Abstract
Caveolae are specialized domains of the plasma membrane. Formation of these invaginations is dependent on the expression of Caveolin-1 or -3 and proteins of the cavin family. In response to stress, caveolae disassemble and cavins are released from caveolae, allowing cavins to potentially interact with intracellular targets. Here, we describe the intracellular (non-plasma membrane) cavin interactome using biotin affinity proteomics and mass spectrometry. We validate 47 potential cavin-interactor proteins using a cell-free expression system and protein-protein binding assays. These data, together with pathway analyses, reveal unknown roles for cavin proteins in metabolism and stress signaling. We validated the interaction between one candidate interactor protein, protein phosphatase 1 alpha (PP1α), and Cavin-1 and -3 and show that UV treatment causes release of Cavin3 from caveolae allowing interaction with, and inhibition of, PP1α. This interaction increases H2AX phosphorylation to stimulate apoptosis, identifying a pro-apoptotic signaling pathway from surface caveolae to the nucleus.
Highlights
Caveolae are specialized domains of the plasma membrane
We tested a number of commonly used cell lines and found that MCF-7 cells (ATCC HTB-22) lack CAV1, Cavin[1], Cavin[2], Cavin[3] at the mRNA level (Supplementary Fig. 1e) and Cavin[1] and CAV1 at the protein level (Supplementary Fig. 1b–d)
Given that PP1α was the only protein identified in multiple approaches (BioID and GFP Trap/MS experiments from A431 and MCF-7 cells expressing Cavin3GFP), and importantly, showed a direct interaction with in vitro synthesized cavins by ALPHAScreen, we focused our attention on the role of this interaction in our model cell systems
Summary
Caveolae are specialized domains of the plasma membrane. Formation of these invaginations is dependent on the expression of Caveolin-1 or -3 and proteins of the cavin family. The role of caveolae in mechanoprotection was first observed when the structural integrity of caveolae was disrupted by increased mechanical tension[8,9] Under these conditions, caveolae flatten at the plasma membrane releasing cavin coat proteins into the cytosol. Expressed cavin proteins in MCF-7 cells exhibit a cytosolic localization that mimics release of cavins from caveolae in cells subjected to increases in plasma membrane tension[8,9]. Using this model system, we have generated a comprehensive list of potential interacting proteins for non-caveolar Cavin[3]. We have complemented this approach by screening in vitro expressed potential interacting proteins using Amplified Luminescent Proximity Homogeneous Assay Screen (ALPHAScreen; refs. 16–18) as well as GFP-Trap pulldowns of Cavin[3] in both MCF-7 and A431 cells
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