Figure 4.

SAC1^mito-mediated PM PI4P turnover is not enhanced at mito:PM contact sites. COS-7 cells were transfected with TagBFP2-Sac1^mito, EGFP-P4C PI4P biosensor, iRFP713-FRB-Fis1^C31 (FRB^mito), and the indicated FKBP-tagged construct, After 18–24 h, they were subject to time-lapse imaging by TIRFM. At time 0, 30 nM PI4KA inhibitor GSK-A1 was added together with 1 µM rapamycin to induce dimerization of FRB and FKBP. FKBP-tagged constructs were: (A) mCherry-FKBP as control; (B) an artificial PM-localized tethering construct, mCherry-FKBP-ELL-CAAX, consisting of the PM-anchored c-terminal tail of H-Ras linked to FKBP by an artificial helical and flexible linker mCherry-ORP5^ΔTMD-FKBP; (C) a double deletion mutant of ORP5 lacking ER-localized transmembrane domain and the ORD, mCherry-ORP5^{ΔORD−ΔTMD}-FKBP; (D) the PI4P-binding deficient mutant, mCherry-ORP5^{H378/9A−ΔTMD}-FKBP; and (E) mCherry-ORP5^ΔTMD-FKBP. For all panels, graphs show the ratio of mCherry-FKBP fluorescence at mitochondria to elsewhere in the TIRF footprint (i.e., PM), the relative intensity of the iRFP-FRB^mito signal, and the total fluorescence intensity of EGFP-P4C in the TIRF footprint over time. Data are grand means ± SE of three experiments, analyzing 10–12 cells per experiment.