Cell-free formation and interactome analysis of caveolae

J Cell Biol. 2018 Jun 4;217(6):2141-2165. doi: 10.1083/jcb.201707004. Epub 2018 May 1.


Caveolae have been linked to the regulation of signaling pathways in eukaryotic cells through direct interactions with caveolins. Here, we describe a cell-free system based on Leishmania tarentolae (Lt) extracts for the biogenesis of caveolae and show its use for single-molecule interaction studies. Insertion of expressed caveolin-1 (CAV1) into Lt membranes was analogous to that of caveolin in native membranes. Electron tomography showed that caveolins generate domains of precise size and curvature. Cell-free caveolae were used in quantitative assays to test the interaction of membrane-inserted caveolin with signaling proteins and to determine the stoichiometry of interactions. Binding of membrane-inserted CAV1 to several proposed binding partners, including endothelial nitric-oxide synthase, was negligible, but a small number of proteins, including TRAF2, interacted with CAV1 in a phosphorylation-(CAV1Y14)-stimulated manner. In cells subjected to oxidative stress, phosphorylated CAV1 recruited TRAF2 to the early endosome forming a novel signaling platform. These findings lead to a novel model for cellular stress signaling by CAV1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Caveolae / metabolism*
  • Caveolae / ultrastructure
  • Caveolins / metabolism
  • Cell Extracts
  • Cell-Free System
  • Endosomes / metabolism
  • Endosomes / ultrastructure
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells / metabolism
  • Humans
  • Leishmania / metabolism
  • Phosphorylation
  • Protein Interaction Mapping*
  • Reproducibility of Results
  • Spectrum Analysis
  • TNF Receptor-Associated Factor 2 / metabolism


  • Caveolins
  • Cell Extracts
  • TNF Receptor-Associated Factor 2
  • Green Fluorescent Proteins