SNAP25 undergoes phase separation to facilitate the assembly of the synaptic vesicle fusion machinery

The assembly of the SNARE complexes drives synaptic exocytosis in neurotransmission. SNAP25 is a key SNARE protein that pairs with syntaxin-1 and VAMP2 in synaptic vesicle fusion. The dysfunction of SNAP25 is associated with neurodevelopmental and neurodegenerative disorders. Here, we discovered that SNAP25 undergoes liquid-liquid phase separation (LLPS) in vitro and in living cells. The linker region connecting the two SNARE motifs is critical for the phase separation of SNAP25, which is regulated by palmitoylation modification. Syntaxin-1 co-condenses with SNAP25 and promotes the LLPS of SNAP25. The SNAP25 condensates recruit syntaxin-1 and VAMP2 to form ternary coacervates, facilitating the assembly of the SNARE complexes. The developmental and epileptic encephalopathy-associated mutation, I67N or I192N, impairs the LLPS of SNAP25 by blocking condensate fusion, thereby suppressing the SNARE assembly. Our findings provide new insights into the mechanisms of SNARE assembly, highlighting the critical role of SNARE phase separation in synaptic vesicle fusion.