Molecular Mechanism For Atlastin-mediated Homotypic Fusion Of Endoplasmic ReticulumMembranes

The homotypic fusion of endoplasmic reticulum (ER), which is required for ER network formation, is catalyzed by the dynamin-like, membrane-bound GTPase atlastin (ATL). ATL consists of an N-terminal cytosolic domain, containing a GTPase module and a three-helix bundle (3HB), followed by two transmembrane segments (TMs) and a C-terminal tail (CT). Here, we have addressed the mechanism of ATL-mediated membrane fusion. The crystal structure of the cytosolic domain of human ATL1wasdetermined in the presence of GDP. Interestingly, we obtained twodifferent structures. This conformational change and biochemical analyses demonstrate thatthe initial contact between ATL molecules in opposing membranes is mediated by the GTPase domains. Upon GTP hydrolysis and phosphate release, the3HBs of the two ATL molecules undergo a major conformational change, which pulls the membranes together. The CT and TMs are also required for efficient fusion. The CT can form an amphipathic helix and facilitates vesicle fusion by perturbing the lipid bilayer. A synthetic peptide corresponding to the helix can restore the fusion activity of tailless ATL. The TMsare more than just membrane anchors, because they cannot be deleted, mutatedor replaced with unrelated TMs.Coimmunoprecipitation experimants prove that TMs can mediate the nucleotide-independent oligomerization of ATL to promote membrane fusion. Finally, we also found at early stage of fusion reaction, continuous GTP hydrolysis is required for ATL to tether vesicles. The linker region between the3HB and the TMsis important for the transition from tethered sta,te to fusion. Taken together, we propose a model in which different domains of ATL cooperate to mediate membrane fusion.Our results alsoprovide some explanations regarding how ATL1mutations cause hereditary spastic paraplegia.