Lee Chanwoo, Lepore Dante, Lee Seung-Hak, Kim Tae Gyun, Buwa Natasha, Lee Jongchan, Munson Mary, Yoon Tae-Young
School of Biological Sciences and Institute for Molecular Biology and Genetics, Seoul National University, Seoul, South Korea.
Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, MA, USA.
Nat Struct Mol Biol. 2025 Jan;32(1):150-160. doi: 10.1038/s41594-024-01388-2. Epub 2024 Sep 6.
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.
外泌体复合体是一种大型多亚基拴系复合体，对于真核细胞中分泌囊泡的靶向和融合至关重要。尽管已提出组装好的外泌体复合体将囊泡拴系到质膜上，并激活可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNAREs）以进行膜融合，但外泌体复合体在SNARE介导的融合中所刺激的关键生化步骤仍未确定。在这里，我们结合单分子和整体荧光测定法，研究纯化的八聚体酵母外泌体复合体在重组酵母外排SNARE组装和囊泡融合系统中的作用。外泌体复合体在从SNARE蛋白激活到二元和三元复合体组装的多个不同步骤中具有刺激作用。重要的是，外泌体复合体在驱动膜融合和囊泡腔的完全内容物混合方面具有下游作用。我们的数据表明，外泌体复合体在SNARE复合体组装和融合的整个过程中提供广泛的分子伴侣功能，从而在多个步骤中控制胞吐作用。
