Fig. 6
PIP selectivity of Osh6p and ORD8. a Intermembrane PI(4)P and PI(4,5)P2 transfer activity of ORD8 measured with donor LB liposomes containing either PI(4)P or PI(4,5)P2 or both PIPs. LB liposomes (200 μM total lipid concentration) made of DOPC and containing either 5% 16:0/16:0-PI(4)P or 5% 16:0/16:0-PI(4,5)P2 or both PIPs were mixed with NBD-PHFAPP (250 nM) in HK buffer at 37 °C. After 1 min, LA liposomes, made of DOPC and containing 2% Rhod-PE (200 μM), were added to the reaction mix. After 3 min, ORD8 was injected (240 nM). Similar experiments were conducted with NBD-PHPLCδ1 (250 nM) and LB liposomes containing only 5% 16:0/16:0-PI(4,5)P2 or also 5% 16:0/16:0-PI(4)P. PIP transport was followed by measurement of the quenching of the fluorescence signal of NBD-PHPLCδ1 or NBD-PHFAPP caused by the translocation of the lipid sensor from LB to LA liposomes and FRET with Rhod-PE. The signal was normalized in term of PI(4)P or PI(4,5)P2 amount delivered into LA liposomes. The injection of ORD8 set time to zero. Each trace is the mean ± s.e.m. of independent kinetics (n = 3). b Initial 16:0/16:0-PIP transfer rates determined with NBD-PHFAPP and NBD-PHPLCδ1. Data are represented as mean ± s.e.m. (error bars, n = 3). c Initial rates of 18:0/20:4-PIP transfer between membranes determined for ORD8 using NBD-PHFAPP and NBD-PHPLCδ1 sensors. Data are represented as mean ± s.e.m. (error bars, n = 3). d LA liposomes (200 μM total lipid), made of DOPC and 16:0/18:1-PS (95:5), were mixed with NBD-C2Lact in HK buffer at 37 °C. After 1 min, LB liposomes (200 μM), consisting of 93% DOPC, 2% Rhod-PE, and 5% 18:0/20:4-PI(4)P or 18:0/20:4-PI(4,5)P2, were injected. Two minutes later, a third population of liposomes (LC, 200 μM) made of DOPC, doped or not with 5% 18:0/20:4-PI(4)P or 18:0/20:4-PI(4,5)P2, was injected. ORD8 (240 nM final concentration) was injected 2 min later. PS delivery into LB liposomes was followed by measurement of the quenching of the fluorescence of NBD-C2Lact provoked by its translocation from LA to LB liposomes and FRET with Rhod-PE. The signal was normalized in term of PS amount transferred into LB liposomes. e Initial rates of ORD8-mediated PS transfer into acceptor LB liposomes, as a function of the presence of PI(4)P and PI(4,5)P2 in acceptor LB and/or LC liposomes. Data are represented as mean ± s.e.m. (error bars, n = 4). f Competition assay. DOPC/NBD-PS liposomes (98:2, 100 μM total lipid) were added to ORD8 or Osh6p (200 nM) in HK buffer at 30 °C. The sample was excited at 280 nm and the emission was measured at 335 nm. Incremental amounts of liposome, containing 5% PI(4)P or PI(4,5)P2, were injected to the reaction mix. The signal was normalized considering the initial Fmax fluorescence, prior to the addition of NBD-PS-containing liposomes, and the dilution effect due to liposome addition. Data are represented as mean ± s.e.m. (n = 3). g Accessibility assay. CPM (4 μM) was mixed with 400 nM Osh6p(noC/S190C) in the presence of pure DOPC liposomes or liposomes doped with 2% 18:0/20:4-PI(4)P or 18:0/20:4-PI(4,5)P2. Intensity bars correspond to the fluorescence measured 30 min after adding CPM (n = 3)