FIGURE 8.

NBD-PS flip does not represent the essential function of the DRS2/DNF subfamily of P-type ATPases. A, early log phase cells (dnf2Δdnf3Δ, HCY285; dnf1Δdnf2Δdnf3Δ, HCY286; dnf1Δdnf2Δdrs2Δ, HCY288; dnf2Δdnf3Δdrs2Δ, HCY291; dnf3Δdrs2Δ, HCY290; dnf1Δdrs2Δ, ZHY2149D; dnf2Δdrs2Δ, ZHY615D2A; dnf1Δdnf3Δdrs2Δ, ZHY708; and dnf1Δdnf3Δ, PFY3272G) grown in YPD were labeled with 10 μm NBD-PS for 1 h at 2 °C and then washed three times with ice-cold SC-azide. Mean fluorescence internalized was measured by flow cytometry. Data are the mean of at least three independent experiments, and the error bars represent the S.D. between experiments. B, ZHY409 (dnf1Δdnf2Δdnf3Δdrs2Δ pRS313::DRS2) and ZHY410-3A (dnf1Δdnf2Δdnf3Δdrs2Δ pRS313::drs2-ts) cells grown in YPD overnight were diluted to early log phase for 2 h at 30 °C, then shifted to 37 °C for 1 h to inactive DRS2 as previously described by (37). Cells were labeled with 10 μm NBD-PS at 2 °C for 1 h or at 30 °C or 37 °C for 30 min, and the mean fluorescence was examined by flow cytometry. Data presented are the mean of at least three independent experiments for 2 °C and two independent experiments for 30 °C and 37 °C. Error bars represent the S.D. between experiments.