P4-ATPase control over phosphoinositide membrane asymmetry and neomycin resistance

Abstract

Neomycin, an aminoglycoside antibiotic, has robust antibacterial properties, yet its clinical utility is curtailed by its nephrotoxicity and ototoxicity. The mechanism by which the polycationic neomycin enters specific eukaryotic cell types remains poorly understood. In budding yeast, NEO1 is required for neomycin resistance and encodes a phospholipid flippase that establishes membrane asymmetry. Here, we show that mutations altering Neo1 substrate recognition cause neomycin hypersensitivity by exposing phosphatidylinositol-4-phosphate (PI4P) in the plasma membrane extracellular leaflet. Human cells also expose extracellular PI4P upon knockdown of ATP9A, a Neo1 ortholog and ATP9A expression level correlates to neomycin sensitivity. In yeast, the extracellular PI4P is initially produced in the cytosolic leaflet of the plasma membrane and then delivered by Osh6-dependent nonvesicular transport to the endoplasmic reticulum (ER). Here, a portion of PI4P escapes degradation by the Sac1 phosphatase by entering the ER lumenal leaflet. COPII vesicles transport lumenal PI4P to the Golgi where Neo1 flips this substrate back to the cytosolic leaflet. Cryo-EM reveals that PI4P binds Neo1 within the substrate translocation pathway. Loss of Neo1 activity in the Golgi allows secretion of extracellular PI4P, which serves as a neomycin receptor and facilitates its endocytic uptake. These findings unveil novel mechanisms of aminoglycoside sensitivity and phosphoinositide homeostasis, with important implications for signaling by extracellular phosphoinositides.

Figure 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 colors (PDB ID 9BS1, this study). The dashed box is the substrate translocation pathway enlarged in (b). b, Transmembrane (TM) segments TM1, TM2, and TM4 form the substrate transport pathway of Neo1, and 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) 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. Two-way ANOVA was performed to test the variance and comparisons with WT Neo1 were made with Tukey’s post hoc analysis (n = 3, ±standard deviation (SD). ****p < 0.000,ns represents non-significant .e, f, Neomycin sensitivity of S. cerevisiae flippase mutants (neo1–1, drs2Δ, dnf1,2Δ, dnf3Δ) and strains deficient for Neo1 regulators (dop1–1, mon2Δ). The data represent growth relative to WT cells in absence of the drug. Two-way ANOVA was performed to test the variance and comparisons with WT Neo1 were made with Tukey’s post hoc analysis (n = 3, ±standard deviation (SD). ns represents non-significant.

Figure 2:. neo1 mutants cells expose PI4P in the plasma membrane extracellular leaflet.

a, Phosphoinositide synthesis in the cytoplasmic leaflet of the plasma membrane and the use of recombinantly purified GFP-SidC_P4C and GFP-PH_PLC to probe for external PI4P and PI(4,5)P₂, respectively. b, Inactivation of neo1–1 at the nonpermissive temperature (38 °C) causes PI4P exposure. Fluorescent images were inverted to black signal on white background for clarity. The right panel is the fluorescence signal intensity of the GFP-probe and left panel shows the DIC panel to display yeast cells. Scale bar = 2 μm c, Quantification of GFP-SidC_P4C and GFP-PLC_PH binding to the cell surface of cells expressing Neo1 variants. One-way ANOVA was performed to test the variance and comparisons with WT Neo1 were made with Tukey’s post hoc analysis (n = 20, ±standard deviation (SD). P<0.0001 is ****. d, The GFP-SidC_P4C(R652Q) PI4P binding defective mutant fails to bind neo1–1 cells. One-way ANOVA was performed to test the variance and comparisons with GFP-SidC_P4C were made with Dunnett’s multiple comparisons test (n = 20, ±standard deviation (SD). P<0.0001 is **** . e, stt4–4 suppresses the neomycin sensitivity of neo1S221L or neo1S452Q at 33°C. g, pik1–83 fails to suppress the neomycin sensitivity of neo1S221L or neo1S452Q at 34°C. The data represents growth relative to cells in the absence of the drug. Two-way ANOVA was performed to test the variance and comparisons were made with Tukey’s post hoc analysis (n = 3, ±standard deviation (SD). **p < 0.01, ns represents non-significant.

Figure 3:. ATP9A knockdown cells are neomycin sensitive and expose PI4P in the plasma membrane extracellular leaflet.

a, Neomycin sensitivity of wild type HeLa cells and ATP9A knockdown cells. The data represent % viable cells relative to the viable cells in absence of the neomycin. One-way ANOVA was performed to test the variance and comparisons with control cells were made with Dunnett’s multiple comparisons test (n = 3, ±standard deviation (SD). **p < 0.01. b, ATP9A knockdown HeLa cells expose PI4P in extracellular leaflets. Hoechst dye was used as nuclear stain. Scale bar = 10 μm c, Quantification of GFP-SidC_P4C and GFP-PLC_PH binding to the cell surface of ATP9A knockdown cells. One-way ANOVA was performed to test the variance and comparisons with control siRNA treated cells were made with Tukey’s post hoc analysis (n = 20, ±standard deviation (SD). P<0.0001 is ****. d, Neomycin sensitivity of wild type HEK293 cells and ATP9A knockdown cells. The data represent % viable cells relative to the viable cells in absence of the neomycin. One-way ANOVA was performed to test the variance and comparisons with WT cells were made with Dunnett’s multiple comparisons test (n = 3, ±standard deviation (SD). **p < 0.01. e, ATP9A knockdown HEK293 cells expose PI4P in extracellular leaflets. Scale bar = 10 μm f, Quantification of GFP-SidC_P4C and GFP-PLC_PH binding to the cell surface of ATP9A knockdown cells. One-way ANOVA was performed to test the variance and comparisons with control siRNA treated cells were made with Tukey’s post hoc analysis (n = 20, ±standard deviation (SD). P<0.0001 is ****.

Figure 4:. Cryo-EM structure of Neo1-PI4P and comparison of PI4P binding in Neo1 and Drs2.

a, ATP hydrolysis by purified WT Neo1 with or without 0.02 mM PI4P or 0.1 mM PE (substrate) or both PI4P+PE. One-way ANOVA was performed to test the variance and comparisons with no substrate or PE were made with Dunnett’s multiple comparisons test (n = 3, ±standard deviation (SD****p < 0.0001, ns-not significant). b, ATP hydrolysis activity of WT Neo1 and Neo1 mutants at increasing PI4P concentration. Data points represent the mean ± SD in triplicate. c, Global views of Neo1-PI4P in E2P state with the 4 key residues in substrate transport pathway shown in sticks and PI4P shown in teal spheres. d, Middle: the structure of Neo1-PI4P is superimposed with Drs2-PI4P (gray, PDB ID 6ROJ) and shown in cartoon (RMSD = 4.0 Å). Note the different binding site of PI4P as an activator in Drs2 (gray spheres) and as a substrate in Neo1 (teal spheres). The PI4P binding site residues in Drs2 are highlighted in red box. Left: enlarged view of the PI4P binding site in Drs2. Right: enlarged view of PI4P binding site in Neo1. PI4P and interacting residues are shown in sticks. Hydrogen bonds between the PI4P headgroup and flippase residues are labeled by dashed black lines. The EM density of PI4P in Neo1 is superimposed on the atomic model and shown as a transparent surface. e, Mutation in PI4P binding residue causes neomycin sensitivity. HE: H472 E475. The data represents growth relative to WT cells in absence of the drug. Two-way ANOVA was performed to test the variance and comparisons with WT Neo1 were made with Tukey’s post hoc analysis (n = 3, ±standard deviation (SD). ****p < 0.0001.

Figure 5:. Non-vesicular and vesicular transport is required for PI4P exposure.

a, A model for the trafficking of PI4P and the role of Neo1 in maintaining PI4P membrane asymmetry. PM: Plasma membrane, ER: Endoplasmic Reticulum, PS: Phosphatidylserine b, sac1Δ cells are neomycin sensitive. c, Overexpression of wild-type NEO1 (2μ NEO1) suppresses sac1Δ neomycin sensitivity. d, sac1Δ cells expose extracellular PI4P and overexpression of NEO1 suppresses PI4P exposure. One-way ANOVA was performed to test the variance, and comparisons with were made with Tukey’s multiple comparisons test (n = 20, ±standard deviation (SD). P<0.001 is ***, P<0.01 is **. e, Deletion of OSH6 (osh6Δ) reduces neomycin sensitivity of neo1 mutants. f, Reducing the rate of vesicular transport with sec23–1, sec12–4, sec18–1, or sec14–1 suppresses neomycin sensitivity of neo1–1 at 26 °C. All the comparisons were calculated via a One-Way ANOVA followed by Tukey’s multiple comparisons test. Colors represent comparison to WT, pairwise comparisons shown with brackets and asterisk. The data represent growth relative to WT cells in absence of the drug (n = 3, ±standard deviation (SD). **p < 0.01, ***p < 0.001, ****p < 0.0001. For b, c, e, and f, cells were grown at 26°C.

Figure 6:. Neomycin binds to exposed PI4P and is endocytosed.

a, Use of neomycin-Texas Red (Neo-TR) to probe PI4P exposed on the extracellular leaflet of the membrane. b, Neo-TR binds neomycin-sensitive neo1 mutants. Scale bar = 2 μm. c, Quantification of mean fluorescence intensity of cells stained with Neo-TR. One-way ANOVA was performed to test the variance and comparisons with WT Neo1 were made with Dunnett’s multiple comparisons test (n = 20, ±standard deviation (SD) ****p < 0.0001). d, LatA blocks Neo-TR internalization. e, Quantification of Neo-TR fluorescence intensity at the PM. For all quantification, data from ~20 cells from three independent experiments were obtained and analyzed. One-way ANOVA was performed to test the variance and comparisons with different time points were made with Tukey’s multiple comparisons test (n = 20, ±standard deviation (SD) ****p < 0.0001) Scale bar = 2 μm. f, Neo-TR binds to the ATP9A knockdown HeLa cells. Hoechst dye was used as a nuclear stain. g, Quantification of total Neo-TR fluorescence intensity of cells (n=20) Scale Bar = 10 μm. h, Neomycin-sensitive HEK293 and ATP9A knockdown cells bind Neo-TR. Scale Bar = 10 μm. i, Quantification of total Neo-TR fluorescence intensity (n=20). One-way ANOVA was performed to test the variance and comparisons with control cells were made with Dunnett’s multiple comparisons test (n = 20, ±standard deviation (SD) ****p < 0.0001).