SGIP1 functions as a selective endocytic adaptor for the internalization of synaptotagmin 1 at synapses

Proper sorting of exocytosed synaptic vesicle (SV) proteins into individual SVs during endocytosis is of the utmost importance for the fidelity of subsequent neurotransmission. Recent studies suggest that each SV protein is sorted into individual SVs by its own dedicated adaptors as well as by association between SV proteins. The SH3-containing GRB2-like protein 3-interacting protein 1 (SGIP1), an ortholog of Fer/Cip4 homology domain-only (FCHo) proteins, contains a -homology domain (HD) and binds AP-2 and Eps15, thus functioning as an endocytic regulator of clathrin-mediated endocytosis (CME). Its longest isoform SGIP1 is predominantly expressed in the brain but the functional significance of SGIP1 in SV recycling remains unknown. Here, we found that SGIP1, a brain-specific long isoform of SGIP1 binds synaptotagmin1 (Syt1) via its HD and promotes the internalization of Syt1 on the neuronal surface. The small hairpin RNA (shRNA)-mediated knockdown (KD) of SGIP1 caused selective impairment of Syt1 internalization at hippocampal synapses and it was fully rescued by coexpression of the shRNA-resistant form of SGIP1 in KD neurons. We further found that the HD of SGIP1 is structurally similar to those of AP-2 and stonin2, and mutations at Trp771 and Lys781, which correspond to Syt1-recognition motifs of AP-2 and stonin2, to Ala bound less efficiently to Syt1 and failed to rescue the endocytic defect of Syt1 caused by KD. Our results indicate that SGIP1 is an endocytic adaptor dedicated to the retrieval of surface-stranded Syt1. Since endocytic sorting of Syt1 is also mediated by the overlapping activities of synaptic vesicle glycoprotein 2A/B (SV2A/B) and stonin2, our results suggest that complementary fail-safe mechanism by these proteins ensures high fidelity of Syt1 retrieval.