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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Highlights d IRE1a forms an ER-stress-independent complex with the Sec61/Sec63 translocon d IRE1a binding to BiP chaperone relies on its association with Sec61/Sec63 d Sec63 attenuates IRE1a signaling by mediating BiP binding to IRE1a during ER stress d IRE1a remains activated in Sec63-deficient cells during prolonged ER stress

A large number of newly synthesized membrane proteins in the endoplasmic reticulum (ER) are assembled into multi-protein complexes, but little is known about the mechanisms required for assembly membrane proteins. It has been suggested that membrane chaperones might exist, akin to the molecular chaperones that stabilize and direct the assembly of soluble protein complexes, but the mechanisms by which these proteins would bring together membrane protein components is unclear. Here, we have identified that the tail length of the C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane proteins in the ER. We found that membrane proteins with C-TMD tails shorter than ~60 amino acids are poorly inserted into the ER membrane, which suggests that translation is terminated before they are recognized by the Sec61 translocon for insertion. These C-TMDs with insufficient hydrophobicity are post-translationally recognized and retained by the Sec61 translocon complex, providing a time window for efficient assembly with TMDs from partner proteins. Retained TMDs that fail to assemble with their cognate TMDs are slowly translocated into the ER lumen and are recognized by the ER-associated degradation (ERAD) pathway for removal. In contrast, C-TMDs with sufficient hydrophobicity or tails longer than ~80 residues are quickly released from the Sec61 translocon into the membrane or the ER lumen, resulting in inefficient assembly with partner TMDs. Thus, our data suggest that C-terminal tails harbor crucial signals for both the insertion and assembly of membrane proteins.

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