Synaptotagmin-1 Is an Antagonist for Munc18-1 in SNARE Zippering

Xiaochu Lou,Jaeil Shin,Yoosoo Yang,Jaewook Kim,Yeon-Kyun Shin

doi:10.1074/jbc.m114.631341

Abstract

In neuroexocytosis, SNAREs and Munc18-1 may consist of the minimal membrane fusion machinery. Consistent with this notion, we observed, using single molecule fluorescence assays, that Munc18-1 stimulates SNARE zippering and SNARE-dependent lipid mixing in the absence of a major Ca(2+) sensor synaptotagmin-1 (Syt1), providing the structural basis for the conserved function of Sec1/Munc18 proteins in exocytosis. However, when full-length Syt1 is present, no enhancement of SNARE zippering and no acceleration of Ca(2+)-triggered content mixing by Munc18-1 are observed. Thus, our results show that Syt1 acts as an antagonist for Munc18-1 in SNARE zippering and fusion pore opening. Although the Sec1/Munc18 family may serve as part of the fusion machinery in other exocytotic pathways, Munc18-1 may have evolved to play a different role, such as regulating syntaxin-1a in neuroexocytosis.

Highlights

The molecular mechanisms of the critical necessity of Munc18-1 protein for neurotransmitter release remain unclear
Our results show that Munc18-1 has the capacity to stimulate SNARE complex formation and SNARE-dependent lipid mixing, Syt1 largely negates such positive effects on membrane fusion, suggesting that Syt1 acts as an antagonist for Munc18-1
To investigate the effects of Munc18-1 on the conformation of the trans-SNAREpin with single molecule FRET (smFRET), we prepared the N-terminal FRET pair VAMP2 Q33C and syntaxin-1a I203C labeled with the donor dye Cy3 and the acceptor dye Cy5, respectively (NN)

Summary

Background

The molecular mechanisms of the critical necessity of Munc protein for neurotransmitter release remain unclear. SNAREs and Munc may consist of the minimal membrane fusion machinery. Consistent with this notion, we observed, using single molecule fluorescence assays, that Munc stimulates SNARE zippering and SNARE-dependent lipid mixing in the absence of a major Ca2؉ sensor synaptotagmin-1 (Syt1), providing the structural basis for the conserved function of Sec1/Munc proteins in exocytosis. Essential to the functional connectivity in the central nerve system is neurotransmitter release at synapses, which requires fusion of vesicles to the presynaptic plasma membrane. The SNARE complex formed between vesicle SNARE (v-SNARE) and target plasma membrane SNARE (t-SNARE) is considered the core of the fusion machine [3,4,5,6,7,8,9,10,11]. We investigated, using single molecule FRET (smFRET), the conformational changes of a trans-SNARE com-

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION