Synaptotagmin 1 oligomerization via the juxtamembrane linker regulates spontaneous and evoked neurotransmitter release

Significance Synaptotagmin 1 (syt1) is a synaptic vesicle (SV) protein that is rapidly activated by Ca2+ influx into presynaptic nerve terminals, triggering SV exocytosis. Syt1 also inhibits exocytosis, prior to Ca2+ influx, and thus helps synchronize evoked exocytosis upon Ca2+ binding. Herein, we identified a cluster of lysine residues, in the oft-ignored juxtamembrane linker region of syt1, that governs homo-multimerization in an anionic lipid-dependent manner. Neutralization of this positively charged region abolished syt1 self-association on phospholipid bilayers in vitro. Subsequently, in neurons, we found mutations that disrupted syt1 self-association were correlated with defects in clamping spontaneous SV release and in triggering and synchronizing evoked exocytosis. Thus, syt1 regulates SV exocytosis as an oligomer via charged residues in the juxtamembrane linker.
Synaptotagmin 1 (syt1) is a Ca2+ sensor that regulates synaptic vesicle exocytosis. Cell-based experiments suggest that syt1 functions as a multimer; however, biochemical and electron microscopy studies have yielded contradictory findings regarding putative self-association. Here, we performed dynamic light scattering on syt1 in solution, followed by electron microscopy, and we used atomic force microscopy to study syt1 self-association on supported lipid bilayers under aqueous conditions. Ring-like multimers were clearly observed. Multimerization was enhanced by Ca2+ and required anionic phospholipids. Large ring-like structures (∼180 nm) were reduced to smaller rings (∼30 nm) upon neutralization of a cluster of juxtamembrane lysine residues; further substitution of residues in the second C2-domain completely abolished self-association. When expressed in neurons, syt1 mutants with graded reductions in self-association activity exhibited concomitant reductions in 1) clamping spontaneous release and 2) triggering and synchronizing evoked release. Thus, the juxtamembrane linker of syt1 plays a crucial role in exocytosis by mediating multimerization.