Phospholipase D1 production of phosphatidic acid at the plasma membrane promotes exocytosis of large dense-core granules at a late stage.
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.
Zeniou-Meyer
Maria
M
Département Neurotransmission & Sécrétion Neuroendocrine, Institut des Neurosciences Cellulaires et Intégratives, CNRS and Université Louis Pasteur, 5 rue Blaise Pascal, Strasbourg, France.
Zabari
Naama
N
Ashery
Uri
U
Chasserot-Golaz
Sylvette
S
Haeberlé
Anne-Marie
AM
Demais
Valérie
V
Bailly
Yannick
Y
Gottfried
Irit
I
Nakanishi
Hideki
H
Neiman
Aaron M
AM
Du
Guangwei
G
Frohman
Michael A
MA
Bader
Marie-France
MF
Vitale
Nicolas
N
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
D008563:Membrane Lipids; D010712:Phosphatidic Acids; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013006:Growth Hormone; D010739:Phospholipase D; C106801:phospholipase D1