ATL2 and ATL3 can form heterodimers. (A) Donor and acceptor liposomes bearing ATL2-1 (K107A) alone (1:500), ATL3 alone (1:2,000), or both ATL2-1 (K107A; 1:500) 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). (B) Donor and acceptor liposomes bearing ATL2-1 (K107A) alone (1:500), ATL2-2 alone (1:4,000), or both ATL2-1 (K107A; 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. The difference between samples was not statistically significant (n.s.), one-way ANOVA with Tukey’s multiple comparisons test. (C) The his6-tagged N-terminal cytosolic domain of ATL2 was incubated with free GST or GST-tagged ATL3 in the presence of GDP or GMP-PNP at 37°C for 1 h. The mixture was then further incubated with Ni-NTA agarose (Qiagen) at 4°C for 30 min, and proteins bound to Ni-NTA agarose were analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining. GST-ATL3, but not free GST, was co-precipitated with the his6-tagged N-terminal cytosolic domain of ATL2 in the presence of GMP-PNP, a non-hydrolyzable GTP analog. (D) GTPase activity is largely impaired in ATL3 (Y192C). The GTPase activity of wild-type ATL3 or ATL3 (Y192C) was determined by measuring the release of inorganic phosphate using a Malachite Green Phosphate Assay Kit (Sigma-Aldrich). Liposomes bearing wild-type ATL3 or ATL3 (Y192C) were incubated in the presence of GTP at 37°C for 30 min. After further incubation with malachite green reagents for 30 min, OD655 was measured, and the readings were normalized using a phosphate standard curve. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. ***P < 0.001, two-tailed unpaired Student’s t test. Source data are available for this figure: SourceData FS6.
Figure S6.

ATL2 and ATL3 can form heterodimers. (A) Donor and acceptor liposomes bearing ATL2-1 (K107A) alone (1:500), ATL3 alone (1:2,000), or both ATL2-1 (K107A; 1:500) 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). (B) Donor and acceptor liposomes bearing ATL2-1 (K107A) alone (1:500), ATL2-2 alone (1:4,000), or both ATL2-1 (K107A; 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. The difference between samples was not statistically significant (n.s.), one-way ANOVA with Tukey’s multiple comparisons test. (C) The his6-tagged N-terminal cytosolic domain of ATL2 was incubated with free GST or GST-tagged ATL3 in the presence of GDP or GMP-PNP at 37°C for 1 h. The mixture was then further incubated with Ni-NTA agarose (Qiagen) at 4°C for 30 min, and proteins bound to Ni-NTA agarose were analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining. GST-ATL3, but not free GST, was co-precipitated with the his6-tagged N-terminal cytosolic domain of ATL2 in the presence of GMP-PNP, a non-hydrolyzable GTP analog. (D) GTPase activity is largely impaired in ATL3 (Y192C). The GTPase activity of wild-type ATL3 or ATL3 (Y192C) was determined by measuring the release of inorganic phosphate using a Malachite Green Phosphate Assay Kit (Sigma-Aldrich). Liposomes bearing wild-type ATL3 or ATL3 (Y192C) were incubated in the presence of GTP at 37°C for 30 min. After further incubation with malachite green reagents for 30 min, OD655 was measured, and the readings were normalized using a phosphate standard curve. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. ***P < 0.001, two-tailed unpaired Student’s t test. Source data are available for this figure: SourceData FS6.

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