P4M localization at the PM depends on PtdIns4P. (A) Rapamycin (rapa)-induced recruitment of PJ-Sac to the PM via dimerization with FRB targeted to the PM causes depletion of PtdIns4P (PI4P) and release of GFP-P4M from the membrane, without effect on the PtdIns(4,5)P₂ (PI(4,5)P₂) probe iRFP-PH-PLCδ1. (B) Recruitment of an INPP5E domain to the PM causes breakdown of PtdIns(4,5)P₂ into PtdIns4P, causing release of iRFP-PH-PLCδ1 and increased PM binding of GFP-P4M. (C) PM recruitment of PJ (containing both Sac and INPP5E domains) causes breakdown of PtdIns(4,5)P₂ and PtdIns4P into PtdIns, resulting in release of both probes from the PM. (D) No effect on either probe is seen when catalytically inactive mutations in the Sac and INPP5E domains are introduced into PJ. (E and F) PM intensity of cells expressing either PtdIns4P-binding GFP-P4M or GFP-PH-OSBP during recruitment of PJ-Sac (E) or INPP5E (F) was measured by TIRF. Images show the FRB domain fused to CFP and the PM-targeted acylated Lyn₁₁ peptide and the resulting binary masks used to measure PM intensity as well as the GFP-P4M and iRFP PH-PLCδ1 domains before and after enzyme recruitment with rapamycin. Graphs are from three (E) or four (A–D and F) independent experiments and are grand means ± SEM. Statistical significance (P < 0.05) is depicted over the indicated range (two-way ANOVA; see Materials and methods). n.s. = i.e., P > 0.05. Bars, 10 µm.