Syndapin I and endophilin I bind overlapping proline-rich regions of dynamin I: role in synaptic vesicle endocytosis

Dynamin I mediates vesicle fission during synaptic vesicle endocytosis (SVE). Its proline-rich domain (PRID) binds the Src-homology 3 (SH3) domain of a subset of proteins that can deform membranes. Syndapin I, amphiphysin I, and endophilin I are its major partners implicated in SVE. Syndapin binding is controlled by phosphorylation at Ser-774 and Ser778 in the dynamin phospho-box. We now define syndapin and enclophilin-bincling sites by peptide competition and sitedirected mutagenesis. Both bound the same region of the dynamin PRID and both exhibited unusual bidirectional binding modes around core PxxP motifs, unlike amphiphysin which employed a class II binding mode. Enclophilin binds to tandem PxxP motifs in the sequence (778)SPTPQRRA PAVPPARPGSR(796) in dynamin, with SPTPQ being an overhang sequence. In contrast, syndapin binding involves two components in the region (772)RRSPTSSPTPQRRAPAVPPARPGSR(796). It required a single PxxP core and a non-PxxP N-terminally anchored extension which bridges the phosphobox and may contribute to binding specificity and affinity. Syndapin binding is exquisitely sensitive to the introduction of negative charges almost anywhere along this region, explaining why it is a highly tuned phospho-sensor. Overexpression of dynamin point mutants that fail to bind syndapin or endophilin inhibit SVE in cultured neurons. Due to overlapping binding sites the interactions between dynamin and syndapin or endophilin were mutually exclusive. Because syndapin acts as a phospho-sensor, this supports its role in depolarization-induced SVE at the synapse, which involves dynamin dephosphorylation. We propose syndapin and endophilin function either at different stages during SVE or in mechanistically distinct types of SVE.