Septin Dynamics Are Essential for Exocytosis

• DOI: 10.1074/jbc.m114.616201
• Publication Date: 2015-01-10T06:24:29Z
• Authors: Elmira Tokhtaeva; Joe Capri; Elizabeth A. Marcus; Julian P. Whitelegge; Venera Khuzakhmetova; Ellya Bukharaeva; Nimrod Deiss-Yehiely; Laura A. Dada; George Sachs; Ester Fernandez-S...; Olga Vagin
• Keywords: Male; Proteomics; 0301 basic medicine; Biomedical and clinical sciences; Proteome; Pyridines; Membrane Fusion; Medical and Health Sciences; PC12 Cells; Madin Darby Canine Kidney Cells; Mice; Inbred BALB C; Cytoskeleton; Microscopy; Mice, Inbred BALB C; Tumor; Microscopy, Confocal; Blotting; Brain; Biological Sciences; Biological sciences; Confocal; Neurological; RNA Interference; Female; Western; Protein Binding; 570; Biochemistry & Molecular Biology; 572; Synaptosomal-Associated Protein 25; 1.1 Normal biological development and functioning; Blotting, Western; Exocytosis; Cell Line; 03 medical and health sciences; Dogs; Cell Line, Tumor; Animals; Humans; Phenylurea Compounds; Neurosciences; Protein Secretion; Cell Biology; Rats; HEK293 Cells; Chemical sciences; Membrane Trafficking; Chemical Sciences; Biochemistry and Cell Biology; Protein Multimerization; Septins

Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis.