Molecular Composition and Ultrastructure of the Caveolar Coat Complex

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. © 2013 ludwig et al.