The juxtamembrane linker of synaptotagmin 1 regulates Ca²⁺ binding via liquid-liquid phase separation

Synaptotagmin (syt) 1, a Ca²⁺ sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca²⁺ via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca²⁺-sensitivity of syt1. Moreover, Ca²⁺ and anionic phospholipids facilitate the observed phase separation, and increases in [Ca²⁺]_i promote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca²⁺ binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS.