FIGURE 1:

Identification of SFK1 as a multicopy suppressor of pap B sensitivity in lem3Δ mutant cells. (A) High sensitivity to pap B and duramycin in lem3Δ and dnf1Δ dnf2Δ cells. Cells were cultured in YPDA medium at 30°C, serially diluted, and spotted onto YPDA plates containing 0.5 μg/ml pap B or 5 μM duramycin, followed by incubation for 1.5 d at 30°C. For the pap B assay including (B), YKT2112 (cdc50Δ) and YKT2000 (drs2Δ) strains were used, whereas YKT249 (cdc50Δ) and YKT745 (drs2Δ) were used for the duramycin assay. (B) Suppression of pap B sensitivity in flippase mutants by overexpression of SFK1. Cells transformed with YEp24 or YEp24-SFK1 were cultured in SDA-U medium at 30°C, and cell growth was examined as in A for 2 d at 30°C. (C) Suppression of duramycin sensitivity in lem3Δ by overexpression of SFK1. lem3Δ cells transformed with YEp24 or YEp24-SFK1 were cultured in SDA-U medium at 30°C, and cell growth was examined as in A for 2 d at 30°C. (D) Staining of exposed PE in the lem3Δ mutant with biotinylated Ro-peptide (Bio-Ro) and Alexa Fluor 488–labeled streptavidin. Arrowheads indicate Alexa Fluor 488 signal on the bud tip. Dashed lines indicate cell edges. Scale bar: 5 μm. (E) PE exposure in lem3Δ is decreased by overexpression of SFK1. Wild-type and lem3Δ cells transformed with YEp24 or YEp24-SFK1 were cultured in SDA-U medium at 30°C and then treated with Bio-Ro. Cells were categorized into three patterns: 1) cells with no signal, 2) those with signal at the bud, and 3) those with signal all over the cell surface. Percentages for each pattern are shown (n > 100 cells). (F) Deletion of SFK1 increases sensitivity to pap B and duramycin in lem3Δ. Cell growth was examined as in A for 2-d at 30°C. (G) Deletion of SFK1 increases exposure of PE in lem3Δ. Cells were cultured in YPDA medium at 30°C, and exposed PE was visualized as in D. Dashed lines indicate cell edges. Scale bar: 5 μm. Cells were categorized as in E (n > 100 cells).