The juxtamembrane linker of synaptotagmin 1 regulates Ca binding via liquid-liquid phase separation.

Synaptotagmin (syt) 1, a Ca sensor for synaptic vesicle exocytosis, functions as a multimer. Syt1 senses Ca 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 Ca-sensitivity of syt1. Moreover, Ca and anionic phospholipids facilitate the observed phase separation, and increases in [Ca] 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 Ca 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.