Cell-free formation and interactome analysis of caveolae

Author:

Jung WooRam1ORCID,Sierecki Emma1ORCID,Bastiani Michele1ORCID,O’Carroll Ailis1,Alexandrov Kirill1,Rae James1,Johnston Wayne1,Hunter Dominic J.B.1ORCID,Ferguson Charles1,Gambin Yann1ORCID,Ariotti Nicholas1ORCID,Parton Robert G.12ORCID

Affiliation:

1. The University of Queensland, The Institute for Molecular Bioscience, Brisbane, Queensland, Australia

2. The University of Queensland, The Centre for Microscopy and Microanalysis, Brisbane, Queensland, Australia

Abstract

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.

Funder

National Health and Medical Research Council of Australia

Australian Research Council

Discovery

Publisher

Rockefeller University Press

Subject

Cell Biology

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