The Essential Neo1 Protein from Budding Yeast Plays a Role in Establishing Aminophospholipid Asymmetry of the Plasma Membrane

Abstract

Eukaryotic organisms typically express multiple type IV P-type ATPases (P4-ATPases), which establish plasma membrane asymmetry by flipping specific phospholipids from the exofacial to the cytosolic leaflet. Saccharomyces cerevisiae, for example, expresses five P4-ATPases, including Neo1, Drs2, Dnf1, Dnf2, and Dnf3. Neo1 is thought to be a phospholipid flippase, although there is currently no experimental evidence that Neo1 catalyzes this activity or helps establish membrane asymmetry. Here, we use temperature-conditional alleles (neo1(ts)) to test whether Neo1 deficiency leads to loss of plasma membrane asymmetry. Wild-type (WT) yeast normally restrict most of the phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the inner cytosolic leaflet of the plasma membrane. However, the neo1-1(ts) and neo1-2(ts) mutants display a loss of PS and PE asymmetry at permissive growth temperatures as measured by hypersensitivity to pore-forming toxins that target PS (papuamide A) or PE (duramycin) exposed in the extracellular leaflet. When shifted to a semi-permissive growth temperature, the neo1-1(ts) mutant became extremely hypersensitive to duramycin, although the sensitivity to papuamide A was unchanged, indicating preferential exposure of PE. This loss of asymmetry occurs despite the presence of other flippases that flip PS and/or PE. Even when overexpressed, Drs2 and Dnf1 were unable to correct the loss of asymmetry caused by neo1(ts) However, modest overexpression of Neo1 weakly suppressed loss of membrane asymmetry caused by drs2Δ with a more significant correction of PE asymmetry than PS. These results indicate that Neo1 plays an important role in establishing PS and PE plasma membrane asymmetry in budding yeast.

Keywords: ATPase; P4-ATPase; duramycin; flippase; membrane lipid; papuamide A; phosphatidylethanolamine; phosphatidylserine; transporter.

FIGURE 1.

neo1^ts cells display a loss of membrane asymmetry. A and B, WT (MTY219RR), neo1-1 (MTY628-15B), neo1-2 (MTY628-34A), and drs2Δ (ZHY615M2D) cells were incubated for 20 h at the permissive growth temperature of 27 °C (A) or the semi-permissive growth temperature of 30 °C (B) with increasing concentrations of papuamide A. C and D, same set of strains was assayed for duramycin sensitivity at 27 °C (C) or 30 °C (D). Growth relative to WT cells in the absence of drug was plotted (n ≥5, error bars ±S.E.M.). Sigmoidal dose-response curve fitting modality was applied when the adjusted R² values are greater than 0.8.

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.

FIGURE 3.

neo1^ts mutants display a dramatic loss of membrane asymmetry at growth temperatures that do not disrupt protein trafficking. A, Rer1-GFP trafficking in wild-type cells (SEY6210) and copI^ts alleles (ret1-1 and sec21-1) at 27 °C. FM4-64 staining was done with wild-type and copI^ts alleles expressing pURA3-GFP-RER1. White arrows indicate the punctate localization for GFP-tagged Rer1. B, quantitative analysis of Rer1-GFP localization by Western blotting analysis for wild-type, copI^ts, and neo1^ts cells grown at 27 °C. Ratios of GFP cleaved versus total GFP were determined for wild-type, copI^ts, and neo1^ts cells; n ≥2. Duramycin and papuamide A sensitivity assays were performed with wild-type and copI^ts cells at 27 °C (C) and at 30 °C (D). Growth relative to wild-type cells (SEY6210) was plotted; n ≥3. Sigmoidal dose-response curve fitting modality yielded adjusted R² values smaller than 0.8. Data points were connected to generate the graphs.

FIGURE 4.

NEO1 exerts an essential function that cannot be provided by DNF1 or DRS2. A, overexpression of DNF1 or DRS2 with their co-chaperones failed to suppress neo1Δ lethality. neo1Δ + pRS416-NEO1 (YWY10)-expressing single copy (cen) NEO1 (pRS313-NEO1), empty vector (pRS313), single copy or multicopy (2μ) DNF1 or DRS2 (pRS313-DNF1, pRS423-DNF1, pRS315-DRS2, or pRS425-DRS2), and their co-chaperones (pRS425-LEM3 or pRS423-CDC50, respectively) were spotted onto minimal media plates (SD), and the pRS416-NEO1 plasmid was counter-selected on 5-FOA. B, neo1^ts mutant (MTY628-15B) was transformed with the same set of plasmids and assayed for growth at the indicated temperatures. C, neo1Δ + pRS416-NEO1 (YWY10)-expressing single copy NEO1 (pRS313-NEO1), empty vector (pRS313), or single copy catalytically dead neo1-D503N (pRS313-neo1-D503N) was spotted onto minimal media plates (SD), and the pRS416-NEO1 plasmid was counter-selected on 5-FOA. D, neo1-1 hypersensitivity to CW at 30 °C was not suppressed by overexpression of DRS2 or DNF1. Images are representative of three independent experiments.

FIGURE 5.

Moderate overexpression of DNF1 or DRS2 failed to suppress loss of membrane asymmetry in neo1-1 cells. Pap A (A) and duramycin (B) sensitivity of neo1-1 cells expressing single copy NEO1, DNF1, DRS2, or empty vector at 30 °C; n ≥4.

FIGURE 6.

NEO1 overexpression weakly suppresses drs2Δ but not dnf1,2,3Δ. A, overexpression of NEO1 failed to suppress dnf1,2,3Δ drs2Δ synthetic lethality. dnf1,2,3Δ drs2Δ pRS416-DNF1 (ZHY704) strains expressing single copy (cen) DRS2 (pRS313-DRS2), empty vector (pRS313), single copy or multicopy (2μ) NEO1 (pRS313-NEO1 or pRS423-NEO1, respectively) were spotted onto minimal media plates (SD), and pRS416-DRS2 was counter-selected on 5-FOA. B, overexpression of NEO1 can partially suppress drs2Δ cold sensitivity. drs2Δ strains (ZHY615M2D) expressing single copy DRS2 (pRS313-DRS2), empty vector (pRS313), or constructs driving increasing NEO1 expression (pRS313-NEO1, pRS413-P_ADH-NEO1, and pRS423-NEO1, respectively) were spotted onto SD plates and incubated at indicated temperatures. C, catalytically dead neo1-D503N failed to suppress drs2Δ cold sensitivity. D, overexpression of NEO1 exacerbated the hyperacidification of vacuoles in dnf1,2,3Δ cells (PFY3273A). Wild-type cells and dnf1,2,3Δ cells transformed with empty vector (pRS313), single copy NEO1 (pRS313-NEO1), or multicopy NEO1 (pRS423-NEO1) were stained with quinacrine, and vacuolar fluorescence was quantified using flow cytometry relative to WT cells (BY4741) (*, p < 0.01; ***, p < 0.0001, Student's t test, n = 4). Images of colony growth are representative of three independent experiments.

FIGURE 7.

Moderate overexpression of NEO1 can partially suppress loss of PE and PS asymmetry in drs2Δ cells but not in dnf1,2Δ cells. A and B, sensitivity of WT (BY4741) and dnf1,2Δ cells (PFY3275F) expressing empty vector (pRS313) or single copy NEO1 (pRS313-NEO1) to Pap A (A) and duramycin (B) at 30 °C. C and D, sensitivity of drs2Δ cells expressing single copy DRS2 (pRS313-DRS2) and empty vector (pRS313) or single copy NEO1 (pRS313-NEO1) to papuamide A (C) or duramycin (D) at 30 °C. Growth relative to WT cells in the absence of drug was plotted (*, p < 0.01; **, p < 0.001; ***, p < 0.0001 Student's t test; n ≥5). Images are representative of four independent experiments.