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.

Exocyst 是一种大型多亚基束缚复合物，对于真核细胞中分泌囊泡的靶向和融合至关重要。尽管组装的外囊复合物已被提议将囊泡束缚在质膜上并激活可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体 (SNARE) 进行膜融合，但外囊在 SNARE 介导的融合中刺激的关键生化步骤尚未确定。在这里，我们结合使用单分子和本体荧光测定来研究纯化的八聚体酵母胞外囊复合物在重建的酵母胞外囊 SNARE 组装和囊泡融合系统中的作用。 Exocyst 在从 SNARE 蛋白激活到二元和三元复合物组装的多个不同步骤中具有刺激作用。重要的是，外囊在驱动囊泡腔的膜融合和全内容物混合方面具有下游作用。我们的数据表明，胞吐在 SNARE 复合物组装和融合的整个过程中提供了广泛的陪伴功能，从而在多个步骤中控制胞吐作用。