Structural and Functional Studies of Munc18 and SNARE Proteins
Release of neurotransmitters is a tightly regulated process and a key event in interneuron communication. Release involves a series of steps, including vesicles docking to the active zone of the plasma membrane, priming to a readily releasable state, and Ca2+-triggered membrane fusion. These steps are tightly controlled by an intricate protein machinery. Essential components of this machinery are proteins from the Sec1/Munc18 (SM) and SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) families. SNAREs function by forming a four-helix bundle called SNARE complex; assembly of the SNARE complex brings two membranes together and is key for membrane fusion. The function of SM proteins is less clear. The neuronal SM proteins Munc18-1 was identified and linked to synaptic vesicle fusion due to its tight binding to syntaxin-1. The strict requirement of Munc18-1 for release is illustrated by the observation that, in mice, deletion of Munc18-1 abolished neurotransmitter release completely. Munc18-1 binds to the closed conformation of syntaxin-1 as well as to assembled SNARE complexes containing open syntaxin-1. Analysis of point mutations on Munc18-1 showed that binding of Munc18-1 to the Habc domain of open syntaxin-1 is critical for synaptic vesicle priming but not for the release step. The fact that Munc18-1 and complexin-1 could bind simultaneously to the SNARE complex suggested Munc18-1 remained bound to a macrocomplex that is poised for Ca2+ triggering of fusion. Analysis using diverse biophysical approaches revealed that Munc18-1 indeed binds to the C-terminus of the synaptobrevin SNARE motif and to the SNARE four-helix bundle. Both interactions have similar affinities and the N-terminal region of syntaxin-1 competes with the SNARE four-helix bundle and synaptobrevin for Munc18-1 binding, suggesting that the interaction between Munc18-1 and the SNARE four-helix bundle involves the same cavity of Munc18-1 that binds to syntaxin-1. To directly test Munc18-1’s role in fusion, I reconstituted v- and t-SNAREs into separate liposomes. Fusion between these proteoliposomes in the presence of Munc18-1 was monitored with a lipid mixing assay. Intriguingly, I found that enhanced lipid mixing caused by rat Munc18-1 alone was observed. Biochemistry studies showed that denaturation of rat Munc18-1 or squid Munc18-1 causes membrane lipid mixing in the absence of SNAREs proteins.