Sequential N- to C-terminal SNARE complex assembly drives priming and fusion of secretory vesicles.
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.
Sørensen
Jakob B
JB
Department of Membrane Biophysics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany. jsoeren@gwdg.de
Wiederhold
Katrin
K
Müller
Emil M
EM
Milosevic
Ira
I
Nagy
Gábor
G
de Groot
Bert L
BL
Grubmüller
Helmut
H
Fasshauer
Dirk
D
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D019439:Chromaffin Cells / Q000166:cytology / Q000378:metabolism; D002942:Circular Dichroism; D004594:Electrophysiology; D005089:Exocytosis; D008561:Membrane Fusion; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009154:Mutation; D050765:Qa-SNARE Proteins / Q000378:metabolism*; D050683:R-SNARE Proteins / Q000378:metabolism*; D022142:Secretory Vesicles / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism*
D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050825:Synaptosomal-Associated Protein 25