Identification of Endonuclease G binding protein using a method which specifically isolates binding partners within subcellular compartments

The elucidation of protein-protein interaction networks is crucial for understanding the regulatory mechanisms and functional roles of proteins. A commonly used technique is to express an epitope-tagged protein (bait) and perform co-immunoprecipitation with the tag antibody to identify proteins bound to the tagged protein. However, when bait proteins that are normally targeted to specific cellular organelles such as mitochondria or the endoplasmic reticulum, are exogenously expressed, a portion of these proteins may fail to localize correctly and instead accumulate in the cytosol or other non-targeted regions. This mis-localization leads to contamination during immunoprecipitation, as cytosolic proteins that bind to the bait are co-precipitated. Therefore, there is a pressing need for a method that can pinpoint organelle-specific binding partners. Proteins entering organelles such as the mitochondria or the endoplasmic reticulum usually possess an N-terminal organelle-targeting sequence that is cleaved off upon entry. In this study, we describe the internal FLAG-M1 antibody (IFM1) technique, a novel method for identifying binding partners by means of the anti-FLAG M1 antibody, which specifically recognizes an internal FLAG tag exposed after the cleavage of the targeting sequence within the organelle. Using the internal FLAG-M1 antibody (IFM1) technique, we identified binding partners of Endonuclease G and TRX2 within mitochondria and discovered novel interactions with Peroxiredoxin 3 and ATP synthase subunits α and β. In conclusion, IFM1 is a reliable method for identifying mitochondria-specific binding partners and has proven to be a straightforward and effective proteomics tool.