Synaptophysin 1 Clears Synaptobrevin 2 from the Presynaptic Active Zone to Prevent Short-Term Depression

• Self-assembly of Syb2 and Syp1 into clusters mediates release site clearance
• Syp1 prevents exocytosed surplus Syb2 from cis-SNARE complex formation
• Loss of Syp1 causes strong frequency-dependent short-term depression
• The G100Y mutation of Syb2 has a dominant-negative effect on release site clearance

SummaryRelease site clearance is an important process during synaptic vesicle (SV) recycling. However, little is known about its molecular mechanism. Here we identify self-assembly of exocytosed Synaptobrevin 2 (Syb2) and Synaptophysin 1 (Syp1) by homo- and hetero-oligomerization into clusters as key mechanisms mediating release site clearance for preventing cis-SNARE complex formation at the active zone (AZ). In hippocampal neurons from Syp1 knockout mice, neurons expressing a monomeric Syb2 mutant, or after acute block of the ATPase N-ethylmaleimide-sensitive factor (NSF), responsible for cis-SNARE complex disassembly, we found strong frequency-dependent short-term depression (STD), whereas retrieval of Syb2 by compensatory endocytosis was only affected weakly. Defects in Syb2 endocytosis were stimulus- and frequency-dependent, indicating that Syp1 is not essential for Syb2 retrieval, but for its efficient clearance upstream of endocytosis. Our findings identify an SV protein as a release site clearance factor.

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