Munc18-1 Is a Master Orchestrator of Synaptic SNARE Assembly




Stepien, Karolina Paulina

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Neurotransmitters are released by tightly controlled synaptic vesicle exocytosis at the active zone of presynaptic nerve terminals. This process requires primed fusion-competent vesicles and coordinated calcium-secretion coupling, and all these steps are governed by a sophisticated protein apparatus. SNAREs and Munc18-1 are the core components of this apparatus: SNAREs form SNARE complexes that bridge the vesicle and plasma membranes and Munc18-1 coordinates SNARE assembly. Productive formation of SNARE complexes is topologically complicated and requires overcoming multiple energy barriers. This work elucidates distinctive molecular mechanisms of how Munc18-1 orchestrates SNARE assembly. Reproducing the steps that lead to neuronal exocytosis with purified proteins and synthetic lipid vesicles has become an irreplaceable tool to decipher mechanistic details underlying synaptic membrane fusion. In one project, I created a new, much slower system by reconstituting a limited number of SNAREs. This new tool not only allowed to study the activity of the calcium sensor, synaptotagmin-1, showing its stimulatory function, but also provided a way to control the number of SNARE complexes. The latter is important as emerging evidence suggests that the number of primed SNARE complexes in neurons is small. My study shows that liposome fusion with few SNAREs requires Munc18-1, synaptotagmin-1, and Munc13-1. While the requirement of Munc13-1 for synaptic fusion can be partially bypassed by SNARE or Munc18-1 mutations, the absence of Munc18-1 has not been rescued by any means so far. In my next project, I elucidated the fundamental mechanism that completely prevents synaptic vesicle fusion unless Munc18-1 is present. Using fluorescence and nuclear resonance spectroscopy, I showed that another protein, αSNAP, completely blocks SNARE-dependent liposome fusion through diverse interactions with SNAREs and that such inhibition can only be overcome by Munc18-1 upon binding to closed syntaxin-1, which in turns initiates SNARE assembly. My third study described another mechanism underlying the essential role of Munc18-1 in SNARE assembly. I solved cryo-electron microscope structures of Munc18-1 bound simultaneously to its two cognate SNAREs, showing structural details of how Munc18-1 organizes formation of productive SNARE complexes. Overall, I described multiple mechanisms of how Munc18-1 functions at virtually every step of SNARE assembly.

General Notes

Pages 114-185 are misnumbered as pages 115-186.

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