The CH of ATL2-1 does not inhibit ATL2-2- or ATL3-mediated liposome fusion. (A) ATL2-1 peptide inhibits ATL2-1-ΔCH-mediated fusion in a dose-dependent manner. Donor and acceptor liposomes bearing ATL2-1-ΔCH at a protein/lipid ratio of 1:1,000 were mixed and incubated in the presence of GTP and synthetic peptides derived from the CH of ATL2-1 at 30°C for 20 min. The kinetics graph (left) represents five independent results, which are presented as a bar graph (right). Data represent the means ± SEM (error bars; n = 5). n refers to the number of independent experiments. ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test. (B) ATL2-2 peptide does not inhibit ATL2-1-ΔCH-mediated fusion. Donor and acceptor liposomes bearing ATL2-1-ΔCH at a protein/lipid ratio of 1:1,000 were mixed and incubated in the presence of GTP and synthetic peptides derived from the CH of ATL2-2 at 30°C for 20 min. The kinetics graph (left) is representative of three independent results, which are presented as a bar graph (right). Data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. p = n.s., one-way ANOVA with Tukey’s multiple comparisons test. (C and D) ATL2-1 peptide does not inhibit ATL2-2- or ATL3-mediated fusion. Donor and acceptor liposomes bearing ATL2-2 (C) or ATL3 (D) at a protein/lipid ratio of 1:1,000 were mixed and incubated in the presence of GTP and synthetic peptides (500 μM) derived from the CH of ATL2-1 at 30°C for 10 min. Data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. p = n.s., one-way ANOVA with Tukey’s multiple comparisons test. (D) The CH of ATL2-1, but not that of ATL2-2, inhibits the GTPase activity of ATL2-1-ΔCH. The GTPase activities of ATL2-1-ΔCH and the full-length ATL2-2 in the absence or presence of the ATL2-1 or ATL2-2 peptide were determined by measuring the release of inorganic phosphate using the Malachite Green Phosphate Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions. Data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. **P < 0.01, one-way ANOVA with Tukey’s multiple comparisons test.
Figure S3.

The CH of ATL2-1 does not inhibit ATL2-2- or ATL3-mediated liposome fusion. (A) ATL2-1 peptide inhibits ATL2-1-ΔCH-mediated fusion in a dose-dependent manner. Donor and acceptor liposomes bearing ATL2-1-ΔCH at a protein/lipid ratio of 1:1,000 were mixed and incubated in the presence of GTP and synthetic peptides derived from the CH of ATL2-1 at 30°C for 20 min. The kinetics graph (left) represents five independent results, which are presented as a bar graph (right). Data represent the means ± SEM (error bars; n = 5). n refers to the number of independent experiments. ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test. (B) ATL2-2 peptide does not inhibit ATL2-1-ΔCH-mediated fusion. Donor and acceptor liposomes bearing ATL2-1-ΔCH at a protein/lipid ratio of 1:1,000 were mixed and incubated in the presence of GTP and synthetic peptides derived from the CH of ATL2-2 at 30°C for 20 min. The kinetics graph (left) is representative of three independent results, which are presented as a bar graph (right). Data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. p = n.s., one-way ANOVA with Tukey’s multiple comparisons test. (C and D) ATL2-1 peptide does not inhibit ATL2-2- or ATL3-mediated fusion. Donor and acceptor liposomes bearing ATL2-2 (C) or ATL3 (D) at a protein/lipid ratio of 1:1,000 were mixed and incubated in the presence of GTP and synthetic peptides (500 μM) derived from the CH of ATL2-1 at 30°C for 10 min. Data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. p = n.s., one-way ANOVA with Tukey’s multiple comparisons test. (D) The CH of ATL2-1, but not that of ATL2-2, inhibits the GTPase activity of ATL2-1-ΔCH. The GTPase activities of ATL2-1-ΔCH and the full-length ATL2-2 in the absence or presence of the ATL2-1 or ATL2-2 peptide were determined by measuring the release of inorganic phosphate using the Malachite Green Phosphate Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions. Data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. **P < 0.01, one-way ANOVA with Tukey’s multiple comparisons test.

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