Stress response and energy metabolism as a function of aging

National Heart, Lung, And Blood Institute

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Abstract

The network of protein-protein interactions is key to understanding the regulation and functions of any protein. One 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, with exogenous expression of bait proteins that normally translocate to a cellular organelle (e.g. mitochondria or endoplasmic reticulum), some fraction of the bait proteins can remain behind in the cytosol or mislocalize. When the bait protein is immunoprecipitated, proteins interacting with it in the cytosol will contaminate the co-immunoprecipitated population of proteins. Avoiding such contamination will require pre-isolation of the organelle wherein the bait protein normally resides, but organelle isolation techniques are technically challenging and generally do not yield pure organelle preparation. Therefore, a methodology that can identify organelle-specific binding-partners without prior organelle isolation will be a valuable tool in molecular and cellular biology. Proteins entering organelles such as the mitochondria or endoplasmic reticulum usually have an organelle-targeting sequence in the N-terminus that is cleaved off during its entry. In the present study, we describe the internal FLAG-M1 antibody (IFM1) technique, a new method to identify binding partners by means of the anti-FLAG M1 antibody, which has specificity for an internal FLAG tag that is exposed after the cleavage of the targeting sequence in the organelle. We use IFM1 to identify binding partners for mitochondrial proteins Endonuclease G (ENDOG) and TRX2 in mitochondria, demonstrating that IFM1 provides a simple and easy-to use proteomics tool to identify mitochondria-specific binding-partners.