PDK1

PIP3

PDK1

Caspase7

Current methods to detect protein-protein interactions are either laborious to implement, not adaptable for mammalian systems or in vitro methods. By adding a peroxisomal targeting signal (PTS) onto one protein, binding partners lacking a targeting signal were co-transported into the peroxisomes in a piggy-back fashion, as visualised by confocal and electron microscopy. A fragment of colicin E2 and its tightly interacting immunity protein, Imm E2, were both expressed in the cytosol. When either one contained a PTS-tag, both proteins were colocalised in the peroxisomes. The cytokine-independent survival kinase (CISK) containing a PTS-tag was not efficiently targeted to the peroxisomes unless the Phox homology (PX domain, attaching the protein to endosomal membranes, was removed. However, PTS-tagged CISK with deleted PX domain was able to directed 3-phosphoinositide-dependent protein kinase-1 (PDK-1) into the peroxisomes. This demonstrates that the two proteins interact in vivo. Mutating Ser486, which is phosphorylated in activated CISK, to Ala prevented the interaction, indicating that CISK and PDK1 interact in a phosphorylation-dependent manner. The method therefore allows assessment of protein-protein interactions that depend on post-translational modifications that are cell-specific or dependent on the physiological state of the cell.