Figure 2.

Model of SV protein reclustering and sorting at the presynapse. (A) After full collapse fusion freely diffusing SV proteins are confined and recaptured by endocytic sorting adaptors at the periactive zone to allow for release site clearance. At the plasma membrane SV proteins might either be clustered by AP-2 and additional cargo-specific adaptor proteins (I), they might interact with each other and thereby self-assemble into clusters (II) or form mixed clusters of self-assembled SV proteins together with sorting adaptors (III). These clusters can directly be endocytosed from the plasma membrane by Clathrin-mediated endocytosis (CME) to reform SVs. However, cargo-specific sorting proteins together with AP-2 and Clathrin can also operate on endosomal-like vacuoles (ELVs) after Clathrin-independent endocytosis (CIE) to recycle SVs with correct protein composition. (B–D) Sorting of individual SV proteins. (B) The precise sorting of Synaptotagmin1 can be accomplished by three possible mechanisms. (a) Synaptotagmin1 associates with AP-2μ2 via its C2B domain. However, this interaction does not suffice for efficient Synaptagmin1 retrieval. The specific adaptor protein Stonin2 is needed to strengthen the link between AP-2 and Synaptotagmin1 by interacting simultaneously with Synaptotagmin1’s C2A domain and the AP-2α ear. (b) Synaptotagmin1 can also be sorted by association with SV2, another SV protein. The N-terminus of SV2 binds to the C2B domain of Synaptotagmin1 and thereby facilitates correct Synaptotagmin1 sorting. (c) As Stonin2 and SV2 interact with distinct C2 domains of Synaptotagmin1, both proteins might bind at the same time to the Ca²⁺ sensor and link it to AP-2 to enable its precise retrieval. (C) Free Synaptobrevin2 can also be sorted by multiple mechanisms. (a) Synaptobrevin2 is recognized by the specific sorting adaptors AP180 and CALM via their ANTH domain that interacts with the SNARE domain of Synaptobrevin2 and with the plasma membrane. Via the unstructured C-terminus AP180 and CALM associate with AP-2 and Clathrin thereby linking Synaptobrevin2 to the CME machinery. (b) Synaptobrevin2 can also be sorted by association with the SV protein Synaptophysin1 via their transmembrane domains. (D) In inner hair cells Otoferlin is directly sorted by AP-2 via the interaction of di-leucine motifs within the linker regions between the C2A-C2B-C2C domains of Otoferlin with AP-2α/σ2. In addition, Otoferlin associates with AP-2μ2. However, the exact interaction sites are not clear yet.