The CH of ATL2-1 has auto-inhibitory activity. (A) Donor and acceptor liposomes bearing the ATL2-1 isoform, the ATL2-2 isoform, or mutant ATL2-1 lacking its CH (ATL2-1-ΔCH) at a protein/lipid ratio of 1:1,000 were mixed and incubated for 30 min at 30°C in the absence or presence of GTP. Fusion is represented as the percentage of total NBD fluorescence. 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. (B) 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 (500 µM) derived from the CH of ATL2-1, the CH of ATL2-2, or the mutant CH of ATL2-1 reported to lack autoinhibitory activity (Crosby et al., 2022). Fusion is represented as the percentage of total NBD fluorescence. The kinetics graph (left) represents 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 < 0.01, *P < 0.05, two-tailed unpaired Student’s t test.