Fig. 1: Separation-of-function mutations in Neo1 that cause neomycin sensitivity do not correlate with loss of PS or PE asymmetry.

a, The structure of S. cerevisiae Neo1 with the major domains displayed in different colours (PDB ID 9BS1, this study). The dashed box is the substrate translocation pathway enlarged in b. b, The transmembrane (TM) segments TM1, TM2 and TM4 form the substrate transport pathway of Neo1, and the mutation of residues shown causes exposure of PS and/or PE on the extracellular leaflet of the plasma membrane. c, Neomycin sensitivity of Neo1 substrate transport pathway mutants at 26 °C on YPD and YPD neomycin (500 µg ml⁻¹) plates, relative to WT and neo1-1 (a temperature-sensitive strain displaying a loss of PS and PE membrane asymmetry at this permissive growth temperature). d, Neomycin sensitivity in liquid culture of strains expressing the indicated Neo1 variants. The data represent growth relative to WT cells in the absence of the drug. The mixed model analysis was performed to test the variance and comparisons with WT Neo1 were made with Tukey’s multiple comparison analysis (n = 3 biologically independent experiments; ****P < 0.0001). The data are the mean ± s.d. e,f, Neomycin sensitivity of S. cerevisiae flippase mutants (neo1-1, drs2∆, dnf1,2∆ and dnf3∆) and strains deficient for Neo1 regulators (dop1-1 and mon2∆) on YPD + neomycin plates (e) and liquid culture (f). The data represent the growth relative to WT cells in absence of the drug. A mixed model analysis was performed to test the variance and comparisons with WT Neo1 were made with Tukey’s multiple comparison analysis (n = 3 biologically independent experiments; ****P < 0.0001). n.s., not significant. The data are the mean ± s.d.

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