SGIP1\u03b1 functions as a selective endocytic adaptor for the internalization of synaptotagmin 1 at synapses

Sang-Eun Lee,Unghwi Lee,Soomin Jeong,Sunghoe Chang

doi:10.1186/s13041-019-0464-1

Abstract

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 μ-homology domain (μHD) and binds AP-2 and Eps15, thus functioning as an endocytic regulator of clathrin-mediated endocytosis (CME). Its longest isoform SGIP1α is predominantly expressed in the brain but the functional significance of SGIP1 in SV recycling remains unknown. Here, we found that SGIP1α, a brain-specific long isoform of SGIP1 binds synaptotagmin1 (Syt1) via its μHD and promotes the internalization of Syt1 on the neuronal surface. The small hairpin RNA (shRNA)-mediated knockdown (KD) of SGIP1α caused selective impairment of Syt1 internalization at hippocampal synapses and it was fully rescued by coexpression of the shRNA-resistant form of SGIP1α in KD neurons. We further found that the μHD of SGIP1α 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 SGIP1α 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.

Highlights

Neurotransmission involves the calcium (Ca2+)-regulated 2+fusion of synaptic vesicle (SV), a process that requires the Ca sensor Syt1 at the active zone, which defines sites of neurotransmitter release [1]
We found that hippocampal neurons endogenously expressed a protein with a molecular mass of about 130 kDa, which was comparable to the size of FLAG-tagged SGIP1α but higher than that of FLAG-tagged SH3containing GRB2-like protein 3-interacting protein 1 (SGIP1) in HEK293T (Fig. 1b)
When we repeated endocytosis assays using other SV proteins, synaptophysin (Fig. 2 g and h) or vesicle associated membrane protein 2 (VAMP2) (Fig. 2) i and j, no endocytic defects were observed. These results suggest that SGIP1α KD hinders the endocytosis of Syt1, but not synaptophysin or VAMP2

Summary

Introduction

Neurotransmission involves the calcium (Ca2+)-regulated 2+fusion of SVs, a process that requires the Ca sensor Syt at the active zone, which defines sites of neurotransmitter release [1]. Post-exocytic SV proteins are retrieved by endocytosis from the plasma membrane to regenerate new SVs [2]. More than forty different integral membrane proteins that are essential for SV trafficking and neurotransmission were identified and they should be incorporated into individual SVs with the correct stoichiometry to be functional for subsequent. [5, 11,12,13,14] Such that each regenerated individual synaptic vesicle will obtain all essential synaptic vesicle proteins with high fidelity. Recent studies indicated that the association between SV proteins is critical for their accurate retrieval and sorting into SVs. For example, synaptophysin and SV2A/B associates with VAMP2 and Syt, respectively, facilitating their endocytic retrieval [15, 16]

Methods

Results

Conclusion