The generation of the tubular network of the endoplasmic reticulum (ER) requires homotypic membrane fusion that is mediated by the dynamin-like, membrane-bound GTPase atlastin (ATL). Here, we have determined crystal structures of the cytosolic segment of human ATL1, which give insight into the mechanism of membrane fusion. The structures reveal a GTPase domain and athree-helix bundle, connected by a linker region. One structure corresponds to a prefusion state, in which ATL molecules in apposing membranes interact through their GTPase domains to form a dimer with the nucleotides bound at the interface. The other structure corresponds to a postfusion state generated after GTP hydrolysis and phosphate release. Compared with the prefusion structure, the three-helix bundles of the two ATL molecules undergo a major conformational change relative to the GTPase domains, which could pull the membranes together. The proposed fusion mechanism is supported by biochemical experiments and fusion assays with wild-type and mutant full-length Drosophila ATL. These experiments also show that membrane fusion is facilitated by the C-terminal cytosolic tails following the two transmembrane segments. Finally, our results show that mutations in ATL1 causing hereditary spastic paraplegia compromise homotypic ER fusion.protein structure | membrane remodeling | organelle shaping | spastic paraplegia gene 3A | endoplasmic reticulum network formation

Generation of the tubular endoplasmic reticulum (ER) network requires homotypic membrane fusion. This is mediated in metazoans by atlastin (ATL), a dynamin‐like GTPase that consists of an N‐terminal cytosolic domain followed by two transmembrane segments (TMs) and a C‐terminal tail (CT). A GTP‐hydrolysis‐induced conformational change in the N‐terminal cytosolic domain is required for fusion, but it is unclear if this alone is sufficient for the fusion reaction. Here, we show using in vitro fusion assays that the CT and TMs are required for efficient fusion. A conserved amphipathic helix in the CT promotes fusion by interacting with and perturbing the lipid bilayer. The TMs not only serve as membrane anchors but also mediate ATL oligomerization. Point mutations in the CT or the TMs also impair ATL's ability to maintain ER membrane morphology in vivo. Our results suggest that protein‐lipid and protein‐protein interactions in the membrane cooperate with the conformational change of the cytosolic domain to achieve fusion and maintain the tubular ER network. These findings are relevant to mechanisms used by other membrane fusion proteins, such as mitofusins/Fzo1p, viral fusogens, and SNAREs.

The crystal structures of the N-terminal cytosolic domain of Sey1p shed light on the mechanism of Sey1p-mediated ER membrane fusion.

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