Biophysical Society Conference | Estes Park 2023

Membrane Fusion and Budding

Sunday Speaker Abstracts

TURBO-CHARGING SYNAPTIC VESICLES FOR EXPLOSIVE RELEASE OF NEUROTRANSMITTERS

James E Rothman 1 ; 1 Yale University, Department of Cell Biology, New Haven, CT, USA

How neurotransmitter release occurs thousands of times faster than other forms of SNARE dependent membrane fusion has been a vexing problem. We have now discovered a simple principle that can explain this involving two layers of SNAREpins. A ring of 6 "central" SNAREpins is released by Ca++ to open the fusion pore. Unlike fusion reactions, we suggest that synaptic vesicles are "turbo-charged" by an outer set of 6 "peripheral SNAREpins" that provides additional force. This model has its origin in cryo-EM tomography that revealed a protein structure with 6-fold symmetry at the interface of each ready-release synaptic vesicle with the active zone plasma membrane. Our current studies involve single molecule counting of SNAREpins in single synthetic ready-release vesicles (using fully-defined reconstitutions of vesicles with suspended bilayers) and novel high resolution cryo-EM structures of the membrane-bound SNARE-assembling chaperone Munc13. Together, the new results suggest that the observed 6-fold symmetry results from the de novo assembly of a series of sequential, symmetrical supra-molecular machines. In this model, the vesicle is initially captured by a hexagonal "basket" of 18 copies of the chaperone Munc13 in their upright conformation. Munc13 then transits thru a lateral hexagon arrangement, moving the vesicle closer to the plasma membrane. The 6 peripheral SNAREpins are coordinately assembled during this transition. The 6 central SNAREpins are assembled later, each by one subunit of the Munc13 hexagon as it flattens onto the plasma membrane to form an outer ring. The central SNAREpins are bound to the inner Ca++-sensitive ring of Synaptotagmin, each paired with a peripheral SNAREpin by a bridging molecule of Complexin in the previously observed trans-clamping arrangement. Binding of Ca++ then releases all 12 SNAREpins simultaneously. This model makes many novel and testable predictions and is consistent with available structural and genetic evidence.

11