ATL3 or a cytosolic factor relieves the autoinhibition by the CH and enables ATL2-1 to support ER fusion in vivo. (A) Donor and acceptor liposomes bearing ATL2-1 alone (1:1,000), ATL3 alone (1:2,000), or both ATL2-1 (1:1,000) and ATL3 (1:2,000) were mixed and incubated in the absence or presence of GTP. Fusion is presented as the percentage of total NBD fluorescence. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. ****P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. (B) Donor and acceptor liposomes bearing ATL2-1 alone (1:500), ATL2-2 alone (1:4,000), or both ATL2-1 (1:500) and ATL2-2 (1:4,000) were mixed and incubated in the absence or presence of GTP. Fusion is presented as the percentage of total NBD fluorescence. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. *P < 0.05, **P < 0.01, two-way ANOVA with Tukey’s multiple comparisons test. (C) Liposomes containing ATL2-1 alone (1:1,000), ATL3 alone (1:2,000), or both ATL2-1 (1:1,000) and ATL3 (1:2,000) were incubated in the presence of GMP-PNP at 37°C for 30 min. Then, liposomes were treated or untreated with BMOE at 37°C for 1 h for crosslinking. Reactions were quenched by adding DTT (50 mM) at room temperature for 15 min. Samples were analyzed by SDS-PAGE followed by silver staining (Thermo Fisher Scientific). (D) Donor and acceptor liposomes bearing ATL2-1 alone (1:500), ATL2-ΔCH alone (1:4,000), or both ATL2-1 (1:500) and ATL2-ΔCH (1:4,000) were mixed and incubated in the absence or presence of GTP. Fusion is presented as the percentage of total NBD fluorescence. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. ***P < 0.001, ****P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. (E) Donor and acceptor liposomes bearing ATL2-1 (1:500) were incubated with GTP in the absence or presence of cytosol (0.5 μg/μl) purified from HEK293T cells and affinity-purified anti-ATL2 antibodies (1 μM). Fusion is represented as the percentage of total NBD fluorescence. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. **P < 0.01, two-way ANOVA with Tukey’s multiple comparisons test. (F) Working model of how the CH-mediated autoinhibition is relieved during ER fusion in vivo. Source data are available for this figure: SourceData F9.
Figure 9.

ATL3 or a cytosolic factor relieves the autoinhibition by the CH and enables ATL2-1 to support ER fusion in vivo. (A) Donor and acceptor liposomes bearing ATL2-1 alone (1:1,000), ATL3 alone (1:2,000), or both ATL2-1 (1:1,000) and ATL3 (1:2,000) were mixed and incubated in the absence or presence of GTP. Fusion is presented as the percentage of total NBD fluorescence. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. ****P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. (B) Donor and acceptor liposomes bearing ATL2-1 alone (1:500), ATL2-2 alone (1:4,000), or both ATL2-1 (1:500) and ATL2-2 (1:4,000) were mixed and incubated in the absence or presence of GTP. Fusion is presented as the percentage of total NBD fluorescence. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. *P < 0.05, **P < 0.01, two-way ANOVA with Tukey’s multiple comparisons test. (C) Liposomes containing ATL2-1 alone (1:1,000), ATL3 alone (1:2,000), or both ATL2-1 (1:1,000) and ATL3 (1:2,000) were incubated in the presence of GMP-PNP at 37°C for 30 min. Then, liposomes were treated or untreated with BMOE at 37°C for 1 h for crosslinking. Reactions were quenched by adding DTT (50 mM) at room temperature for 15 min. Samples were analyzed by SDS-PAGE followed by silver staining (Thermo Fisher Scientific). (D) Donor and acceptor liposomes bearing ATL2-1 alone (1:500), ATL2-ΔCH alone (1:4,000), or both ATL2-1 (1:500) and ATL2-ΔCH (1:4,000) were mixed and incubated in the absence or presence of GTP. Fusion is presented as the percentage of total NBD fluorescence. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. ***P < 0.001, ****P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. (E) Donor and acceptor liposomes bearing ATL2-1 (1:500) were incubated with GTP in the absence or presence of cytosol (0.5 μg/μl) purified from HEK293T cells and affinity-purified anti-ATL2 antibodies (1 μM). Fusion is represented as the percentage of total NBD fluorescence. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. **P < 0.01, two-way ANOVA with Tukey’s multiple comparisons test. (F) Working model of how the CH-mediated autoinhibition is relieved during ER fusion in vivo. Source data are available for this figure: SourceData F9.

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