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Sequential N‐ to C‐terminal SNARE complex assembly drives priming and fusion of secretory vesicles
Author(s) -
Sørensen Jakob B,
Wiederhold Katrin,
Müller Emil M,
Milosevic Ira,
Nagy Gábor,
de Groot Bert L,
Grubmüller Helmut,
Fasshauer Dirk
Publication year - 2006
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1038/sj.emboj.7601003
Subject(s) - biology , synaptobrevin , exocytosis , snare complex , lipid bilayer fusion , vesicle , microbiology and biotechnology , vesicle fusion , biophysics , fusion , munc 18 , snap25 , secretory vesicle , syntaxin , secretion , synaptic vesicle , biochemistry , membrane , linguistics , philosophy
During exocytosis a four‐helical coiled coil is formed between the three SNARE proteins syntaxin, synaptobrevin and SNAP‐25, bridging vesicle and plasma membrane. We have investigated the assembly pathway of this complex by interfering with the stability of the hydrophobic interaction layers holding the complex together. Mutations in the C‐terminal end affected fusion triggering in vivo and led to two‐step unfolding of the SNARE complex in vitro , indicating that the C‐terminal end can assemble/disassemble independently. Free energy perturbation calculations showed that assembly of the C‐terminal end could liberate substantial amounts of energy that may drive fusion. In contrast, similar N‐terminal mutations were without effects on exocytosis, and mutations in the middle of the complex selectively interfered with upstream maturation steps (vesicle priming), but not with fusion triggering. We conclude that the SNARE complex forms in the N‐ to C‐terminal direction, and that a partly assembled intermediate corresponds to the primed vesicle state.