| Description |
Vesicle fusion is driven by the concerted action of Unc18 and SNARE (soluble NSF-attachment protein receptor) proteins. Fusion is mediated by the assembly of the SNARE proteins: synaptobrevin, syntaxin, and SNAP25. However, the precise function of Unc18 in fusion is still unclear. Unc18 forms a clamp on syntaxin, locking it in a closed state where the SNARE motif would be inaccessible to the fusion machinery. This interaction is necessary for trafficking syntaxin to the synapse. However, this inhibitory function does not explain the positive functions of Unc18 in vitro and in vivo. In this dissertation, I investigated Unc18 function in SNARE-mediated fusion utilizing the nematode C. elegans. In Chapter 2, I focused on the Unc18 binding partner, syntaxin. Specifically, I exchanged different domains of the worm syntaxin with the respective region from yeast. I observed that syntaxin Habc domain plays an essential role in fusing synaptic vesicles. In the third chapter, I exchanged both Unc18 proteins and the Habc domain of syntaxin with the respective proteins from organisms ranging from yeast to trichoplax. I observed that when the syntaxin Habc domain and Unc18 came from the same species, locomotory behaviors and synaptic transmission were partially restored, suggesting a direct interaction between Unc18 and syntaxin Habc. To further explore this interaction, I used an in silico approach to probe for potential interaction surfaces. Charge swap mutations at one of the predicted salt-bridges provided in vivo validation for a novel interaction face between syntaxin Habc and Unc18. In Chapter 4, I reconstituted the yeast fusion machinery in C. elegans that supported neurotransmission. I identified that Unc18 interacts with the SNARE complex and synergistically function with the Habc domain to regulate fusion. Furthermore, I engineered yeast Unc18 with worm residues predicted to participate in SNARE complex assembly, based on a distant SM protein family member, showed restoration of synaptic transmission only when there was a species match with syntaxin Habc domain, supporting a role of Unc18 in SNARE assembly. In total, my findings provide, in the context of a whole organism, an important advancement in understanding the role of Unc18 in neurotransmission. |
