Nat. Methods 15, 715–722 (2018)

Methods to study protein–protein interactions (PPIs) such as affinity pulldown or proximity-tagging systems are typically more effective for cytosolic PPIs than for those at the membrane. To expand the scope of these methods for the study of membrane PPIs, Liu and Zheng et al. developed the proximity-tagging approach PUP-IT (pupylation-based interaction tagging), which uses the small bacterial protein Pup to label proteins of interest. Analogous to ubiquitination, the Pup ligase PafA phosphorylates the C-terminal Glu of Pup and then conjugates it to a lysine side chain on its target protein. When fused to a protein of interest, PafA ligates Pup to any nearby lysine residues on either the fused protein or its interaction partners. After labeling with Pup–biotin, tagged proteins are enriched and identified by mass spectrometry. The authors applied this method to identify new interaction partners of CD28 and also integrated PUP-IT with the FRB/FKBP dimerization system to label cells expressing certain ligands. PUP-IT can detect weak interactions (Kd up to 200 μM) in cells, and exhibits high specificity because the activated Pup intermediate does not diffuse from the enzyme. Furthermore, as lysine is present in most human proteins, this approach should be applicable to a wide variety of protein targets.

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Nature Methods