Claudins constitute a family of tetraspan transmembrane proteins that are ubiquitously expressed in epithelial tissues. They are the major components of tight junctions (TJs), defining the barrier properties in epithelia and regulating the paracellular transport of water and solutes. Claudins can also be found outside the TJ along the basolateral plasma membrane of epithelial cells. It is not yet well understood how claudins regulate the formation of TJs and which functions they exert outside them. The role of their extracellular loops in the regulation of paracellular transport has been widely studied. In contrast, it is not clear how their long and unstructured intracellular C-terminal regions intervene in their functions beyond interacting with tight junction protein ZO-1 (TJP1). Although protein interaction studies can help to answer these two key questions, the disordered nature of the cytosolic tail of claudins makes it challenging. In this work, a large-scale study is presented combining two complementary pull-down techniques followed by mass spectrometry to create an interaction landscape of the claudin protein family. Co-immunoprecipitation (CoIP) of recombinant claudins overexpressed in MDCK-C7 epithelial cells provided information about interactions beyond the already known TJ proteins. Protein interaction screen on a peptide matrix (PRISMA) allowed for the mapping of interactions along the disordered cytosolic C-terminal region of claudins and enables to study the effect of numerous post-translational modifications (PTMs) in these interactions. We confirmed newly identified claudin tail interactors from our PRISMA approach by proximity ligation assays (PLA) and revealed their possible implication in spatially separated biological processes in epithelial cells. By combining these two complementary approaches, we were able to create the Claudinome, the first comprehensive map of interactors for the entire human claudin protein family, which constitutes a valuable resource to improve our understanding of functional connections and regulatory processes for all claudins.
