P4M domain localization is PtdIns4P dependent at the Golgi, which contains low levels of PtdIns(4,5)P₂. (A) Rapamycin (rapa)-induced recruitment of PJ-Sac to the FRB-tagged tail domain (residues 3,140–3,269) from human giantin (GOLGB1) induces dephosphorylation of PtdIns4P (PI4P) to PtdIns (PI), releasing GFP-P4M from the Golgi. No change is induced by the catalytically inactive PJ-Dead construct. Graphs show normalized intensity of GFP-P4M or PH-PLCδ1-GFP at the Golgi (defined by the FRB-giantin^tail mask) after treatment with 1 µM rapamycin. Data are grand means ± SEM of eight independent experiments. Significant (P < 0.05) changes are denoted as the largest p-value over the indicated range (two-way ANOVA; see Materials and methods). Images show representative cells with the Golgi localization of the FRB-mCherry-giantin^tail recruiter and its conversion to a binary mask for quantification of GFP-P4M fluorescence. Also shown is the localization of mRFP-tagged PJ-Sac and GFP-P4M before and after addition of 1 µM rapamycin. (B) Rapamycin-induced recruitment of FKBP-PIP5K to FRB-giantin^tail stimulates phosphorylation of PtdIns4P to produce PtdIns(4,5)P₂ (PI(4,5)P₂), recruiting PH-PLCδ1-GFP. No effect is observed for the catalytically inactive D253A mutant. Data are grand means of three or four independent experiments ± SEM. Insets depict an enlarged view of the regions depicted by hashed boxes. Bars: (main images) 10 µm; (insets) 5 µm.