Localization of the tubby domain, a PI(4,5)P2 biosensor, to E-Syt3-rich ER-PM junctions.

The phospholipid PI(4,5)P2 acts as a signaling lipid at the plasma membrane (PM) with pleiotropic regulatory actions on multiple cellular processes. Signaling specificity may result from spatiotemporal compartmentalization of the lipid and from combinatorial binding of PI(4,5)P2 effector proteins to additional membrane components. Here, we analyzed the spatial distribution of tubbyCT, a paradigmatic PI(4,5)P2 binding domain, in live cells by TIRF microscopy and molecular dynamics simulations. We found that unlike other well-characterized PI(4,5)P2 recognition domains, tubbyCT segregates into distinct domains within the PM. TubbyCT enrichment occurred at contact sites between PM and ER (ER-PM junctions) as shown by co-localization with ER-PM markers. Localization to these sites was mediated in a combinatorial manner by binding to PI(4,5)P2 and by interaction with a cytosolic domain of Extended Synaptotagmin 3 (E-Syt3), but not other E-Syt isoforms. Selective localization to these structures suggests tubbyCT as a novel selective reporter for a ER-PM junctional pool of PI(4,5)P2. Finally, we found that association with ER-PM junctions is a conserved feature of tubby-like proteins (TULPs) suggesting an as yet unknown function of TULPs.