Yeast 2-hybrid assay for investigating the interaction between the centrosome proteins PLK4 and STIL

Centrosomes act as a major microtubule-organizing center in many animal cells. This non-membranous cell organelle is involved in a wide range of cell functions like division, migration, and signaling. Centrosome houses hundreds of proteins that interact in a regulated manner to carry out their tasks. Most of these centrosome proteins have either the coiled-coil or disordered regions, making them challenging to purify for functional analysis. Yeast 2-hybrid (Y2H) is an in vivo based method for studying protein-protein interactions by exploiting the modular nature of yeast's GAL4 transcription factor. This method provides an alternative strategy for investigating bi-molecular interactions, especially when dealing with otherwise difficult proteins to purify by conventional biochemical approaches. We present a detailed protocol for showing the interaction between the two core centrosome proteins, i.e., PLK4 and STIL, using the Y2H assay.