Fast vesicle fusion in living cells requires at least three SNARE complexes.
Exocytosis requires formation of SNARE [soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] complexes between vesicle and target membranes. Recent assessments in reduced model systems have produced divergent estimates of the number of SNARE complexes needed for fusion. Here, we used a titration approach to answer this question in intact, cultured chromaffin cells. Simultaneous expression of wild-type SNAP-25 and a mutant unable to support exocytosis progressively altered fusion kinetics and fusion-pore opening, indicating that both proteins assemble into heteromeric fusion complexes. Expressing different wild-type:mutant ratios revealed a third-power relation for fast (synchronous) fusion and a near-linear relation for overall release. Thus, fast fusion typically observed in synapses and neurosecretory cells requires at least three functional SNARE complexes, whereas slower release might occur with fewer complexes. Heterogeneity in SNARE-complex number may explain heterogeneity in vesicular release probability.
Mohrmann
Ralf
R
Department of Membrane Biophysics, Max-Planck Institute for Biophysical Chemistry, Göttingen, Germany. Ralf.Mohrmann@uks.eu
de Wit
Heidi
H
Verhage
Matthijs
M
Neher
Erwin
E
Sørensen
Jakob B
JB
D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D019439:Chromaffin Cells / Q000502:physiology; D022162:Cytoplasmic Vesicles / Q000502:physiology; D005089:Exocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D008561:Membrane Fusion / Q000502:physiology*; D051379:Mice; D009154:Mutation; D050600:SNARE Proteins / Q000502:physiology; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000502:physiology*
D050600:SNARE Proteins; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins