Human atlastins support proteoliposome fusion. (A) Human atlastins (ATL1, ATL2, and ATL3) are sufficient to drive liposome fusion. ATL1, ATL2, or ATL3 recombinantly expressed and purified from E. coli was reconstituted into liposomes with a protein-to-lipid ratio of 1:1,000 as described in the Materials and methods. Donor and acceptor proteoliposomes bearing ATL1, ATL2, or ATL3 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 40 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 < 0.0001, two-tailed unpaired Student’s t test. (B) Human atlastin-mediated fusion requires GTP hydrolysis. Fusion assays were performed in the absence or presence of GTP, GDP, GTPγS, or GMPPNP for 40 min. Data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. (C) Human atlastins have comparable GTPase activities. The GTPase activities of human atlastins were determined by measuring the release of inorganic phosphate using the Malachite Green Phosphate Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, proteoliposomes bearing ATL1, ATL2, or ATL3 were incubated with 0.25 mM GTP at 37°C. After incubation for 30 min with malachite green reagents, OD₆₅₅ was measured and the readings were normalized using a phosphate standard curve. 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.), two-way ANOVA with Tukey’s multiple comparisons test.