Intracellular Vesicle Fusion Requires a Membrane-Destabilizing Peptide Located at the Juxtamembrane Region of the v-SNARE

Rathore, Shailendra S.; Liu, Yinghui; Yu, Haijia; Wan, Chun; Lee, MyeongSeon; Yin, Qian; Stowell, Michael H.B.; Shen, Jingshi

The function of the juxtamembrane motif requires both polar and nonpolar residues • Linker insertions between SNARE and juxtamembrane motifs impair vesicle fusion (A) Sequence alignment of the juxtamembrane motifs of WT VAMP2 and a VAMP2 mutant in which the juxtamembrane motif was mutated into alanines. Asterisks indicate the conserved residues mutated in the VAMP2 mutant. Lysine 94 (K94) was not mutated because this basic residue demarcates the boundary of the TMD. Lysine 91 (K91) was not mutated because it is not evolutionarily conserved. Nevertheless, identical results were observed when K91 was also mutated ( Figure 6 ). (Yu et al., 2013a) . Biotin-labeled WT t-SNARE liposomes were anchored to avidin agarose beads and used to bind rhodamine-labeled v-SNARE liposomes (WT or mutant). The VAMP2 mutant is depicted in Figure 2A . The binding reactions were carried out at 4°C for 1 h in the absence or presence of 5 μM Munc18-1. Biotin-labeled protein-free liposomes were used as a negative control to obtain the background fluorescent signal, which was subtracted from other binding reactions to alculate SNARE-dependent liposome docking. The data are presented as average fluorescence intensity of rhodamine bound to the beads based on three independent experiments. Error bars indicate SD. (B) Measurements of SNARE-Munc18-1 association using ITC. The ternary SNARE complexes were assembled from the cytoplasmic domains of v-and t-SNAREs: VAMP2 (residues 1-95, WT or mutant), syntaxin-1 (residues 1-265), and full-length SNAP-25 (Yu et al., 2013a) . The dissociation constant of the SNARE-Munc18-1 complex was calculated by fitting the data with a nonlinear least-squares routine using the MicroCal Origin software. (C) Representative immunoblots showing the binding of WT or mutant SNARE complexes to GST-Munc18-1. GST-Munc18-1 proteins bound to glutathione Sepharose beads were used to precipitate full-length ternary SNARE complexes using a previously established procedure . Protein complexes in the precipitates were resolved on SDS-PAGE and analyzed by immunoblotting using the indicated antibodies. See also Figure S3 . (B) Sulforhodamine B-loaded protein-free liposomes were incubated with buffer or the indicated peptides at 37°C for 60 min, and sulforhodamine B fluorescence during the incubation was measured. Liposome leakage leads to sulforhodamine B dequenching and increases in its fluorescence. Each peptide was added to a final concentration of 100 μM. At the end of the incubation, 10 μL of 10% CHAPSO was added to lyse the liposomes to obtain the maximum fluorescence. The data are shown as percentage of maximum fluorescence. (C) Sulforhodamine B fluorescence at the end of the 60-min incubation shown in (B) . Data are presented as the average percentage of maximum fluorescence based on three independent experiments. Error bars indicate SD. (D) Representative electron micrographs of liposomes incubated with buffer or the indicated peptides. Scale bars, 100 nm. See also Figure S4 .