma membrane are poorly understood as compared to the situation of glyco- proteins but are now under intense investigations particularly in animal cells (Van Meer 1989, Voelker 1991, Koval and Pagano 1991).

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

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 4xa0days 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 24xa0h 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.