In C. elegans neurons, UNC-18 is necessary for evoked synaptic vesicle release, but its mechanism is unknown. Prior experiments suggest that UNC-18 acts either before or after vesicle docking. We propose UNC-18 has both pre- and post-docking functions: 1) it is a molecular chaperone that traffics syntaxin to the synapse in a non-active (closed) conformation and 2) a facilitator of vesicular release independent of closed syntaxin binding. Furthermore, we propose phosphorylation of UNC-18 provides the molecular switch converting UNC-18 to a form that can bind assembled SNARE complexes. Molecular chaperone:
unc-18 null mutants have a severe locomotion defect. One model suggests that UNC-18 acts via the SNARE protein syntaxin. UNC-18 exhibits high affinity for syntaxin in the closed state. In immunoprecipitation pull-down assays, anti-GFP beads recover high levels of both UNC-18::GFP and syntaxin, indicating the two proteins physically interact. The mutant protein UNC-18(R39C) does not bind syntaxin with high affinity.
unc-18 mutants exhibit abnormal localization of syntaxin to neuronal cell bodies. These defects are completely rescued by an UNC-18::GFP expression construct, which suggest UNC-18 is required for normal syntaxin localization. In a UNC-18(R39C)::GFP point mutant construct, pull-down recovery of syntaxin is nearly abolished (400 x decrease). Additionally, the R39C construct partially retains a cell body staining defect of syntaxin localization. These results indicate the tight-binding of UNC-18 to syntaxin facilitates syntaxin transport to the synapse. Release facilitator: Electron microscopy of
unc-18 null shows a 2.5 fold decrease in docked synaptic vesicles and electrophysiology reveals the remaining docked vesicles are incompetent for evoked release. UNC-18(R39C) rescues the Unc phenotype yet a docking defect remains. These results indicate UNC-18 has a post-docking role that promotes vesicle release.