Figure 3.
Elevating cellular PA levels leads to the recruitment of Chm7 to NE/ER membranes independently of Heh1. (A) The recruitment of Chm7 to NE/ER membranes in the context of elevated cellular PA is dependent on the hydrophobic stretch and not Heh1. Deconvolved fluorescence micrographs of Chm7-GFP (top left panels) or chm7-(W3AV1A)-GFP (top right panels) with vacuole membranes stained with FM4-64 and merged images. GFP fluorescence is inverted. Arrowheads point to Chm7-GFP at NE/ER membranes as confirmed in Fig. S2 A. Scale bar, 5 µm. (B) Plot of the percentage of cells from experiments as in A showing the relative distribution of Chm7-GFP or chm7-W3AV1A-GFP between NE/ER and vacuole membranes in the indicated genetic backgrounds. The mean and SD from three independent experiments of >50 cells is shown. P values were calculated from a two-way ANOVA with Dunnett’s post hoc test where ns (not significant) is P > 0.05; ****, P < 0.0001. (C) Cartoon of the PA metabolism pathway, which can be modulated to control both lipid storage and membrane growth as shown. Nem1/Spo7 dephosphorylate and activate Pah1, which in turn dephosphorylates PA to produce DAG. Thus, deletion of PAH1 and NEM1 elevates cellular PA levels. Dgk1 phosphorylates DAG to produce PA; therefore, there is less PA in dgk1Δ cells. TAG is triacylglycerol lipids stored in lipid droplets. Brown lipids represent DAG, and red lipids represent PA. (D)CHM7 is required to maintain fitness of pah1Δ cells. 10-fold serial dilutions of the indicated strains grown at 23°C for at least 48 h before imaging. (E) Nuclear-cytoplasmic compartmentalization is perturbed in pah1Δ and chm7Δpah1Δ cells. Deconvolved fluorescence micrographs of NLS-GFP, Nup170-mCherry, and merged images in the indicated strains cultured at 23°C. Scale bar, 5 µm. (F) Violin plot of the distribution of the mean nuclear (nuc) to cytosolic (cyt) fluorescence intensity of individual cells with median (solid line) quartiles (dotted line) from three independent experiments (50 cells/strain/experiment) of an NLS-GFP reporter from cells of the indicated genotype. The width of each plot represents the relative frequency distribution of cells. P values were calculated from a one-way ANOVA with Tukey’s post hoc test where ns is P > 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Elevating cellular PA levels leads to the recruitment of Chm7 to NE/ER membranes independently of Heh1. (A) The recruitment of Chm7 to NE/ER membranes in the context of elevated cellular PA is dependent on the hydrophobic stretch and not Heh1. Deconvolved fluorescence micrographs of Chm7-GFP (top left panels) or chm7-(W3AV1A)-GFP (top right panels) with vacuole membranes stained with FM4-64 and merged images. GFP fluorescence is inverted. Arrowheads point to Chm7-GFP at NE/ER membranes as confirmed in Fig. S2 A. Scale bar, 5 µm. (B) Plot of the percentage of cells from experiments as in A showing the relative distribution of Chm7-GFP or chm7-W3AV1A-GFP between NE/ER and vacuole membranes in the indicated genetic backgrounds. The mean and SD from three independent experiments of >50 cells is shown. P values were calculated from a two-way ANOVA with Dunnett’s post hoc test where ns (not significant) is P > 0.05; ****, P < 0.0001. (C) Cartoon of the PA metabolism pathway, which can be modulated to control both lipid storage and membrane growth as shown. Nem1/Spo7 dephosphorylate and activate Pah1, which in turn dephosphorylates PA to produce DAG. Thus, deletion of PAH1 and NEM1 elevates cellular PA levels. Dgk1 phosphorylates DAG to produce PA; therefore, there is less PA in dgk1Δ cells. TAG is triacylglycerol lipids stored in lipid droplets. Brown lipids represent DAG, and red lipids represent PA. (D)CHM7 is required to maintain fitness of pah1Δ cells. 10-fold serial dilutions of the indicated strains grown at 23°C for at least 48 h before imaging. (E) Nuclear-cytoplasmic compartmentalization is perturbed in pah1Δ and chm7Δpah1Δ cells. Deconvolved fluorescence micrographs of NLS-GFP, Nup170-mCherry, and merged images in the indicated strains cultured at 23°C. Scale bar, 5 µm. (F) Violin plot of the distribution of the mean nuclear (nuc) to cytosolic (cyt) fluorescence intensity of individual cells with median (solid line) quartiles (dotted line) from three independent experiments (50 cells/strain/experiment) of an NLS-GFP reporter from cells of the indicated genotype. The width of each plot represents the relative frequency distribution of cells. P values were calculated from a one-way ANOVA with Tukey’s post hoc test where ns is P > 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal