Membrane fusion is fundamental to biology. Studies on viral and SNARE fusion protein catalysts have revealed a common strategy by which proteins anchored in opposing membranes undergo favorable protein-folding reactions that draw the membranes into close apposition and drive the lipid rearrangements necessary for fusion. More recently, a new fusion paradigm has arisen with discovery that atlastin (ATL) a membrane-anchored dynamin related GTPase can trigger fusion of synthetic vesicles and is required for the branched morphology of the ER. ATL is distinct from previously studied fusion proteins because it couples fusion catalysis to the hydrolysis of GTP. Importantly, while substantial progress has been made on the ATL fusion mechanism, a consensus has yet to be reached. In the presence of GTP, the N-terminal cytosolic domain of ATL undergoes trans dimerization through the GTPase domain and a crossover conformational change hypothesized to draw membranes sufficiently close together to drive fusion. However, no fusion is observed in the absence of an amphipathic helix within the C-terminal cytosolic tail of ATL, suggesting a sequential model in which crossover formation constitutes an upstream step for membrane docking, and the tail functions subsequently to drive lipid mixing. On the other hand, our recent work suggests that crossover dimerization provides the energy for fusion but does not explain the role of the tail. Thus, whether crossover serves primarily to mediate docking, or whether it drives fusion, needs to be resolved. Similarly, how GTP hydrolysis energizes the fusion reaction cycle remains under debate. Prevailing models have held that the hydrolysis of GTP powers formation of the ATL crossover dimer directly for fusion. However, our recent work suggests that GTP hydrolysis serves to disassemble the crossover dimer after fusion for the purpose of subunit recycling. In aim 1, we will use FRET probes to distinguish whether crossover dimerization can be uncoupled from fusion, or whether it plays a central role and therefore is inseparable from fusion. In aim 2, we will extend our analysis of the GTP hydrolysis reaction cycle from the soluble phase to the context of membranes to ascertain whether the hydrolysis of GTP, as suggested by our recent work, functions only after the completion of fusion for the purpose of subunit recycling. Finally, previous studies on the ATL fusion mechanism have relied on use of the Drosophila ATL ortholog, the only ATL for which in vitro reconstitution of fusion activity has been achieved. This has limited our understanding of the role and potential regulation of the multiple ATL paralogs ATL1-3 present in humans. Thus, a third major goal is to reconstitute the fusion activity of the human paralogs. Altogether, the proposed studies promise to reveal the core principles of how GTP-dependent fusion proteins catalyze membrane fusion and to reveal sorely lacking insights into the mammalian ATL1-3 paralogs. Becau...