Department of Physiology and Biomedical Sciences, Neuroscience Research Institute, Seoul National University College of Medicine, Seoul, 03080, South Korea.
Mol Brain. 2019 May 3;12(1):41. doi: 10.1186/s13041-019-0464-1.
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.
正确地将胞吐的突触小泡 (SV) 蛋白分拣到内吞作用过程中的各个 SV 中，对于随后的神经传递的保真度至关重要。最近的研究表明，每个 SV 蛋白都通过其自身的专用衔接蛋白以及 SV 蛋白之间的相互作用进行分拣到各个 SV 中。含有 SH3 结构域的 GRB2 样蛋白 3 相互作用蛋白 1（SGIP1）是 Fer/Cip4 同源结构域仅（FCHo）蛋白的同源物，它含有 μ 同源结构域（μHD），并与 AP-2 和 Eps15 结合，因此作为网格蛋白介导的内吞作用（CME）的内吞调节因子发挥作用。其最长的同种型 SGIP1α 主要在大脑中表达，但 SGIP1 在 SV 再循环中的功能意义尚不清楚。在这里，我们发现 SGIP1α，SGIP1 的大脑特异性长同种型，通过其 μHD 与突触融合蛋白 1（Syt1）结合，并促进神经元表面上 Syt1 的内化。通过小发夹 RNA（shRNA）介导的 SGIP1α 敲低（KD）导致海马突触处 Syt1 内化的选择性损害，并且在 KD 神经元中表达 shRNA 抗性形式的 SGIP1α 完全挽救了这种损害。我们进一步发现，SGIP1α 的 μHD 在结构上与 AP-2 和 stonin2 相似，并且 Trp771 和 Lys781 处的突变，对应于 AP-2 和 stonin2 的 Syt1 识别基序，突变为 Ala 与 Syt1 的结合效率降低，并且不能挽救由 KD 引起的 Syt1 内吞缺陷。我们的结果表明，SGIP1α 是一种专门用于回收表面 Syt1 的内吞衔接蛋白。由于 Syt1 的内吞分选也由突触小泡糖蛋白 2A/B（SV2A/B）和 stonin2 的重叠活性介导，我们的结果表明，这些蛋白的互补故障安全机制确保了 Syt1 回收的高保真度。
