FIGURE 3.

Dnf1 suppressors of drs2Δ flip diacyl PS. A, localization of the PS-specific probe GFP-LactC2 in the WT strain or PS-deficient strain (cho1Δ). In WT yeast, PS recruits GFP-LactC2 to the plasma membrane, whereas in the cho1Δ strain, GFP-LactC2 is present primarily in the cytosol. The plot of pixel intensity across the lines in the images indicates that GFP-Lac2 is present at the plasma membrane in WT cells but not cho1Δ. B, a cho1Δdrs2Δ PS-deficient strain expressing GFP-LactC2 and the indicated Dnf1 variants were incubated with 10 μm monoacyl lyso-PS or 100 μm DLPS at 4 °C for the times indicated. The ability of each strain to translocate unlabeled PS substrate across the membrane is indicated by recruitment of GFP-LactC2 from the cytosol to the inner leaflet of the plasma membrane. Dnf1 N550S and Dnf1 T254A can flip dually acylated PS (DLPS) to the cytosolic leaflet, but WT Dnf1 and Dnf1[Y618F] cannot. The right column is a plot of pixel intensity along the lines drawn across cell buds at 150 min. Scale bars = 10 μm.