Abstract
Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.
Highlights
The cascade of reactions and proteins conferring regulated exocytosis needs to be characterized
In which VAMP2-small unilamellar vesicles (SUVs) and t-sensitive factor attachment protein receptor (SNARE)-giant unilamellar vesicles (GUVs) were preincubated in the presence of Munc18-1 for 1 h on ice, were subsequently transferred to a 96-well plate at room temperature to delay the onset of fusion
VAMP2 and synaptotagmin 1 (Syt1) were reconstituted at a protein to lipid ratio of 1/200 and 1/800, respectively, corresponding to the physiological concentrations found in synaptic vesicles [58]
Summary
The cascade of reactions and proteins conferring regulated exocytosis needs to be characterized. Cpx stabilizes partially assembled SNAREpins and blocks further SNAREpin zippering To release this block and to couple the reaction to a Ca2ϩ signal, Syt, which is anchored via its N-terminal transmembrane domain in the vesicular membrane, binds with its two C2 domain anionic phospholipids in a Ca2ϩ-dependent manner. A SM protein, is essential for regulated exocytosis and has several functions in SNAREpin assembly (30 –35) It stabilizes syntaxin 1, contributes to the transport of syntaxin 1 from the ER to the plasma membrane, and keeps syntaxin 1 in a closed conformation that blocks SNAP-25 binding and t-SNARE assembly (36 – 45). In the simplest model Munc, Syt, CpxII, and the neuronal SNAREs would be the basic machinery generating a readily releasable pool of docked vesicles, which immediately responds to a Ca2ϩ signal by membrane fusion
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