The phospho‐regulated amphiphysin/endophilin interaction is required for synaptic vesicle endocytosis

The multidomain adaptor protein amphiphysin‐1 (Amph1) is an important coordinator of clathrin‐mediated endocytosis in non‐neuronal cells and synaptic vesicle (SV) endocytosis at central nerve terminals. Amph1 contains a lipid‐binding N‐BAR (Bin/Amphiphysin/Rvs) domain, central proline‐rich (PRD) and clathrin/AP2 (CLAP) domains, and a C‐terminal SH3 domain. Amph1 interacts with both lipids and proteins, with all of these interactions required for SV endocytosis, with the exception of the Amph1 PRD. The Amph1 PRD associates with the endocytosis protein endophilin A1, however, the role of this interaction in SV endocytosis has not been investigated. In this study, we set out to determine whether the Amph1 PRD and its interaction with endophilin A1 was essential for efficient SV endocytosis at typical small central synapses. To achieve this, domain‐specific interactions of Amph1 were validated using in vitro GST pull‐down assays, with the role of these interactions in SV endocytosis determined in molecular replacement experiments in primary neuronal culture. Using this approach, we confirmed important roles for CLAP and SH3 domain interactions of Amph1 in the control of SV endocytosis. Importantly, we identified the interaction site for endophilin A1 within the Amph1 PRD and exploited specific binding mutants to reveal a key role for this interaction in SV endocytosis. Finally, we determined that the formation of the Amph1‐endophilin A1 complex is dependent on the phosphorylation status of Amph1‐S293 within the PRD and that the phosphorylation status of this residue is essential for efficient SV regeneration. This work, therefore, reveals a key role for the dephosphorylation‐dependent Amph1‐endophilin A1 interaction in efficient SV endocytosis. The synaptic vesicle endocytosis protein amphiphysin‐1 (Amph1) has several domains that share essential interactions with clathrin, AP2 and dynamin‐1 (Dyn1). It also has a proline‐rich domain (PRD) of unknown function that is phosphorylated on serine‐293 at central synapses. The Cousin laboratory revealed that phosphorylation of this site controls PRD interactions with the endocytosis protein endophilin A1, using phospho‐null (S293A) and mimetic (S293E) Amph1 mutants. Since S293 is dephosphorylated during neuronal activity, this suggests that endophilin A1 is recruited by Amph1 during endocytosis and then released after activity terminates via Amph1 rephosphorylation.