title: Phospholipase D1 production of phosphatidic acid at the plasma membrane promotes exocytosis of large dense-core granules at a late stage.
abstract: Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, however, the biosynthetic pathway, the dynamic location, and actual function of PA in secretory cells remain unknown. Using a short interference RNA strategy on chromaffin and PC12 cells, we demonstrate here that phospholipase D1 is activated in secretagogue-stimulated cells and that it produces PA at the plasma membrane at the secretory granule docking sites. We show that phospholipase D1 activation and PA production represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. Finally, we show that the PLD1 short interference RNA-mediated inhibition of exocytosis can be rescued by exogenous provision of a lipid that favors the transition of opposed bi-layer membranes to hemifused membranes having the outer leaflets fused. Our findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway. We propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion.
authors.affiliation: Département Neurotransmission & Sécrétion Neuroendocrine, Institut des Neurosciences Cellulaires et Intégratives, CNRS and Université Louis Pasteur, 5 rue Blaise Pascal, Strasbourg, France.
authors.forename: Maria
authors.initials: M
authors.lastname: Zeniou-Meyer
authors.forename: Naama
authors.initials: N
authors.lastname: Zabari
authors.forename: Uri
authors.initials: U
authors.lastname: Ashery
authors.forename: Sylvette
authors.initials: S
authors.lastname: Chasserot-Golaz
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Haeberlé
authors.forename: Valérie
authors.initials: V
authors.lastname: Demais
authors.forename: Yannick
authors.initials: Y
authors.lastname: Bailly
authors.forename: Irit
authors.initials: I
authors.lastname: Gottfried
authors.forename: Hideki
authors.initials: H
authors.lastname: Nakanishi
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Neiman
authors.forename: Guangwei
authors.initials: G
authors.lastname: Du
authors.forename: Michael A
authors.initials: MA
authors.lastname: Frohman
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Bader
authors.forename: Nicolas
authors.initials: N
authors.lastname: Vitale
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D019439:Chromaffin Cells / Q000502:physiology; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure; D004594:Electrophysiology; D005089:Exocytosis / Q000502:physiology*; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D008563:Membrane Lipids / Q000096:biosynthesis; D008564:Membrane Potentials; D016253:Microscopy, Immunoelectron; D016716:PC12 Cells; D010712:Phosphatidic Acids / Q000096:biosynthesis*; D010739:Phospholipase D / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D014162:Transfection
chemical_list: D008563:Membrane Lipids; D010712:Phosphatidic Acids; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013006:Growth Hormone; D010739:Phospholipase D; C106801:phospholipase D1