Molecular composition and ultrastructure of the caveolar coat complex

• Published in: PLoS biology 2013-08, Vol.11 (8), p.e1001640-e1001640
• Main Authors: Ludwig, Alexander, Howard, Gillian, Mendoza-Topaz, Carolina, Deerinck, Thomas, Mackey, Mason, Sandin, Sara, Ellisman, Mark H, Nichols, Benjamin J
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing - metabolism; Animals; Binding sites; Biology; Biosynthesis; Carrier Proteins - metabolism; Caveolae; Caveolae - metabolism; Caveolae - ultrastructure; Caveolin 1 - metabolism; Cell membranes; Cellular signal transduction; Experiments; HeLa Cells; Humans; Mass spectrometry; Membranes; Microscopy; Microscopy, Electron; Molecular weight; Permeability; Physiological aspects; Proteins; Tomography
• ISSN: 1545-7885; 1544-9173; 1545-7885
• DOI: 10.1371/journal.pbio.1001640

Caveolae are an abundant feature of the plasma membrane of many mammalian cell types, and have key roles in mechano-transduction, metabolic regulation, and vascular permeability. Caveolin and cavin proteins, as well as EHD2 and pacsin 2, are all present in caveolae. How these proteins assemble to form a protein interaction network for caveolar morphogenesis is not known. Using in vivo crosslinking, velocity gradient centrifugation, immuno-isolation, and tandem mass spectrometry, we determine that cavins and caveolins assemble into a homogenous 80S complex, which we term the caveolar coat complex. There are no further abundant components within this complex, and the complex excludes EHD2 and pacsin 2. Cavin 1 forms trimers and interacts with caveolin 1 with a molar ratio of about 1∶4. Cavins 2 and 3 compete for binding sites within the overall coat complex, and form distinct subcomplexes with cavin 1. The core interactions between caveolin 1 and cavin 1 are independent of cavin 2, cavin 3, and EHD2 expression, and the cavins themselves can still interact in the absence of caveolin 1. Using immuno-electron microscopy as well as a recently developed protein tag for electron microscopy (MiniSOG), we demonstrate that caveolar coat complexes form a distinct coat all around the caveolar bulb. In contrast, and consistent with our biochemical data, EHD2 defines a different domain at the caveolar neck. 3D electron tomograms of the caveolar coat, labeled using cavin-MiniSOG, show that the caveolar coat is composed of repeating units of a unitary caveolar coat complex.