Cholesterol and PE are critical for ATL2-mediated liposome fusion. Liposome fusion was assayed as in Fig. 1 A using ATL2-proteoliposomes bearing various sets of lipids (complete ER mix, complete ER mix without cholesterol, and complete ER mix without PE and cholesterol). The omission of cholesterol only or cholesterol/PE markedly reduces ATL2-driven liposome fusion. Donor and acceptor proteoliposomes were mixed in the presence of Mg²⁺ and incubated for 10 min at 30°C. After the addition of GTP, NBD fluorescence was measured every minute for 20 min. To determine total fluorescence, β-octylglucoside was added at the end of the reaction and NBD fluorescence was measured. Fusion is expressed as the percentage of total NBD fluorescence. 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 < 0.05, **P < 0.01, one-way ANOVA with Tukey’s multiple comparisons test.