Structural Organization of Caveolin-1 8S Oligomers Determined by Cryo-Electron Microscopy

Caveolae are cholesterol-rich membrane invaginations found on the surface of many cell types. They play a critical role in membrane buffering, mechanotransduction, and cellular signaling. The integral membrane protein caveolin-1 (Cav1) is essential for caveolae formation in eukaryotic cells and can even drive the formation of caveolae-like structures in bacteria. Cav1 contains an unusual hairpin-like intramembrane domain predicted to bend caveolar membranes by a wedging mechanism, and must oligomerize to form 8S complexes that function as the fundamental building blocks of caveolae. However, the molecular architecture of these complexes is currently unknown. To fill this gap in knowledge, we used negative staining and single particle cryo-electron microscopy to determine the structure of Cav1 8S complexes. By negative staining we show that Cav1 complexes forms discs ∼ 5 nm wide that are flat on one side, contain a central protrusion on the other, and vary in diameter. The diameters differ by a fixed amount, suggesting that they contain different numbers of Cav1 monomers. Using Venus as a fiduciary marker, we show the flat side of the disc corresponds to the membrane-facing side of the protein and the C-terminus of Cav1 is localized to the central protrusion. We also generated a ∼10 Å resolution structure of the 8S Cav1 complexes using cyoelectron microscopy. At this resolution the discs are reminiscent of a wheel, consisting of an outer ring connected by spokes to an inner protruding ring. Interestingly, the intramembrane domains of Cav1 monomers are localized to the outermost portion of the wheel. Current work is focused on improving the resolution of 8S complex structure in order to build its detailed molecular model. Ultimately, these studies should provide a structural framework for understanding how caveolae assemble and control cellular functions.