FIGURE 2.

Inactivation of Neo1 does not lead to significant changes in the distribution or expression of Drs2/Dnf P4-ATPases but does increase Dnf1 and Dnf2 plasma membrane flippase activity. A, Western blotting analysis of wild-type and neo1^ts cells expressing HA-tagged Dnf1 (MTYD1-219RRL and MTYD1-62815BL) and HA-tagged Dnf2 (MTYD2-219RRL and MTYD2-62815BL) at 27 and 30 °C. Equal amounts of total protein (0.75 mg) were loaded from each sample and probed with anti-HA, anti-Drs2, and anti-Arf1. WT* (untagged) and drs2Δ were used as specificity controls. Arf1 levels were used to demonstrate equal loading of the samples. These images are representative of at least three biological replicates. B, quantification of Dnf1-HA, Dnf2-HA, and Drs2 levels in wild-type and neo1^ts cells at 27 and 30 °C. Intensity values for neo1^ts cells were normalized to those values of wild-type cells at corresponding temperatures (n ≥3, error bars ±S.E.). C, subcellular fractionation was done with wild-type and neo1^ts cells expressing Dnf1-HA and Dnf2-HA (same strains as used for A). Kar2 and Pma1 were probed as ER and plasma membrane markers, respectively. Dnf1-HA, Dnf2-HA, Drs2, Kar2, and Pma1 levels for each fraction were plotted as the percentage of total intensity for each protein in all seven fractions. (For example, Dnf1-HA in fraction 7 was divided by the sum of Dnf1-HA in all seven fractions.) The percent in plasma membrane fractions (% PM) is the sum of the percentages in fractions 5–7. D, inactivation of neo1^ts alleles leads to increased Dnf1/Dnf2 activities at the plasma membrane. Lipid uptake assays were performed with WT (MTY219RR), neo1-1 (MTY628-15B), and neo1-2 (MTY628-34A) cells grown at 30 °C using fluorescent-labeled (NBD) phospholipids. Lipid uptake activities were plotted as percentage of NBD-PC uptake for wild-type cells. SM, sphingomyelin; ns, not significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001; Student's t test, n ≥9; error bars ±S.E.