Figure 2.

PM binding of ORP5 depends on both PI4P and PI(4,5)P₂. (A) Kinetics of PI(4,5)P₂ resynthesis after AngII stimulation in ORP5- or ORP8-expressing cells. HEK293-AT1 cells transfected with mCherry-tagged ORP5, ORP8, or mCherry were analyzed with BRET using PM-anchored Venus and PLCδ-PH–fused luciferase. After baseline measurement, cells were treated with vehicle or AngII (100 nM). Relative change in PI(4,5)P₂ level by AngII (AngII/vehicle) was normalized to the mean values gained from baseline measurements (the baseline values showed a slight increase both with ORP5 and ORP8 expression as shown in the insert). Grand means ± SEM are shown from three independent experiments performed in triplicate. (B) Quantitation of PI4P changes after PM recruitment of FKBP12-fused ORP5/8. HEK293-AT1 cells were transfected with mCherry or mCherry-tagged FKBP-ORP5 (FK-ORP5) or -ORP8 (FK-ORP8) in addition to PM-anchored FRB (PM2-FRB). PM PI4P levels were quantitated with BRET analysis using PM-anchored Venus and P4M(2x)-fused luciferase. After baseline measurement, cells were treated with DMSO or rapamycin (100 nM) for recruiting FK-ORP5 or -ORP8 to the PM. Relative PI4P level (rapamycin/DMSO) was normalized to mean value of baseline measurement. Grand means ± SEM are shown from three independent experiments performed in triplicate. (C) ORP5-PM contacts in PM PI4P- or PI(4,5)P₂-depleted cells. COS-7 cells were transfected with GFP-tagged ORP5 and mCherry-tagged FKBP-PJ, -PJ-Sac, -PJ-Dead, or FKBP-INPP5E. Lyn₁₁-FRB-iRFP was transfected as a recruiter. 1 d after transfection, cells were imaged by time-lapse TIRF microscopy, inducing acute depletion of PI4P, PI(4,5)P₂, or both with the addition of 1 µM rapamycin at time 0. Representative images from the time-lapse 0, 2.5, 5, 7.5, and 10 min of rapamycin treatment (left). The graph shows normalized GFP-ORP5 fluorescence intensity in the evanescent field for 28–34 cells imaged across three independent experiments (means ± SEM). Bars, 10 µm.