Abstract

Flotillin-1 and flotillin-2 form hetero-oligomers to create flotillin membrane microdomains essential for endocytosis and protein sorting. However, the mechanisms of flotillin oligomerization and microdomain organization remain incompletely understood. Here, we present the cryo-EM structure of human flotillin complex, showing that flotillin-1 and -2 form a 44-mer, membrane attached, and dome-shaped structure that defines a 30-nm circular membrane domain. The cryo-ET data demonstrates that while attached to the cytoplasmic leaflet, flotillin complexes possess intrinsic structural plasticity in situ on the native membrane. Each flotillin complex may represent a fundamental unit of membrane microdomains, with their clustering enabling the formation of larger and more elaborate domains. We further reveal that phosphorylation at residues Y160 (flotillin-1) and Y163 (flotillin-2) may act as a molecular switch to modulate complex assembly, potentially regulating its function in endocytosis. These findings demonstrate the molecular mechanism of flotillin-mediated membrane segregation and microdomain formation, and suggest a previously unrecognized role of flotillin in sequestrating membrane proteins.