Fig. 4
Relationship between the affinity of Osh6p for PS and PI(4)P species and its capacity to transfer them. a Principle of the NBD-PS-based competition assays. The tryptophan (W) fluorescence of Osh6p and ORD8 is quenched when they host an NBD-PS molecule. The replacement of NBD-PS by unlabelled PS restores the fluorescence of these proteins. b Competition assays with different PS species. Liposomes (100 μM total lipid, final concentration), made of DOPC and doped with 2% NBD-PS, were added to Osh6p (240 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 a given PS species at 5%, were added to the sample. The fluorescence was normalized considering the initial Fmax fluorescence, prior to the addition of NBD-PS-containing liposomes, and the dilution effects due to liposome addition. Data are represented as mean ± s.e.m. (n = 3). c Competition assays with liposomes containing either 5% 16:0/16:0-PI(4)P, 16:0/18:1-PI(4)P, or 18:0/20:4-PI(4)P. Data are represented as mean ± s.e.m. (n = 4 for 16:0/18:1-PI(4)P, n = 3 for other PI(4)P species). d Melting curves of Osh6p loaded with different PS species or 16:0/16:0-PI(4)P. In a typical measurement, a sample containing 5 μM of protein and 5× SYPRO Orange in HK buffer was heated and fluorescence was measured at λem = 568 nm (λex = 545 nm). A control experiment with Osh6p incubated with DOPC liposomes devoid of lipid ligands is shown (DOPC only). Only a few curves corresponding to representative ligands are shown for clarity. e Melting temperatures (Tm) determined for Osh6p pre-incubated with pure DOPC liposome or loaded with diverse PS and PI(4)P subspecies. Data are represented as mean ± s.e.m (n = 3–5). Pairwise comparison by unpaired Mann–Whitney U test of Tm rate measured with Osh6p in apo form (DOPC only) and Osh6p loaded with saturated or unsaturated PS species, or a PI(4)P species; *p < 0.05, ns: not significant. f Initial transfer rates determined in non-exchange contexts for PS and PI(4)P subspecies with Osh6p (shown in Figs. 1b,c, 2, and 3) as a function of 1/[L]50 values determined for each lipid subspecies. g PS transfer rates in non-exchange conditions as a function of Tm values determined with Osh6p. h Acceleration factors determined for PS and PI(4)P from experiments shown in Fig. 1b and c1b and c, as a function of the 1/[L]50 values determined for each PS subspecies. The 1/[L]50 value determined with 16:0/16:0-PI(4)P is indicated