Septin dynamics are essential for exocytosis

J Biol Chem. 2015 Feb 27;290(9):5280-97. doi: 10.1074/jbc.M114.616201. Epub 2015 Jan 9.

Abstract

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.

Keywords: Cell Biology; Cytoskeleton; Exocytosis; Membrane Fusion; Membrane Trafficking; Protein Secretion; Septins.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Brain / drug effects
  • Brain / metabolism*
  • Cell Line
  • Cell Line, Tumor
  • Dogs
  • Exocytosis*
  • Female
  • HEK293 Cells
  • Humans
  • Madin Darby Canine Kidney Cells
  • Male
  • Mice, Inbred BALB C
  • Microscopy, Confocal
  • PC12 Cells
  • Phenylurea Compounds / pharmacology
  • Protein Binding / drug effects
  • Protein Multimerization
  • Proteome / metabolism*
  • Proteomics
  • Pyridines / pharmacology
  • RNA Interference
  • Rats
  • Septins / chemistry
  • Septins / genetics
  • Septins / metabolism*
  • Synaptosomal-Associated Protein 25 / metabolism

Substances

  • Phenylurea Compounds
  • Proteome
  • Pyridines
  • Synaptosomal-Associated Protein 25
  • Sept2 protein, mouse
  • Septins
  • N-(2-chloro-4-pyridyl)-N'-phenylurea