Fig. 5.
ORD8 can extract and transport PtdIns(4,5)P 2. a Bar graph showing the percentage of PtdInsP extracted from liposomes by Osh6p, ORD8, and ORD8 (H514A, H515A) mutant. Error bars indicate s.d.; n = 3. b Schematic of the assay employed to examine lipid transport by ORPs. See text for details. c, d Brain PtdIns(4)P c and PtdIns(4,5)P 2 d transport assay. Donor liposomes (LA) were incubated with NBD–PHFAPP followed by addition of acceptor liposomes (LB) doped or not with PtdSer. After 3 min, the protein was injected. The broken line signifies NBD–PHFAPP signal upon complete PtdIns(4)P and PtdIns(4,5)P 2 equilibration between liposomes. e Plot of initial brain PtdIns(4)P and PtdIns(4,5)P 2 transport rates by Osh6p and ORP8 ORD. Error bars indicate s.d.; n = 3. f PtdIns(4,5)P 2 detected by PH-PLC–GFP in HeLa cells overexpressing ORP5A or ORP5AΔPH. g Quantitation of intensity in f, including the ratio of GFP fluorescence of the PM vs. cytosol, and the ratio of GFP signals detected by the TIRF microscopy vs. total epifluorescence (mean + s.d.; ***P < 0.001; *P < 0.05, t test, n = 8 – 15 cells). h Western blot confirming the efficiency of ORP5 and ORP8 double knock-down in HeLa cells transfected with both GFP–ORP5A and mCherry–ORP8L. i Confocal microscopy images showing PtdIns(4,5)P 2 distribution as detected by PH-PLC–GFP in HeLa cells deficient in both ORP5 and ORP8. j Quantitation of intensity in i, including the ratio of GFP fluorescence of the PM vs. cytosol, and the ratio of GFP signals detected by the TIRF microscopy vs. total epifluorescence (mean + s.d.; ***P < 0.001; **P < 0.01, t test, n = 8 – 15 cells). Bar = 10 μm