Fig. 8
From: An electrostatic switching mechanism to control the lipid transfer activity of Osh6p
Mutations in the D/E-rich motif impact Osh6p activity. a Sequence of the D/E-rich motif with the anionic residues colored in red. Substitutions by alanines or glycines in the 4A and 5A2G mutant are in bold. b Electrostatic features of the surface of Osh6p and mutants. c Percentage of accessible PS or PI4P extracted by Osh6p, Osh6p-4A or Osh6p-5A2G (3 µM) from liposomes (80 µM lipids) with 2% POPS or diC16:0-PI4P (n = 3–8, *P < 0.05, **P < 0.01, Mann–Whitney test). d Flotation assay. Osh6p, Osh6p-4A or Osh6p-5A2G was mixed with DOPC/POPS/diC16:0-PI4P (66/30/4 mol/mol) liposomes (n = 3). e Initial PI4P transport rates of Osh6p and of 4A and 5A2G mutants (200 nM) determined with LB liposomes containing 5% diC16:0-PI4P and either 25% diphytanoyl-PC (orange bar) or –PS (brown bar) and LA liposomes containing 5% POPS (n = 3–9). f Ratio between the initial PI4P transport rate, measured for Osh6p and mutants when LA and LB liposomes are weakly anionic (with 5% POPS and 5% PI4P, respectively) and the transport rate measured with LB liposomes that are more anionic (with 25% of diphytanoyl-PS instead of diphytanoyl-PC (as in (e), brown bar, n = 3–9) or the rate measured with LA and LB liposomes that are both more anionic, doped respectively with 15% PI and 4% POPA (at the expense of DOPC), and 10% PI, 4% POPA and 25% diphytanoyl-PS instead of diphytanoyl-PC (purple bars, n = 4). g Growth of osh1Δ-7Δ yeast, containing a plasmid-borne copy of a temperature-sensitive allele of OSH4 and the indicated version of OSH6, at permissive (25 °C) or non-permissive (37 °C) temperature for osh4-1ts. h Redistribution of C2Lact-GFP in PS-depleted yeast cells (cho1Δosh6Δosh7Δ) expressing Osh6p-mCherry or the 5A2G mutant after adding 18:1 lyso-PS. Images were taken every 2 min, with the first panel (t = 0*) showing the last time-point before the onset of C2Lact-GFP signal transition. The absolute timing of this time-point varied between experiments (15–25 min after lyso-PS injection) due to instability of the lyso-PS suspension18. Scale bar: 5 μm. Error bars correspond to s.e.m. Source data are provided as a Source Data file
