Abstract
Exocyst is a large multisubunit tethering complex essential for targeting and fusion of secretory vesicles in eukaryotic cells. Although the assembled exocyst complex has been proposed to tether vesicles to the plasma membrane and activate soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) for membrane fusion, the key biochemical steps that exocyst stimulates in SNARE-mediated fusion are undetermined. Here we use a combination of single-molecule and bulk fluorescence assays to investigate the roles of purified octameric yeast exocyst complexes in a reconstituted yeast exocytic SNARE assembly and vesicle fusion system. Exocyst had stimulatory roles in multiple distinct steps ranging from SNARE protein activation to binary and ternary complex assembly. Importantly, exocyst had a downstream role in driving membrane fusion and full content mixing of vesicle lumens. Our data suggest that exocyst provides extensive chaperoning functions across the entire process of SNARE complex assembly and fusion, thereby governing exocytosis at multiple steps.