Extended Data Fig. 2: Neomycin sensitive neo1 substrate transport pathway mutants expose PI4P in extracellular leaflets.
From: P4-ATPases control phosphoinositide membrane asymmetry and neomycin resistance
a, neo1-1, dop1-1 and mon2∆ mutant exposes PI4P on extracellular leaflets probed using recombinantly purified GFP-SidCP4C. The right panel is the fluorescence signal intensity of the GFP-probe and left panel shows the DIC panel to display yeast cells. Scale bar = 2 μm. b, Quantification of GFP-SidCP4C binding to the cell surface of the WT cells and flippase mutant strains. Two-way ANOVA was performed to test the variance and comparisons with WT cells were made with Tukey’s multiple comparison test. (n = 20 cells from three biologically independent experiments, Data are mean ± (SD). **** P < 0.001). c, Intact live cells expose PI4P on the extracellular leaflet. Scale bar = 2 μm. d, Quantification of the percentage of the cells exposing PI4P stained with PI. Two-way ANOVA was performed to test the variance and comparisons with WT Neo1 cells were made with Å Ãdák’s multiple comparisons test. (n = 3 biologically independent experiments, Data are mean ±(SD).**** P < 0.001). e, Perturbation of plasma membrane phosphoinositide asymmetry was tested in neo1 mutant strains. (n = 20 cells from three biologically independent experiments). Scale bar = 2 μm. f, Only GFP-SidCP4C binds to neomycin-sensitive mutant cells at the rim of cells, suggesting exposure of PI4P on extracellular leaflet of the plasma membrane. (n = 20 cells from three biologically independent experiments). Scale bar = 2 μm. g, HeLa cells were fixed, permeabilized and stained with recombinant biosensors GFP-SidCP4C or GFP-PLCPH. (n = 20 cells from three biologically independent experiments). Scale bar = 10 μm.
