Synaptic transmission requires the fusion of neurotransmitter containing vesicles with the neuron's plasma membrane in a temporally restrictive manner. In Drosophila, this challenge is accomplished in part by the SNARE protein neuronal-Synaptobrevin (n-Syb). The juxtamembrane region of this molecule, linking the cytosolic SNARE motif and transmembrane region, is hypothesized to play a functional role in facilitating membrane fusion. This short, 10 amino acid, segment contains numerous charged residues and one conserved tryptophan residue. Its short rigid structure may be important in transducing force during SNARE complex assembly.
Tryptophan residues, common in membrane proteins, are often observed at the membrane-water interface. It was hypothesized that this conserved tryptophan residue was important for anchoring and positioning n-Syb in the membrane. Proteins produced with tryptophan mutated were tested for anchoring and stability in a membrane model using NMR spectroscopy. Experiments testing depth of insertion using exposure to oxygen, a paramagnetic species, and exchange with deuterium demonstrated that tryptophan anchored n-Syb in the membrane.
To test a potential functional role for the juxtamembrane region of n-Syb in synaptic transmission, a reverse genetic approach was employed. Wild-type and mutant P-element clones were made using the genomic sequence of n-syb including the endogenous promoter. n-Syb was found to be expressed, integrate and orient correctly in the membrane of Drosophila S2 cells. Transgenic Drosophila, produced via P-element transformation, were also found to produce transgenic protein. Transgenic expression of wild-type n-syb was found to restore an n-syb hypomorphic mutant from severe motor impairment and limited lifespan to wild-type levels. Synaptic transmission was assessed in 3rd instar larval preparations of mutant and wild-type transgenics. Mutation of the tryptophan residue and insertion of a short flexible linker were both found to inhibit synaptic transmission, while insertion of a long flexible linker was not.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/43544 |
| Date | 08 January 2014 |
| Creators | DeMill, Colin Don Malcolm |
| Contributors | Stewart, Bryan |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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