Synaptotagmin-1-, Munc18-1-, and Munc13-1-dependent liposome fusion with a few neuronal SNAREs

Neurotransmitter release is governed by eight central proteins among other factors: the neuronal SNAREs syntaxin-1, synaptobrevin, and SNAP-25, which form a tight SNARE complex that brings the synaptic vesicle and plasma membranes together; NSF and SNAPs, which disassemble SNARE complexes; Munc18-1 and Munc13-1, which organize SNARE complex assembly; and the Ca²⁺ sensor synaptotagmin-1. Reconstitution experiments revealed that Munc18-1, Munc13-1, NSF, and α-SNAP can mediate Ca²⁺-dependent liposome fusion between synaptobrevin liposomes and syntaxin-1-SNAP-25 liposomes, but high fusion efficiency due to uncontrolled SNARE complex assembly did not allow investigation of the role of synaptotagmin-1 on fusion. Here, we show that decreasing the synaptobrevin-to-lipid ratio in the corresponding liposomes to very low levels leads to inefficient fusion and that synaptotagmin-1 strongly stimulates fusion under these conditions. Such stimulation depends on Ca²⁺ binding to the two C₂ domains of synaptotagmin-1. We also show that anchoring SNAP-25 on the syntaxin-1 liposomes dramatically enhances fusion. Moreover, we uncover a synergy between synaptotagmin-1 and membrane anchoring of SNAP-25, which allows efficient Ca²⁺-dependent fusion between liposomes bearing very low synaptobrevin densities and liposomes containing very low syntaxin-1 densities. Thus, liposome fusion in our assays is achieved with a few SNARE complexes in a manner that requires Munc18-1 and Munc13-1 and that depends on Ca²⁺ binding to synaptotagmin-1, all of which are fundamental features of neurotransmitter release in neurons.