Co-Immunoprecipitation of Membrane-Bound Receptors

Abstract

The study of cell-surface receptor dynamics is critical for understanding how cells sense and respond to changing environments. Therefore, elucidating the mechanisms by which signals are perceived and communicated into the cell is necessary to understand immunity, development, and stress. Challenges in testing interactions of membrane-bound proteins include their dynamic nature, their abundance, and the complex dual environment (lipid/soluble) in which they reside. Co-Immunoprecipitation (Co-IP) of tagged membrane proteins is a widely used approach to test protein-protein interaction in vivo. In this protocol we present a method to perform Co-IP using enriched membrane proteins in isolated microsomal fractions. The different variations of this protocol are highlighted, including recommendations and troubleshooting guides in order to optimize its application. This Co-IP protocol has been developed to test the interaction of receptor-like kinases, their interacting partners, and peptide ligands in stable Arabidopsis thaliana lines, but can be modified to test interactions in transiently expressed proteins in tobacco, and potentially in other plant models, or scaled for large-scale protein-protein interactions at the membrane.

RIN4 recruits the exocyst subunit EXO70B1 to the plasma membrane

“…We then used a µMACS GFP Isolation Kit (Miltenyi) for co-IP. We used a modified extraction buffer based on the Miltenyi kit Lysis Buffer that contained only 0.1% Triton X-100 (compared with 1% in kit buffer), as we experienced non-specific binding of proteins to the magnetic beads at high Triton X-100 concentration as previously described ( Avila et al , 2015 ). Eluates were resolved on a 10% polyacrylamide gel and blotted onto a nitrocellulose membrane.…”

Simultaneous CRISPR/Cas9‐mediated editing of cassava eIF4E isoforms nCBP‐1 and nCBP‐2 reduces cassava brown streak disease symptom severity and incidence

“…We investigated CBSV VPg association with cassava eIF4E isoforms through co-immunoprecipitation (co-IP) experiments. Several attempts to co-express and co-IP tagged forms of CBSV VPg and cassava eIF4E isoforms in Nicotiana benthamiana were unsuccessful, due possibly to very low levels of VPg expression or competitive interference from endogenous eIF4E isoforms (Avila et al, 2015;Monger et al, 2001). As an alternative, tests for interaction were done using bacterially expressed 6xHIS-VPg-6xHIS-3xFLAG protein mixed with lysates of N. benthamiana expressing YFP-eIF4E isoforms.…”

Perception of double‐stranded RNA in plant antiviral immunity

“…The RNAs are then released into endosomes, where they are recognized by Toll‐like receptors to induce immune responses (Baglio et al ., ; Brencicova and Diebold, ; Dreux et al ., ; Kouwaki et al ., , ; Matsumoto et al ., ; Okamoto et al ., ). In analogy to the mammalian system, it is known that bacterial PAMP receptor‐complexes in plants re‐localize from the plasma membrane to endosomes (Avila et al ., ; Beck et al ., ; Frescatada‐Rosa et al ., ; Russinova et al ., ) and that plant receptor proteins can signal from endosomes during immunity and development (Geldner et al ., ; Irani et al ., ; Mbengue et al ., ; Sharfman et al ., ). Thus, it is conceivable that the putative membrane‐localized PRR receptors for dsRNA may cycle between the plasma membrane and endosomal compartments, and that recognition may take place during membrane fusion events between receptor‐containing endosomes and viral RNA‐containing vesicles or viral replication compartments (VRCs, Fig.…”