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: Ano1; SILAC; epithelia; interactome; membrane protein; salivary gland.

Figure 1. Membrane topology of Ano1. Model of mouse Ano1 topology. This model is based on data from Yu et al. (2012). In this model the sequence 628–638 forms the outer vestibule of the channel and amino acids in the first and third intracellular loops are involved in Ca-dependent gating. See Yu et al. for further details.

Figure 2. Subcellular localization of the Ano1 signaling complex. This complex is shown here by the colocalization of Ano1 (green), the acting binding signaling protein ezrin (red) and actin (blue) in the apical membranes of acinar and duct cells of the submaxillary gland of mouse. The white strings represent the canalicullar lumen highlighted by the overlapping fluorescent signals. The landmark asterisk in the merged panel is enlarged to the right. Cell diagram depicts a submaxillary secreting cell and its relative position in the acinus. A yellow band on the apical domain marks the localization of ezrin and Ano1. Diagram was modified from Lentz. Bar 10 microns.

Figure 3. Flow diagram of the strategy to generate an in vivo membrane protein interactome using SILAC mass spectrometry. See text for details and Perez-Cornejo et al. (2012).