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

Abstract

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.

Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering

“…These studies initially showed that the zippering of concentrated SNAREs can drive slow fusion ( Weber et al, 1998 ; Fukuda et al, 2000 ). As proteoliposome SNARE levels were lowered toward physiological levels, reconstituted vacuolar and neuronal fusion reactions required additional factors ( Zick and Wickner, 2016 ; Stepien and Rizo, 2021 ). In addition to SNAREs, reconstituted neuronal fusion requires Munc18, Munc13, calcium, NSF, and αSNAP ( Ma et al, 2013 ; Lai et al, 2017 ) while reconstituted vacuole fusion needs HOPS ( Stroupe et al, 2006 ), the Rab Ypt7 ( Stroupe et al, 2009 ; Ohya et al, 2009 ), and specific lipid head-group composition and fatty acyl fluidity ( Zick and Wickner, 2016 ).…”

Control of neurotransmitter release and presynaptic plasticity by re-orientation of membrane-bound Munc13-1

“…Research for over three decades has led to major advances in our understanding of the basic mechanisms underlying the priming and fusion of synaptic vesicles to release neurotransmitters, including the notions that Munc18-1 and Munc13-1 coordinate assembly of SNARE complexes in an NSF-αSNAP-resistant manner (Ma et al, 2013;Prinslow et al, 2019) and that this pathway enables the multiple modes of regulation of release probability during presynaptic plasticity that depend on Munc13-1 (Park et al, 2017;Sitarska et al, 2017;Stepien et al, 2019). This large multiple domain protein plays fundamental roles in this process by accelerating opening of the syntaxin-1 closed conformation (Ma et al, 2011;Yang et al, 2015) and bridging the vesicle and plasma membranes (Liu et al, 2016;Quade et al, 2019).…”

“…Electrophysiological data were analyzed offline using Axograph X software version 1. in E. coli were described previously (Chen et al, 2006;Dulubova et al, 2007;Dulubova et al, 1999;Liu et al, 2016;Ma et al, 2013;Prinslow et al, 2019;Sitarska et al, 2017;Stepien and Rizo, 2021). A plasmid for bacterial expression of Rattus Norvegicus His6-Munc13-1 residues 529-1735 lacking a large variable loop (residues 1408-1452) to improve solubility (Ma et al, 2011) was described previously (Quade et al, 2019).…”

“…Liposome fusion assays. Liposome lipid and content mixing assays were performed as previously described (Liu et al, 2016;Liu et al, 2017;Stepien and Rizo, 2021). To prepare the phospholipid vesicles, ).…”

“…Marina Blue DHPE, synaptobrevin (1:10,000 P:L ratio) and synaptotagmin-1(57-421) C74S/C75A/C77S/C79I/C82L/C277S (P:L ratio 1,1000), as described (Stepien and Rizo, 2021).…”