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. 2011 Oct 27;286(52):44669–44678. doi: 10.1074/jbc.M111.285734

FIGURE 1.

FIGURE 1.

Screen for PtdInsP-interacting PDZ domains. A, the screen was based on a fluorescence microscopy assay, where we evaluated the ability of the tested PDZ domain to assist syntenin-1 PDZ1 fused to eYFP (eYFP-S1 PDZ1) in targeting PtdIns(4,5)P2-rich subcellular regions of MCF-7 cells (i.e. the plasma membrane, nuclear speckles, and/or nucleoli). Neither the individual syntenin-1 PDZ1 (top) nor syntenin-2 PDZ2 (S2 PDZ2) (10) was able to target eYFP to these regions. However, eYFP-S1 PDZ1-S2 PDZ2 localized to PtdIns(4,5)P2-rich pools (middle and bottom). Note that the length of the linker region had no impact on the subcellular enrichment. B, confocal micrographs of selected Drosophila PDZ domains fused to eYFP-S1 PDZ1 in MCF-7 cells. C, Confocal micrographs showing the enrichment of eYFP-PH PLCδ and eYFP-S1 PDZ1-Pyd PDZ2 around the PtdIns(4,5)P2-rich membranes of macropinosomes. The vesicular structures were induced by co-expression of a constitutively active Arf6-Q67L mutant (28). D, time lapse wide-field micrographs showing the delocalization of eYFP-S1 PDZ1-Pyd PDZ2 from the plasma membrane upon ionomycin-induced PtdIns(4,5)P2 breakdown. E, time lapse confocal micrographs of MCF-7 cells co-expressing eYFP-S1 PDZ1-Pyd PDZ2, mRFP-FKBP12-PtdInsPs 5′-phosphatase, and palmitoylated eCFP-FRB-PM (PM-target). eYFP-S1 PDZ1-Pyd PDZ is enriched at the plasma membrane and at cell-cell contacts. Note the reduction of this enrichment 5 min after the addition of rapamycin, which induces plasma membrane translocation of the 5′-phosphatase (right).