α-Synuclein (αSYN), a pivotal synaptic protein implicated in synucleinopathies such as Parkinson’s disease and Lewy body dementia, undergoes protein phase separation. We reveal that vesicle-associated membrane protein 2 (VAMP2) orchestrates αSYN phase separation both in vitro and in cells. Electrostatic interactions, specifically mediated by VAMP2 via its juxtamembrane domain and the αSYN C-terminal region, drive phase separation. Condensate formation is specific for R-SNARE VAMP2 and dependent on αSYN lipid membrane binding. Our results delineate a regulatory mechanism for αSYN phase separation in cells. Furthermore, we show that αSYN condensates sequester vesicles and attract complexin-1 and -2, thus supporting a role in synaptic physiology and pathophysiology.

α-突触核蛋白 (αSYN) 是一种与帕金森病和路易体痴呆等突触核蛋白病有关的关键突触蛋白，会经历蛋白质相分离。我们揭示了囊泡相关膜蛋白 2 (VAMP2) 在体外和细胞内协调 αSYN 相分离。静电相互作用，特别是由 VAMP2 通过其近膜结构域和 αSYN C 末端区域介导，驱动相分离。冷凝物的形成是 R-SNARE VAMP2 特异的，并且依赖于 αSYN 脂质膜结合。我们的结果描绘了细胞中 αSYN 相分离的调节机制。此外，我们还发现 αSYN 会凝结隔离囊泡并吸引复合蛋白-1 和 -2，从而支持其在突触生理学和病理生理学中的作用。