Fig. 2: neo1 mutant cells expose PI4P in the plasma membrane extracellular leaflet.
From: P4-ATPases control phosphoinositide membrane asymmetry and neomycin resistance
a, PIP synthesis in the cytoplasmic leaflet of the plasma membrane and the use of recombinantly purified GFP–SidCP4C and GFP–PHPLC to probe for external PI4P and PI(4,5)P2, respectively. PM, plasma membrane; PI, phosphatidylinositol. b, Inactivation of neo1-1 at the non-permissive temperature (38 °C) causes PI4P exposure. The fluorescent images were inverted to black signal on white background for clarity. The fluorescence signal intensity of the GFP probe (left) and the differential interference contrast (DIC) panel to display yeast cells (right) are shown. Scale bar, 2 μm. c, A quantification of GFP–SidCP4C and GFP–PLCPH binding to the cell surface of cells expressing Neo1 variants. Two-way ANOVA was performed to test the variance and comparisons with WT Neo1 were made with Dunnett’s multiple analysis (n = 20 cells from three biologically independent experiments; P < 0.0001). The data are the mean ± s.d. d, The GFP–SidCP4C(R652Q) PI4P binding defective mutant fails to bind neo1-1 cells. A one-way ANOVA was performed to test the variance and comparisons with WT Neo1 were made with Dunnett’s multiple analysis (n = 20 cells from three biologically independent experiments; ****P < 0.0001). The data are the mean ± s.d. e, stt4-4 suppresses the neomycin sensitivity of neo1S221L or neo1S452Q at 33 °C. f, pik1-83 fails to suppress the neomycin sensitivity of neo1S221L or neo1S452Q at 34 °C. The data represent growth relative to cells in the absence of the drug. A mixed effect model was performed to test the variance and comparisons with WT Neo1 were made with Tukey’s multiple comparison test (n = 3 biologically independent experiments; **P = 0.0069, 0.0068). n.s., not significant. The data are the mean ± s.d.
