DogTag – a novel genetically encoded proximity labeling strategy for problematic protein-protein interactions

Receptor-Like kinases are responsible for the perception of almost all extracellular stimuli in plants. Two well characterized receptor like kinases are Flagellin Sensitive 2, a receptor involved in plant pathogen immune responses and Brassinosteroid Insensitive 1, a receptor involved in hormone perception and plant growth. Both of these plant proteins have been shown to possess the post translational S-acylation, a dynamic fatty acid modification catalysed by protein S-acyl transferase (PAT) enzymes. Protein-protein interactions are a key component of cellular processes. Including the interaction between receptor like kinases and PATs. These interactions have been difficult to characterize using traditional protein-protein interaction techniques on account of weak transient interactions between membrane proteins. Through this work I have developed a new proximity labelling technique, DogTag, based on the Pup-IT system that is suitable for tricky membrane interactions in plants. I have optimised aspects of the machinery to enhance sensitivity and improve accuracy. I have been successful in demonstrating that the DogTag Technique can be used for the study of pairwise interactions, with the successful identification of some promising receptor like kinase and PAT interactions. Including PATs 8, 9 and 12. I have also demonstrated that DogTag works for protein discovery with mass spectrometry. Successfully identifying a number of interesting novel potential BRI1 interacting proteins, including members of the HSP90 family.