Fig. 3

The binding of Osh6p to anionic membranes depends on its conformational state. a Accessibility CPM assay. Osh6p(noC/S190C) has a unique cysteine which is solvent-accessible only if the lid is open. Osh6p(noC/S190C) or its L69D counterpart (400 nM) was incubated in HK buffer at 30 °C with DOPC liposomes (400 µM lipids) including or not 2% POPS or diC16:0-PI4P. CPM (4 µM) was added and the apparition of fluorescence was measured over time. Bars correspond to the fluorescence (measured at λ = 470 nm) 30 min after adding CPM (n = 3). b Chemical structure of POPS and diphytanoyl-PS. c PS extraction assay. Fluorescence spectra of NBD-C2_Lact (250 nM) (λ_ex = 460 nm), mixed with DOPC liposomes (80 µM lipid) incorporating 2% POPS or 2% diphytanoyl-PS, were recorded before and after adding Osh6p (3 µM). A control experiment was done with pure DOPC liposomes. Normalized signal provides the fraction (in percentage) of extracted PS (n = 4 with POPS, n = 3 with diphytanoyl-PS). d Accessibility assay. Osh6p(noC/S190C) (400 nM) was incubated with DOPC liposomes doped or not with 2% POPS or diphytanoyl-PS and mixed with CPM (4 µM). Intensity bars correspond to the fluorescence measured 30 min after adding CPM (n = 3). e Flotation assay. Osh6p (750 nM) was incubated with pure DOPC liposomes (750 µM lipids) or liposomes containing 30% POPS, diphytanoyl-PC or diphytanoyl-PS at the expense of DOPC. The bars represent the fraction of protein bound to each type of liposomes (n = 3–4). f Emission spectra of NBD-labeled Osh6p (200 nM, excitation at 460 nm) in HK buffer measured in the absence (red spectrum) or presence of liposomes (400 µM total lipids) made only of DOPC or including 30% POPS, diphytanoyl-PC or diphytanoyl-PS (at the expense of DOPC). The contribution of buffer alone or of light scattering from liposomes to the fluorescence signal was subtracted. Error bars correspond to s.e.m. Source data are provided as a Source Data file