Syp1 Acts As A Clearance Factor For Syb2 At The Presynapse

Fusion of synaptic vesicles (SVs) during synaptic transmission is mediated by SNARE complex assembly formed by coiled-coiling of the SNARE proteins synaptobrevin2 (Syb2), syntaxin-1A and SNAP-25. In order to maintain neurotransmission, exocytosed SV components need to be retrieved from the surface by compensatory endocytosis. Clathrin-mediated endocytosis (CME) is thought to be the predominant mechanism. However, it might be too slow for fast SV recycling. Therefore, a pre-sorted and pre-assembled surface pool of SV proteins at the presynapse, the ‘readily retrievable pool’ (RRetP), might support a first wave of fast CME.Using fluorescence nanoscopy of labeled SV proteins in combination with quantitative k-nearest neighbour and paired correlation analysis, we were able to resolve the spatial distribution of the surface pool at the presynaptic membrane and identified dimerization of Syb2 as a first important step in self-assembly of surface nanodomains. Moreover, we show that the SV protein synaptophysin1 (Syp1) coordinates and sequesters these dimers in larger nanodomains containing a few ten molecules of both Syb2 and Syp1. Both, dimerization and co-expression of Syp1 are sufficient for correct targeting of Syb2 to presynaptic structures. These results were confirmed using shRNA-mediated knockdown of Syp1 or Syp1 knockout mice. Using pHluorin-fusion constructs expressed in hippocampal neurons, we show that dimerization of Syb2 is necessary for proper sorting into endocytosing SVs, since Syb2 monomers stranded at the plasma membrane cause frequency-dependent short term depression (STD) as a result of compromised Syb2 clearance away from release sites. This finding is corroborated in Syp1 knockout neurons that display strong frequency-dependent STD. Our results demonstrate the functional role of the Syb2-Syp1 interaction in sequestering Syb2 into the RRetP for efficient release site clearance, thereby preventing cis-SNARE complex formation.