title: Effects of synaptotagmin 2 on membrane fusion between liposomes that contain SNAREs involved in exocytosis in mast cells.
abstract: Mast cells play a pivotal role in allergic responses. Antigen stimulation causes elevation of the intracellular Ca(2+) concentration, which triggers the exocytotic release of inflammatory mediators such as histamine. Recent research, including our own, has revealed that SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins such as syntaxin-3, -4, SNAP-23, and VAMP-8 are involved in exocytosis. Although exocytosis in mast cells is Ca(2+) dependent, the target molecule that interacts with Ca(2+) is not clear. Synaptotagmin is a Ca(2+) sensor and regulates exocytosis in neuronal cells. However, the role of synaptotagmin 2, a member of the synaptotagmin family, in exocytosis in mast cells remains controversial. In this study, we investigated the role of synaptotagmin 2 by a liposome-based fusion assay. SNARE proteins (SNAP-23, syntaxin-3, VAMP-8) and synaptotagmin 2 were expressed in Escherichia coli and purified as GST-tagged or His-tagged fusion proteins. These SNARE proteins were incorporated by a detergent dialysis method. Membrane fusion between liposomes was monitored by fluorescence resonance energy transfer between fluorescent-labeled phospholipids. In the presence of Ca(2+), low synaptotagmin 2 concentration inhibited membrane fusion between SNARE-containing liposomes, while high synaptotagmin 2 concentration enhanced membrane fusion. This enhancement required phosphatidylserine as a membrane component. These results suggest that synaptotagmin 2 regulates membrane fusion of SNARE-containing liposomes involved in exocytosis in mast cells, and that this regulation is dependent on synaptotagmin 2 concentration, Ca(2+), and phosphatidylserine.
authors.affiliation: Nagoya City University, Nagoya, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Nagai
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tadokoro
authors.forename: Hiroki
authors.initials: H
authors.lastname: Sakiyama
authors.forename: Naohide
authors.initials: N
authors.lastname: Hirashima
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis / Q000502:physiology*; D008081:Liposomes; D008407:Mast Cells / Q000378:metabolism*; D008561:Membrane Fusion / Q000502:physiology*; D050861:Synaptotagmin II / Q000502:physiology*
chemical_list: D008081:Liposomes; D050861:Synaptotagmin II