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

Metabolic energy is stored in cells primarily as triacylglycerols in lipid droplets (LDs), and LD dysregulation leads to metabolic diseases. The formation of monolayer-bound LDs from the endoplasmic reticulum (ER) bilayer is poorly understood, but the ER protein seipin is essential to this process. In this study, we report a cryo–electron microscopy structure and functional characterization of Drosophila melanogaster seipin. The structure reveals a ring-shaped dodecamer with the luminal domain of each monomer resolved at ∼4.0 Å. Each luminal domain monomer exhibits two distinctive features: a hydrophobic helix (HH) positioned toward the ER bilayer and a β-sandwich domain with structural similarity to lipid-binding proteins. This structure and our functional testing in cells suggest a model in which seipin oligomers initially detect forming LDs in the ER via HHs and subsequently act as membrane anchors to enable lipid transfer and LD growth.

Carotenoids and their metabolic derivatives serve critical functions in both prokaryotic and eukaryotic cells, including pigmentation, photoprotection and photosynthesis as well as cell signaling. These organic compounds are also important for visual function in vertebrate and non-vertebrate organisms. Enzymatic transformations of carotenoids to various apocarotenoid products are catalyzed by a family of evolutionarily conserved, non-heme iron-containing enzymes named carotenoid cleavage oxygenases (CCOs). Studies have revealed that CCOs are critically involved in carotenoid homeostasis and essential for the health of organisms including humans. These enzymes typically display a high degree of regio- and stereo-selectivity, acting on specific positions of the polyene backbone located in their substrates. By oxidatively cleaving or isomerizing specific double bonds, CCOs generate a variety of apocarotenoid isomer products. Recent structural studies have helped illuminate the mechanisms by which CCOs mobilize their lipophilic substrates from biological membranes to perform their characteristic double bond cleavage and/or isomerization reactions. In this review, we aim to integrate structural and biochemical information about CCOs to provide insights into their catalytic mechanisms.

Triglycerides (triacylglycerols, TGs) store metabolic energy in organisms and have industrial uses for foods and fuels. Excessive accumulation of TGs in humans causes obesity and is associated with metabolic diseases 1 . TG synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes 2 – 4 whose structures and catalytic mechanisms are unknown. Here we determined the structure of dimeric human DGAT1, a member of the membrane-bound O -acyltransferase (MBOAT) family, by cryo-electron microscopy at 3.0-Å resolution. DGAT1 forms a homodimer through N-terminal segments and a hydrophobic interface, with putative active sites within the membrane region. A structure obtained with oleoyl-CoA substrate resolved at 3.2-Å shows that the CoA moiety binds DGAT1 on the cytosolic side and the acyl group lies deep within a hydrophobic channel, positioning the acyl-CoA thioester bond near an invariant catalytic histidine residue. The reaction center is located inside a large cavity, which opens laterally to the membrane bilayer, providing lipid access to the active site. A lipid-like density, possibly an acyl-acceptor molecule, is located within the reaction center, orthogonal to acyl-CoA. Insights provided by the DGAT1 structures, together with mutagenesis and functional studies, give rise to a model of catalysis for DGAT’s generation of TGs.