Sequential N- to C-terminal SNARE complex assembly drives priming and fusion of secretory vesicles

TY - JOUR

T1 - Sequential N- to C-terminal SNARE complex assembly drives priming and fusion of secretory vesicles

AU - Sørensen, Jakob B

AU - Wiederhold, Katrin

AU - Müller, Emil M

AU - Milosevic, Ira

AU - Nagy, Gábor

AU - de Groot, Bert L

AU - Grubmüller, Helmut

AU - Fasshauer, Dirk

N1 - Keywords: Amino Acid Sequence; Amino Acid Substitution; Animals; Binding Sites; Cell Membrane; Chromaffin Cells; Circular Dichroism; Electrophysiology; Exocytosis; Membrane Fusion; Mice; Mice, Knockout; Molecular Sequence Data; Mutation; Qa-SNARE Proteins; R-SNARE Proteins; Secretory Vesicles; Sequence Homology, Amino Acid; Synaptosomal-Associated Protein 25

PY - 2006

Y1 - 2006

N2 - 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.

AB - 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.

U2 - 10.1038/sj.emboj.7601003

DO - 10.1038/sj.emboj.7601003

M3 - Journal article

C2 - 16498411

SN - 0261-4189

VL - 25

SP - 955

EP - 966

JO - E M B O Journal

JF - E M B O Journal

IS - 5

ER -