Spt23 and Mga2 sense changes in the membrane lipid environment induced by edelfosine. (A) Schematic illustrating the off-on states of the membrane packing sensors Mga2 and Spt23 and the release of the transcription factor N-end in response to changes in membrane environment (created with BioRender.com, adapted from Ballweg et al., 2020). (B and C) Representative images of ^GFPSpt23 expressed from a centromeric plasmid under the constitutive GPD promoter in spt23Δ cells (B) or ^GFPMga2 expressed from a centromeric plasmid under the constitutive GPD promoter in mga2Δ cells (C) were imaged after growth in SD-Leu+Ade and 30 min in the presence of edelfosine, the vehicle, or 0.1% methyl methane sulfonate (MMS). Quantification of cells displaying nucleoplasmic signal is shown beside the microscopy images. Nuclear localization was confirmed using the ER marker ^DsRedHDEL (Fig. S5). Circles represent the percentage of cells displaying nucleoplasmic signal in each experiment (Chi-square test gave a P value <0.0001 for each experiment [n> 65 cells per treatment]). The bar represents the mean of the three experiments ± SD. ** indicates a P value <0.01 and **** indicates a P value <0.0001 as determined by standard one-way ANOVA with Tukey’s multiple comparisons test (N = 3). Schematics illustrate what was classified as nucleoplasmic signal for B and C. (D and E) Western blot of lysates from cells (W303) expressing ^GFPSpt23 (D) or ^GFPMga2 (E) from centromeric plasmids under the constitutive GPD promoter treated with edelfosine for the indicated times (D) or for 60 min (E) Bottom panels show protein loading visualized by TCE. P indicates precursor species (148.2 kD for Spt23, 153.9 kD for Mga2), while N indicates nucleoplasmic species (∼90 kD + EGFP). For all panels: Ctrl = vehicle; EDLF = edelfosine; scale bars represent 2 µm. Source data are available for this figure: SourceData F6.