Depletion of AP180 and CALM causes surface accumulation of synaptobrevin 2. (A) Efficient siRNA knockdown of AP180 or CALM expression. HEK293 cells transiently expressing rat AP180 were transfected with siRNA specific for rat AP180 (Left) or CALM (Right) and analyzed by immunoblotting. Expression levels of AP180 or CALM are efficiently down-regulated. (B and C) SV exocytosis probed by FM4-64 in AP180-, CALM-, or AP180- and CALM-depleted neurons. (B) Exocytosis kinetics measured by FM4-64 unloading. (Upper) Scheme of the protocol used to load and unload FM4-64. (Lower) Normalized kinetic traces of FM4-64 dye release from synaptic boutons transfected with control siRNA, siRNA directed against AP180 (AP180 KD), CALM (CALM KD), or siRNAs against both proteins. No significant changes in exocytic release kinetics were observed in neurons depleted of AP180, CALM, or of both proteins. (C) Relative amount of FM4-64 internalized and released upon stimulation (ΔF). Data were normalized to the ΔF of nontransfected terminals and represent mean ± SEM. (D) Vesicular vs. surface pools of synaptobrevin 2-pHluorin assessed by acid-base quenching in hippocampal neurons. Synaptobrevin 2-pHluorin accumulates on the neuronal surface following depletion of AP180 (P = 0.0172, n = 15 neurons), CALM (P = 0.0022, n = 27 neurons), or both proteins (P < 0.0001, n = 28 neurons). (E) Same as in D but using vGLUT1-pHluorin as a reporter. (F) Dispersion of synaptobrevin 2-pHluorin along the axon of AP180/CALM-depleted neurons. Selective mislocalization of pHluorin-tagged synaptobrevin 2, but not vGLUT1, along the axon was observed in neurons depleted of AP180 and CALM. (Left) Profiles from linescan analyses of neurons. (Scale bar, 5 μm.) (G and H) Morphometric ultrastructural analysis of cumulative SV size frequency (G) and SV density (H) by electron microscopy. The average size of SVs is significantly increased in terminals from neurons depleted of AP180 or of both AP180 and CALM.

NMR spectroscopic analyses of structural changes associated with binding synaptobrevin 2 to CALM-ANTH. (A–C) Sequences of synaptobrevin 2 indicating residues influenced by CALM-ANTH binding as detected in ¹H-¹⁵N-HSQC spectra of solutions in aqueous buffer (Upper sequence and spectrum in B) and in the presence of DPC (Lower sequence and spectrum in C). Secondary structures are indicated above and below the sequences. In the Upper sequence, all residues are shown in orange whose signals disappear upon addition of CALM-ANTH (cf. also data for AP180-ANTH in Fig. S3). The chemical shift changes observed upon addition of CALM-ANTH in buffer containing DPC were classified into three categories (strong: purple; medium: red; weak: yellow) and displayed in the Lower sequence in A. (D) Structure of synaptobrevin 2 (PDB ID code 2KOG) showing residues 30–92 and the residues affected by CALM-ANTH binding in DPC buffer according to the color code in A.

Recognition of the SNARE motif of synaptobrevin 2 by the ANTH domains of AP180 and CALM. (A) AP180 associates with synaptobrevin 2 via its ANTH domain. HEK293 cells coexpressing synaptobrevin 2-FLAG together with empty vector, full-length AP180 (FL), the ANTH domain alone (ANTH), or a mutant lacking the ANTH domain (ΔANTH) were subjected to immunoprecipitation using anti-FLAG antibodies. Samples were analyzed by immunoblotting. Input: 1% of the starting material. Note that the immunoblot representing input material was exposed longer to allow proper visualization of all bands. (B) Synaptobrevin 2 interacts with AP180 via the N-terminal half of its SNARE motif. HEK293 cells coexpressing AP180 and FLAG-tagged truncation mutants of synaptobrevin 2 were subjected to immunoprecipitation as in A. Complex formation was disrupted by deletion of residues from the N-terminal half of the SNARE motif of synaptobrevin 2 (Δ1–50). (C) Delineation of the minimal AP180-ANTH binding site within synaptobrevin 2 using peptide SPOT arrays. Peptides displaying nonspecific binding are boxed in gray and were excluded from the analysis. Binding peptides are boxed in red and the sequence of these peptides is listed below. (D) Direct binding of synaptobrevin 2 to the ANTH domains of CALM and AP180. (Left) Immobilized GST-ANTH domains derived from CALM or AP180 (10 μg) were incubated with His₆-synaptobrevin 2 (1–96) for 1 h at 4 °C. Following extensive washes, samples were analyzed by SDS/PAGE and immunoblotting. (Right) Interaction between GST-synaptobrevin 2 (33–54) comprising the N-terminal half of its SNARE motif immobilized on beads and incubated with His₆-ANTH domains. Samples were analyzed by SDS/PAGE and immunoblotting. (E) Dose-dependent binding of purified CALM-ANTH to immobilized synaptobrevin 2 (1–96) analyzed by surface plasmon resonance. Shown are binding curves from a representative experiment after background substraction. RU, resonance units.

SNARE motif-dependent endocytic sorting of synaptobrevin 2. (A) Mutational analysis of the ANTH domain-binding interface within the SNARE motif of synaptobrevin 2. GST-CALM-ANTH was immobilized on beads and incubated with lysates from HEK293 cells expressing synaptobrevin 2-FLAG carrying the indicated mutations. Samples were analyzed by SDS/PAGE and immunoblotting. (B) Quantification of CALM-ANTH binding to synaptobrevin 2 M46A or D44A as depicted in A. Binding to CALM-ANTH is abolished significantly by M46A and increased by D44A mutations (P < 0.0001 and P = 0.1415, respectively; n = 3 independent experiments). (C) Exo-endocytic cycling of mutant synaptobrevin 2-pHluorin. M46A is seen to accumulate on the neuronal surface (P < 0.0001, n = 9 neurons), whereas D44A shows decreased surface pools (P = 0.0073, n = 19 neurons). (D) Axonal dispersion of synaptobrevin 2 (M46A). Wild-type and D44A synaptobrevin 2-pHluorin display a pronounced concentration at presynaptic boutons, whereas M46A is dispersed along the axon. The experiment was carried out as described in Fig. 1F. (Scale bar, 5μm.)