Structure of the Flotillin Complex in a Native Membrane Environment

In this study we used cryo-electron microscopy to determine the structures of the Flotillin protein complex, part of the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) superfamily, from cell-derived vesicles without detergents. It forms a right-handed helical barrel consisting of 22 pairs of Flotillin1 and Flotillin2 subunits, with a diameter of 32 nm its wider end and 19 nm at its narrower end. Oligomerization is stabilized by the C-terminus, which forms two helical layers linked by a {beta}-strand, and coiled-coil domains that enable strong charge-charge inter-subunit interactions. Flotillin interacts with membranes at both ends; through its SPFH1 domains at the wide end and the C-terminus at the narrow end, facilitated by hydrophobic interactions and lipidation. The inward tilting of the SPFH domain, likely triggered by phosphorylation, suggests its role in membrane curvature induction, which could be connected to its proposed role in clathrin-independent endocytosis. The structure suggests a shared architecture across the family of SPFH proteins and will promote further research into Flotillins roles in cell biology. Significance statementIt is well known that many biochemical processes in cells must occur in localized regions. There are many different ideas about how cells keep processes localized. In this study we demonstrate that Flotillin1 and Flotillin2 co-assemble to form a large, truncated cone shaped cage whose wide end is always attached to a membrane surface and whose narrow end is sometimes attached to a separate membrane. The entire wall of the cage is without holes and is likely impervious even to small molecules, forming a diffusion barrier that can connect membrane systems. The Flotillin cage is thus well suited to isolate biochemical processes. Through membrane attachment, it also alters local membrane curvature, which could influence endocytic and mechanosensory processes.