A comprehensive strategy to identify stoichiometric membrane protein interactomes

Abstract

There are numerous experimental approaches to identify the interaction networks of soluble proteins, but strategies for the identification of membrane protein interactomes remain limited. We discuss in detail the logic of an experimental design that led us to identify the interactome of a membrane protein of complex membrane topology, the calcium activated chloride channel Anoctamin 1/Tmem16a (Ano1). We used covalent chemical stabilizers of protein-protein interactions combined with magnetic bead immuno-affinity chromatography, quantitative SILAC mass-spectrometry and in silico network construction. This strategy led us to define a putative Ano1 interactome from which we selected key components for functional testing. We propose a combination of procedures to narrow down candidate proteins interacting with a membrane protein of interest for further functional studies.

Keywords: :

• membrane protein
• Ano1
• interactome
• SILAC
• epithelia
• salivary gland

Acknowledgments

Supported by grants from the NIH GM60448 (H.C.H.), EY014852 (H.C.H.), NS42599 (V.F.), GM077569 (V.F.), Emory University Research Committee (H.C.H.), NEI training grant 5T32EY007092–25 (C.D.) and NIH FIRST program fellowship K12 GM000608 (A.G.). Additional support was provided by the Microscopy Core of the Emory Neuroscience NINDS Core Facilities Grant P30NS055077 and NEI Core Grant P30EY006360.