SGIP1α functions as a selective endocytic adaptor for the internalization of synaptotagmin 1 at synapses

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.

Keywords: Clathrin-mediated endocytosis; SGIP1α; Synaptic vesicle; synaptotagmin 1.

Fig. 1

SGIP1α is expressed in cultured primary neurons and binds to Syt1. a Domain structures of SGIP1 and SGIP1α. SGIP1α contains additional 28 amino acids in the MP domain and 20 amino acids between the PRD and μHD. b Whole cell lysates from HEK293T, HEK293T with FLAG-SGIP1 and FLAG-SGIP1α and cultured hippocampal neurons were resolved by SDS-PAGE and transferred to nitrocellulose and immunoblotted with SGIP1 antibody. The red dotted line indicates the size of FLAG-SGIP1. c, d Hippocampal neuron lysate was pull-downed with purified GST-SGIP1α (c) or purified GST- SGIP1α-μHD (d) immobilized on glutathione-sepharose. Bound proteins were eluted from the beads by boiling and resolved by SDS-PAGE and either stained with Coomassie blue or transferred to nitrocellulose. Replicate blots were probed with Syt1 antibody as indicated. The asterisk indicates the band of Syt1. e Purified GST-Syt1-C2AB fusion protein immobilized on beads were incubated with cell extracts derived from HEK293T cells transfected with HA-C1 or HA-SGIP1α. Bound proteins were detected by immunoblotting for HA. f GST-Syt1-C2AB fusion protein immobilized on beads were incubated with cell extracts derived from HEK293T cells transfected with HA-C1 or HA-SGIP1α-μHD or HA- SGIP1α ΔμHD. Bound proteins were detected by immunoblotting for antibody against HA. IB, Immunoblot; IP, Immunoprecipitation; TCL, Total cell lysates

Fig. 2

SGIP1α is expressed at the presynaptic terminals of cultured hippocampal neurons and SGIP1α KD causes the selective defects in the internalization of Syt1. a Rat brain hippocampal neurons were fixed at DIV 21 and immunostained with specific antibodies against SGIP1α (red) and Syt1 (green). Scale bar, 5 μm. b Pearson’s coefficient of colocalization between SGIP1α and Syt1 (n = 10). (c and d) Primary cultured neurons at DIV 6 were infected with AAV-shRNA-SGIP1α, and the KD efficiency was confirmed by western blotting with anti-SGIP1 antibody at DIV 21 (n = 3 blots). e-j Average Syt1-pHluorin (e, control: n = 8, KD: n = 12), synaptophysin-pHluorin (g, control: n = 14, KD: n = 17), or VAMP2-pHluorin (I, control: n = 11, KD: n = 19) fluorescence intensity profiles in neurons expressing empty vector or shRNA-SGIP1α, plotted as ΔF/F₀ against time, after stimulation with 300 APs at 10 Hz (dark bar). Fluorescence values were normalized to the maximal fluorescence signal in each experimental condition. f, h, j τ values for the decay of Syt1-pHluorin (f), synaptophysin-pHluorin (h), or VAMP2-pHluorin (j) after stimulation fitted by a single exponential. Data are means ± SEM; *p < 0.05, ***p < 0.001 by Student’s t-test, N.S., not significant

Fig. 3

a Average traces of Syt1-pHluorin signal in control and SGIP1α KD during pH exchange experiments. Non-permeable pH 5.5 acid solution (Q1 and Q2 period) rapidly quenches all surface Syt1-pHluorin. After 50 s, the solution was changed back to pH 7.4. After 300 APs at 10 Hz, the extracellular solution was changed again to pH 5.5 for 50 s then back to pH 7.4. Net Syt-pH fluorescence changes were normalized to the initial values for individual experiments and averaged. Steady and stimulation represents the surface fraction of Syt1-pHluorin probe before and after stimulation, respectively. b, c Bar graphs indicate the surface fraction of Syt1-pHluorin probe before stimulation (b, control, 0.58 ± 0.03, n = 4; KD, 0.58 ± 0.06, n = 4) and after stimulation (c, control, 0.57 ± 0.03, n = 4; KD, 0.80 ± 0.07, n = 4). Data are presented as mean ± SEM. *p < 0.05

Fig. 4

SGIP1α mutant W771A/K781A binds less efficiently to Syt1 and fails to rescue the endocytic defect of Syt1 caused by SGIP1α KD. a Ribbon models of the crystal structures of AP-2 μ subunit, μHD of stonin2 and SGIP1α. The β-sheets of the μHDs are colored lime green and circled in dotted line. b An enlarged representation of the μHDs of AP-2 and SGIP1α. The μHDs and peptides are shown as ribbon models and sticks, respectively. Comparing overall structural similarity, W771 of SGIP1α was identified as the residue corresponding to Y344 of AP-2. c Multiple protein sequence alignment of SGIP1α and orthologues from chimpanzee, cattle, chicken, zebrafish, frog, human, rat, mouse. Numbers refer to the amino acid residue within the predicted β strand. Conserved residues are colored in red. (below) Corresponding sequences of WT SGIP1α and a double mutant of SGIP1α W771A/K781A (SGIP1α-mut). d Purified GST-Syt1-C2AB fusion protein immobilized on beads were incubated with cell extracts derived from HEK293T cells transfected with HA-C1 or HA-SGIP1α or HA-SGIP1α mutant W771A/K781A. Bound proteins were detected by immunoblotting for antibody against HA. Coomassie blue staining of the gels revealed that similar amounts of GST fusion proteins were used. e Relative band intensity of bound Syt1 (1 ± 0 for SGIP1α, 0.20 ± 0.07 for SGIP1α-mut, n = 3 independent blots). f Average Syt1-pHluorin fluorescence intensity profiles in neurons expressing shRNA-resistant SGIP1α (KD + SGIP1α, n = 6) or shRNA-resistant SGIP1α-W771A/K781A (KD + SGIP1α mut, n = 5) with shRNA-SGIP1 and Syt1-pHluorin after stimulation with 300 APs at 10 Hz (dark bar). Fluorescence values were normalized to the maximal fluorescence signal in each experimental condition. g τ values for the decay of fluorescence fitted by a single exponential (KD + SGIP1α-res.: n = 6, KD + SGIP1α- res.mut: n = 5). Data are presented as means ± SEM; *p < 0.05, ***p < 0.001 by Student’s t-test