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bioRxiv · 10.1101/2023.01.15.524101

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

Kontaxi, C.·Cousin, M. A.
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Abstract

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. All domains interact with either lipids or SV endocytosis proteins, with all of these interactions required for SV endocytosis, apart from the Amph1 PRD. In this study, we determined this role and confirmed requirements for established Amph1 interactions in SV endocytosis at typical small central synapses. 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 in the control of SV endocytosis. Furthermore, we identified an interaction site for the endocytosis protein endophilin A1 in the Amph1 PRD and revealed a key role for this interaction in SV endocytosis. Finally, we discovered that the phosphorylation status of Amph1-S293 within the PRD dictates the formation of the Amph1-endophilin A1 complex and is essential for efficient SV regeneration. This work therefore identifies an activity-dependent dephosphorylation-dependent interaction that is key for efficient SV endocytosis.

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Kontaxi, C., Cousin, M. A.. 2023-01-18. The phospho-regulated amphiphysin/endophilin interaction is required for synaptic vesicle endocytosis. https://doi.org/10.1101/2023.01.15.524101

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