The UNC-18/Munc18 protein family is implicated in several stages of exocytosis. Here we present evidence for two UNC-18-dependent synaptic vesicle (SV) targeting steps termed tethering (near the plasma membrane) and docking (contact with the plasma membrane). Docked SVs arise primarily from UNC-13-dependent priming, aprocess leading to SNARE complex assembly and SV fusion competence. The loss of docked SVs in
unc-18 mutants, therefore, supports a growing view that UNC-18 functions in this process. Although docked SVs are depleted in
unc-13 mutants, tethered SVs accumulate, suggesting UNC-18-dependent tethering occurs in the absence of SV priming. UNC-18 is thought to associate with the plasma membrane via syntaxin interactions. Consistent with this notion, membrane bound UNC-18 is abolished in syntaxin (
unc-64) mutant synapses. We, therefore, asked whether SV targeting is impacted by loss of syntaxin. In
unc-64 mutant synapses, tethered and docked SVs are eliminated, indicating both SV steps require UNC-18 and syntaxin. Since Munc18-1 is known to interact with closed and open syntaxin conformations, we next examined synapses in which syntaxin was rendered constitutively open. Open syntaxin reduced SV tethering but docking was enhanced, indicating that UNC-18/closed syntaxin binding is required for SV tethering,where as UNC-18/open syntaxin is required for SV docking. We have previously shown that C. elegans tomosyn (TOM-1) negatively regulates SV priming.Vertebrate tomosyn is known to compete with Munc18-1 for syntax inbinding. Similarly, we found increased membrane-associated TOM-1 in
unc-18 mutants and conversely, increased membrane-bound UNC-18 in
tom-1 mutants. We therefore, predicted that loss of tomosyn would enhance UNC-18/syntaxin interactions at C. elegans synapses, resulting in increased SV tethering and docking. Analysis of
tom-1 mutants confirmed this prediction, indicating that TOM-1 negatively regulates both UNC-18-dependent SV-targeting steps. We next demonstrated that in
tom-1;
unc-18 double mutants, SV docking but not tethering was partially restored resulting in an increased primed SV pool. Together, these data provide evidence for two molecularly distinct UNC-18-dependent SV-targeting mechanisms; tethering, requiring UNC-18/closed syntaxin and docking, in which UNC-18/open syntaxin promotes SV priming.TOM-1 negatively regulates both UNC-18 dependent steps, thereby limiting the number of fusion-competent SVs.