Abstract
The vesicle fusion reaction in regulated exocytosis requires the concerted action of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core fusion engine and a group of SNARE-binding regulatory factors. The regulatory mechanisms of vesicle fusion remain poorly understood in most exocytic pathways. Here, we reconstituted the SNARE-dependent vesicle fusion reaction of GLUT4 exocytosis in vitro using purified components. Using this defined fusion system, we discovered that the regulatory factor synip binds to GLUT4 exocytic SNAREs and inhibits the docking, lipid mixing, and content mixing of the fusion reaction. Synip arrests fusion by binding the target membrane SNARE (t-SNARE) complex and preventing the initiation of ternary SNARE complex assembly. Although synip also interacts with the syntaxin-4 monomer, it does not inhibit the pairing of syntaxin-4 with SNAP-23. Interestingly, synip selectively arrests the fusion reactions reconstituted with its cognate SNAREs, suggesting that the defined system recapitulates the biological functions of the vesicle fusion proteins. We further showed that the inhibitory function of synip is dominant over the stimulatory activity of Sec1/Munc18 proteins. Importantly, the inhibitory function of synip is distinct from how other fusion inhibitors arrest SNARE-dependent membrane fusion and therefore likely represents a novel regulatory mechanism of vesicle fusion.
Highlights
Synip is a sensitive factor attachment protein receptor (SNARE)-binding regulatory factor whose molecular mechanism remains unclear
When added as a soluble protein, SNAP-23 readily assembled with syntaxin-4 to form the binary target membrane SNARE (t-SNARE) complex on the membrane (Fig. 1B)
We found that synip binding did not prevent the pairing of syntaxin-4 with SNAP-23 to form the binary t-SNARE complex (Fig. 1B)
Summary
Synip is a SNARE-binding regulatory factor whose molecular mechanism remains unclear. We reconstituted the SNARE-dependent vesicle fusion reaction of GLUT4 exocytosis in vitro using purified components Using this defined fusion system, we discovered that the regulatory factor synip binds to GLUT4 exocytic SNAREs and inhibits the docking, lipid mixing, and content mixing of the fusion reaction. The inhibitory function of synip is distinct from how other fusion inhibitors arrest SNARE-dependent membrane fusion and likely represents a novel regulatory mechanism of vesicle fusion. A Novel Inhibitory Mechanism in Vesicle Fusion Regulation the regulatory factor synip binds to GLUT4 exocytic SNAREs and arrests fusion at an intermediate stage. The inhibitory function of synip is distinct from how other fusion inhibitors arrest SNARE-dependent membrane fusion and represents a novel regulatory mechanism of vesicle fusion
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