Dynamic Proximity Tagging in Living Plant Cells with Pupylation-Based Interaction Tagging

Identification of protein–protein interactions (PPIs) and protein kinase substrates is fundamental for understanding how proteins exert biological functions with their partners and targets. However, it is still technically challenging, especially for transient and weak interactions involved in most cellular processes. The proximity-tagging systems enable capturing snapshots of both stable and transient PPIs. In this chapter, we describe in detail the methodology of a novel proximity-based labeling approach, PUP-IT (pupylation-based interaction tagging), to identify PPIs using a protoplast transient expression system. We have successfully identified potential kinase substrates by targeted screening and tandem mass spectrometry analysis.