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