title: 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.
authors.affiliation: Department of Cell Biology; Emory University School of Medicine; Atlanta, GA USA.
authors.forename: Avanti
authors.initials: A
authors.lastname: Gokhale
authors.forename: Patricia
authors.initials: P
authors.lastname: Perez-Cornejo
authors.forename: Charity
authors.initials: C
authors.lastname: Duran
authors.forename: H Criss
authors.initials: HC
authors.lastname: Hartzell
authors.forename: Victor
authors.initials: V
authors.lastname: Faundez
keywords: Ano1; SILAC; epithelia; interactome; membrane protein; salivary gland