Journal
JOURNAL OF CELL BIOLOGY
Volume 217, Issue 6, Pages 2141-2165Publisher
ROCKEFELLER UNIV PRESS
DOI: 10.1083/jcb.201707004
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Funding
- National Health and Medical Research Council of Australia [APP1037320, APP1058565, APP569542, APP1045092, APP1100771]
- Australian Research Council (Centre of Excellence in Convergent Bio-Nano Science and Technology)
- Australian Research Council [FT110100478, DP 150100505, DP130102396]
- Australian Research Council [FT110100478] Funding Source: Australian Research Council
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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.
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