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Title: Syntaxin-munc18 interaction
Author: McDonald, Angela
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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Regulated exocytosis is spatially and temporally regulated by a cascade of protein-protein interactions. The minimal machinery required for membrane fusion is the SNARE complex, formed between three membrane proteins: syntaxin, synaptobrevin (VAMP) and SNAP-25. The conformation of syntaxin la, which exists in 'open' and 'closed' states, is believed to play a role in SNARE complex assembly in neuronal and neuroendocrine cells, through regulation of the interconversion between its different conformational states by the cytoplasmic protein muncl8-l. Recent work has also demonstrated that phosphorylation of muncl8-l by protein kinase C on serines 306 and 313 weakens its affinity for syntaxin la in vitro. To further understand the role of the syntaxin la - muncl8-l complex, the interaction of these two proteins was studied in vitro and in vivo. These studies utilized muncl8-l and syntaxin la chimeras containing the fluorescent proteins EGFP and EYFP. Use of radiolabelled muncl8-l, EGFP-muncl8-l, muncl8-lR39c (mutated in a residue that is important for interaction with syntaxin la and for maintaining muncl8-ls conformation), EGFP-muncl8-lR39c, EYFP-muncl8-lS306E:S3i3E(a phosphomimetic mutant) and EYFPmuncl8- lR39C:S306E:S3i3Ein in vitro binding studies revealed that the addition of the fluorescent proteins to syntaxin la and muncl8-l had little effect on their interaction in comparison with wild-type proteins. The muncl8-lR39c and the phosphomimetic muncl8-l mutants had a reduced affinity for syntaxin la. In order to study protein interaction within cells, using a cell line with no endogenous protein background, muncl8-l and syntaxin la variants were expressed in HEK293 cells and imaged using confocal laser scanning microscopy. These studies showed that phosphorylation of muncl8-l reorganised the cellular localisation of the syntaxin la - muncl8-l complex from the cytoplasm to the plasma membrane. Finally FRET and FLIM analysis of the interaction between ECFP-syntaxin la and EYFP-muncl8- 1, and its mutants, revealed that the conformation of the syntaxin la- munc 18-1 complex depended on its cellular location, and that PKC phosphorylation of muncl8-l occurred in a spatially restricted manner in HEK293 cells, altering the intracellular conformation. Also the syntaxin la - muncl8-l complex can assume at least one other spatially defined conformation on the plasma membrane.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available