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Title: Protein-lipid interactions in synaptic vesicle exocytosis
Author: Connell, E. J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2009
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The fusion of neurotransmitter-filled synaptic vesicles with the pre-synaptic membrane in response to calcium influx is exquisitely regulated. Synaptic vesicle exocytosis is energetically demanding and the neuronal SNARE proteins syntaxin, SNAP25 and synaptobrevin have come to prominence as the driving engines behind this process. Resident on both vesicular and pre-synaptic membranes they form a stable four-helical bundle, the assembly of which contributes to membrane fusion. However, SNAREs do not act in isolation during synaptic vesicle exocytosis but are instead regulated by a complex web of interactions with other proteins including synaptotagmin, a calcium-sensing component of the vesicle itself, and Munc18, a highly-conserved cytosolic protein. In addition, changes in the lipid environment surrounding the SNAREs play a critical role. In this thesis I report the results of two lines of investigation, into both synaptotagmin’s and Munc18’s action. Firstly, I consider the significance of the cytoplasmic double C2 domain structure of synaptotagmin. Using several strategies including a novel real-time absorbance assay, I show that these tandem C2 domains, but neither domain alone, rapidly cross-link lipid membranes in the presence of calcium. This property is conserved. Cross-linking ability can be masked in full-length synaptotagmin, via an electrostatic interaction with the membrane in which it is embedded. Finally, I address the mechanism of arachidonic acid action on syntaxin/Munc18, showing that this lipid activates Munc18-bound syntaxin and that a Munc18/syntaxin/SNAP25 assembly exists in brain. Arachidonic acid also activates free syntaxin, defining a molecular target for the reported role of this lipid in the promotion of vesicle fusion. My data are incorporated into a revised model of the protein-lipid interactions underlying synaptic vesicle exocytosis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available