Labeling of human erythrocyte membrane proteins by photoactivatable radioiodinated phosphatidylcholine and phosphatidylserine A search for the aminophospholipid translocase

AbstractWe have synthesized radioiodinated photoactivatable phosphatidylcholine (125-N3-PC) and phosphatidylserine (125I-N3-PS). After incubation with red blood cells in the dark, the labeled PC could be extracted but not the corresponding PS molecule, indicating that the latter was transported by the aminophospholipid translocase, but not the former. When irradiated immediately after incorporation, N3-PS, but not N3-PC, partially blocked subsequent translocation of spin-labeled aminophospholipids. Analysis of probe distribution by SDSpolyacrylamide gel electrophoresis revealed that 125I-N3-PS labeled seven membrane bound components with molecular masses between 140 and 27 kDa: one (or several) of these components should correspond to the aminophospholipid translocase

Plant phosphoinositide-dependent phospholipases C: Variations around a canonical theme

International audiencePhosphoinositide-specific phospholipase C (PI-PLC) cleaves, in a Ca 2þ-dependent manner, phosphati-dylinositol-4,5-bisphosphate (PI-4,5-P 2) into diacylglycerol (DAG) and inositol triphosphate (IP 3). PI-PLCs are multidomain proteins that are structurally related to the PI-PLCzs, the simplest animal PI-PLCs. Like these animal counterparts, they are only composed of EF-hand, X/Y and C2 domains. However, plant PI-PLCs do not have a conventional EF-hand domain since they are often truncated, while some PI-PLCs have no EF-hand domain at all. Despite this simple structure, plant PI-PLCs are involved in many essential plant processes, either associated with development or in response to environmental stresses. The action of PI-PLCs relies on the mediators they produce. In plants, IP 3 does not seem to be the sole active soluble molecule. Inositol pentakisphosphate (IP 5) and inositol hexakisphosphate (IP 6) also transmit signals, thus highlighting the importance of coupling PI-PLC action with inositol-phosphate kinases and phosphatases. PI-PLCs also produce a lipid molecule, but plant PI-PLC pathways show a peculiarity in that the active lipid does not appear to be DAG but its phosphorylated form, phosphatidic acid (PA). Besides, PI-PLCs can also act by altering their substrate levels. Taken together, plant PI-PLCs show functional differences when compared to their animal counterparts. However, they act on similar general signalling pathways including calcium homeostasis and cell phosphoproteome. Several important questions remain unanswered. The cross-talk between the soluble and lipid mediators generated by plant PI-PLCs is not understood and how the coupling between PI-PLCs and inositol-kinases or DAG-kinases is carried out remains to be established

Phytosphingosine-phosphate is a signal for AtMPK6 activation and Arabidopsis response to chilling

Long-chain bases (LCBs) are pleiotropic sphingolipidic signals in eukaryotes. We investigated the source and function of phytosphingosine-1-phosphate (PHS-P), a phospho-LCB rapidly and transiently formed in Arabidopsis thaliana on chilling.PHS-P was analysed by thin-layer chromatography following in vivo metabolic radiolabelling. Pharmacological and genetic approaches were used to identify the sphingosine kinase isoforms involved in cold-responsive PHS-P synthesis. Gene expression, mitogen-activated protein kinase activation and growth phenotypes of three LCB kinase mutants (lcbk1, sphk1 and lcbk2) were studied following cold exposure. Chilling provoked the rapid and transient formation of PHS-P in Arabidopsis cultured cells and plantlets. Cold-evoked PHS-P synthesis was reduced by LCB kinase inhibitors and abolished in the LCB kinase lcbk2 mutant, but not in lcbk1 and sphk1 mutants. lcbk2 presented a constitutive AtMPK6 activation at 22°C. AtMPK6 activation was also triggered by PHS-P treatment independently of PHS/PHS-P balance. lcbk2 mutants grew comparably with wild-type plants at 22 and 4°C, but exhibited a higher root growth at 12°C, correlated with an altered expression of the cold-responsive DELLA gene RGL3. Together, our data indicate a function for LCBK2 in planta. Furthermore, they connect PHS-P formation with plant response to cold, expanding the field of LCB signalling in plants

Etude de la perception et de la transduction du signal froid chez Arabidopsis thaliana (Implication des voies de signalisation lipidique)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Etude de la perception et de la transduction du signal froid chez Arabidopsis thaliana (Implication des voies de signalisation lipidique)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Phospholipid signalling in the salicylic acid pathway of Arabidopsis thaliana

Chez les plantes, l acide salicylique (SA) a un rôle central dans la réponse à de nombreuses contraintes environnementales et lors du développement. Cependant les événements de signalisation précoces qu il déclenche sont peu connus. Nous montrons, par marquage métabolique au 33Pi sur une suspension cellulaire d Arabidopsis thaliana, que le SA induit une diminution rapide et précoce d un pool de phosphatidylinositol (PI). Celle-ci est accompagnée d une accumulation de PI 4-phosphate et PI 4,5-bisphosphate. Ces changements sont inhibés par de la wortmannine à 30 M mais pas à 1 M, ce qui implique une activation de PI 4-kinase de type III. C est pourquoi une étude des effets de la wortmannine sur les modifications de transcriptome par le SA a été menée à l aide de la puce Complete Arabidopsis Transcriptome MicroArray (CATMA). Sur 773 gènes régulés par le SA, 112 sont sensibles à 30 M de wortmannine. En parallèle, nous voyons que l acide phosphatidique issu de la phospholipase D (PLD) est important pour la réponse génique précoce au SA. Une expérience de puces menée pour identifier les gènes régulés par la PLD en réponse au SA a révélé que parmi 1327 gènes régulés par le SA, 97 gènes sont régulés positivement, et 117 gènes négativement, par la PLD. Les régulons de la voie sensible à la wortmannine et de la voie PLD se chevauchent fortement, ce qui suggère que les deux activités agissent en synergie dans la même voie de signalisation en réponse au SASalicylic acid (SA) has a pivotal role in many plant stress and developmental responses but little is known about the early signalling events triggered by this molecule. Using Arabidopsis thaliana suspension cells, it was shown by in vivo metabolic phospholipid labelling with 33Pi that SA induced rapid and early decrease in a pool of phosphatidylinositol (PI). The decrease was accompanied by an increase in PI 4-phosphate and PI 4,5-bisphosphate contents. These changes could be inhibited by 30 M wortmannine, but not by 1 M wortmannine, implying that a type III PI 4-kinase was activated in response to SA. Therefore, a study of wortmannin effects on SA transcriptome was undertaken. Using the Complete Arabidopsis Transcriptome MicroArray (CATMA) chip, 773 SA-regulated genes were identified. Among these, the SA response of 112 was inhibited by 30 M wortmannine, but not by 1 M wortmannin. In parallel, it was discovered that phospholipase D (PLD) derived phosphatidic acid was important for the early SA-regulated gene expression. Microarray experiment aimed to identify genes regulated by PLD in response to SA revealed that out of 1327 genes regulated by SA, 97 genes were positively and 117 genes were negatively regulated by PLD. The wortmannin-sensitive pathway and PLD pathway regulons share an important overlap implying that the two enzyme activities act synergistically in the same signalling pathway in response to SA. Key words: Arabidopsis thaliana, phosphatidylinositol 4-kinase, phPARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceCzech RepublicFRC