title: Molecular composition and ultrastructure of the caveolar coat complex.
abstract: 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.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: Alexander
authors.initials: A
authors.lastname: Ludwig
authors.forename: Gillian
authors.initials: G
authors.lastname: Howard
authors.forename: Carolina
authors.initials: C
authors.lastname: Mendoza-Topaz
authors.forename: Thomas
authors.initials: T
authors.lastname: Deerinck
authors.forename: Mason
authors.initials: M
authors.lastname: Mackey
authors.forename: Sara
authors.initials: S
authors.lastname: Sandin
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Nichols
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D021941:Caveolae / Q000378:metabolism* / Q000648:ultrastructure; D051242:Caveolin 1 / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D051242:Caveolin 1; C406446:EHD2 protein, human; C120714:PACSIN2 protein, human