Characterization of Membrane and Protein Factors Modulating SNARE-Mediated Membrane Fusion

Eukaryotic cells maintain their complex intracellular architecture of membrane-bound organelles through a dynamic system of membrane fission and fusion. Members of the Soluble N-ethylmaleimide Sensitive Factor Attachment Protein Receptor (SNARE) protein superfamily catalyze most intracellular membrane fusion events, including exocytosis of cargo into the extracellular space. Perhaps no exocytosis event is more tightly regulated than exocytosis of neurotransmitter at the neuronal synapse, which must occur with high precision and accuracy to ensure high-fidelity signaling across the entire nervous system. The SNARE proteins that catalyze fusion of synaptic vesicles with the neuronal plasma membrane are Syntaxin-1a and SNAP25 on the plasma membrane and Synaptobrevin-2 on the vesicle membrane. Together, they bridge the two membranes and assemble into a highly stable bundle that pulls the membranes together and promotes a membrane fusion reaction. In neurotransmitter release, this central reaction is assisted by a complex network of regulatory proteins that aid in controlling the speed, specificity, and efficiency of membrane fusion by SNAREs, ensuring signaling only takes place when biologically triggered and is suppressed in the absence of the trigger. Adding to the complexity, the membranes themselves are not only substrates of the fusion reaction, but actively shape it through both general biophysical properties and individual lipid-protein interactions.

Though much progress has been made on elucidating a complete molecular model of SNARE-mediated exocytosis at the synapse, many questions remain about how these many components work together to catalyze regulated fusion. The goal of this work was to explore protein and non-protein factors that can alter the efficiency and other properties of SNARE-mediated membrane fusion. In Chapter 3, we demonstrate that the plasma membrane SNARE protein SNAP25 intrinsically regulates SNARE assembly through interdomain interactions of its two distinct SNARE motif regions. In Chapter 4, we explore the effects of the alcohols ethanol and methanol on fusion and find that altered membrane properties are responsible for significant changes in fusion efficiency under doses of ethanol that can be achieved by alcohol consumption. Finally, in Chapter 5, we examine the relationship between the SNARE regulator protein Complexin-1 and the anionic plasma membrane lipid phosphatidylinositol 4,5-bisphosphate, showing that the lipid is required to potentiate the action of Complexin-1 in preventing untriggered fusion. In summary, these findings demonstrate that various regulatory factors, both protein and non-protein, contribute to the subtle balance of efficiency and control that characterizes fusion and exocytosis during neurotransmitter release.