Sui et al. report the cryo-EM structure of the conserved luminal domain of the lipid droplet (LD)-formation protein seipin. The structure reveals key features of this domain and suggest a new model for seipin's role in LD formation.
SUMMARYMetabolic 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. Here, we report a cryo-electron microscopy structure and functional characterization of D. melanogaster seipin. The structure reveals a ringshaped dodecamer, with the luminal domain of each monomer resolved at ~4.0 Å. Each luminal domain monomer exhibits two distinctive features: a hydrophobic helix positioned towards the ER bilayer, and a b-sandwich domain that has structural similarity with lipidbinding proteins. This structure, and our functional testing in cells, suggest a model in which seipin oligomers initially detect forming LDs in the ER via hydrophobic helices and subsequently act as membrane anchors to enable lipid transfer and LD growth. Wolinski et al., 2015). Previous studies of seipin led to different models for its molecular function, including acting as a regulator of the ER Ca 2+ pump SERCA, a molecular scaffold and regulator of lipid metabolism enzymes, and a structural protein facilitating LD growth at ER-LD contact sites (Bi et al., 2014;Pagac et al., 2016;Sim et al., 2012;Talukder et al., 2015;Wang et al., 2016). However, despite considerable efforts, the molecular function of seipin in LD biogenesis remains unclear.As a step toward unraveling seipin's function, we sought to elucidate its molecular structure. Utilizing cryo-electron microscopy, we report here a structural model of Drosophila melanogaster seipin, solved for the luminal domain at ~4.0 Å resolution. This structure reveals seipin to form a dodecamer that positions multiple hydrophobic helices near the ER bilayer and a b-sandwich folded domain with similarity to lipid-binding proteins. We use several approaches to validate this structure, and we test several of its key features in vitro and in cells. Our results suggest a functional model for how seipin functions to detect forming LDs and promote their growth.
RESULTS
Determination of a cryo-electron microscopy map of Drosophila melanogaster seipinWe purified recombinant D. melanogaster seipin in detergents, and gel-filtration chromatography revealed it to be an oligomer (Fig. S1, A and B), similar to what was reported in other species (Binns et al., 2010;Sim et al., 2014). Consistently, negativestain electron microscopy (EM) analyses showed that seipin particles were monodisperse, and two-dimensional (2D) averages demonstrated distinct views with round or multi-layer barrel shapes (Fig. S1 C).We next analyzed purified seipin by cryo-electron microscopy (cryo-EM). The 2D averages of cryo-EM particle images (Fig. 1, B and C) appeared similar to those ...
