The juxtamembrane linker of synaptotagmin 1 regulates Ca 2+ binding via liquid-liquid phase separation.

Synaptotagmin (syt) 1, a Ca 2+ sensor for synaptic vesicle exocytosis, is thought to act in vivo as a multimer. It senses Ca 2+ 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). Unexpectedly, these interactions negatively regulate the Ca 2+ -sensitivity of the tandem C2-domains of syt1. Ca 2+ and anionic phospholipids promote the observed phase separation. This potentially creates a feedback loop that might serve to finetune the ability of syt1 to trigger release, via alterations in Ca 2+ 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.