Effects of synaptotagmin 2 on membrane fusion between liposomes that contain SNAREs involved in exocytosis in mast cells

• Published in: Biochimica et biophysica acta Vol. 1808; no. 10; pp. 2435 - 2439
• Main Authors: Nagai, Yumiko, Tadokoro, Satoshi, Sakiyama, Hiroki, Hirashima, Naohide
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2011
• Subjects: Blotting, Western; Cell Line; Electrophoresis, Polyacrylamide Gel; Exocytosis; Exocytosis - physiology; Liposome; Liposomes; Mast cell; Mast Cells - metabolism; Membrane fusion; Membrane Fusion - physiology; SNARE; Synaptotagmin; Synaptotagmin II - physiology; Liposome; Membrane fusion; Mast cell; Synaptotagmin; Exocytosis; SNARE
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/j.bbamem.2011.07.003

Mast cells play a pivotal role in allergic responses. Antigen stimulation causes elevation of the intracellular Ca2+ 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 Ca2+ dependent, the target molecule that interacts with Ca2+ is not clear. Synaptotagmin is a Ca2+ 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 Ca2+, 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, Ca2+, and phosphatidylserine. ► Mast cell is a key player in allergic responses. ► Mast cell secretes inflammatory mediators by SNARE-dependent exocytosis. ► We investigated the role of synaptotagmin 2 in exocytosis, using liposomes. ► High concentration of synaptotagmin 2 enhanced the SNARE-mediated liposome fusion. ► Low concentration of synaptotagmin 2 inhibited the liposome fusion.