Fig. 4

Osh6p dissociates from anionic membranes when it extracts a lipid ligand. a PS and PI4P extraction assay. Percentage of accessible PS or PI4P extracted by 3 µM Osh6pΔ35 or Osh6pΔ35(L69D) from liposomes (80 µM lipids, with 2% POPS or diC16:0-PI4P) using NBD-C2_Lact or NBD-PH_FAPP, respectively. Values are obtained by normalizing the signal at 536 nm (n = 5–6 for PS extraction assays, n = 4 for PI4P extraction assays, **P < 0.01, *P < 0.05, Mann–Whitney test). b Flotation assay. Osh6p, Osh6pΔ35, Osh6pΔ69 or Osh6p(L69D) (750 nM) was added to liposomes (750 µM lipids) with 70 mol% DOPC and 30 mol% POPS (n = 3–4). c Binding ability of NBD-labeled wild-type Osh6p (black bar) or Δ35 mutant (grey bar) to liposomes, as function of their composition, was given by the ratio of the NBD fluorescence intensity, F and F₀, measured at 536 nm in the presence or absence of liposomes, respectively (n = 4, *P < 0.05, Mann–Whitney test). d Dissociation of NBD-labeled Osh6p WT or Δ35 from anionic liposomes (DOPC/diphytanoyl-PS 7/3 mol/mol) upon adding extractable PS. Each protein (100 nM) was pre-mixed with liposomes (400 µM lipids) in HK buffer at 30 °C. Then, a small volume (1 µL) of a stock solution of diC12:0-PS in methanol or of pure methanol was injected. NBD fluorescence was followed at 535 nm (λ_ex = 460 nm) over 10 min and normalized. Each trace is representative of several experiments. Control measurements were performed with NBD-C2_Lact (250 nM) instead of NBD-labeled Osh6p. e Flotation assay. Osh6p, Osh6pΔ35, Osh6p(L69D) or Osh6pΔ35(L69D) at 750 nM was incubated with DOPC/diphytanoyl-PS (7/3 mol/mol) liposomes (750 µM lipids) containing increasing amount of POPS (0, 2, 5 or 10%). Alternatively, each protein was incubated with DOPC/diphytanoyl-PS (7/3 mol/mol) membranes with 0, 2, 5 or 10% diC16:0-PI4P (n = 4, *P < 0.05, Mann–Whitney test, comparison with the data measured with no PS or PI4P). Error bars correspond to s.e.m. Source data are provided as a Source Data file