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. 2022 Oct 27;41(23):e110771. doi: 10.15252/embj.2022110771

Figure 2. PC deficiency specifically compromises the autophagic pathway.

Figure 2

  1. Representative images of GFP‐Atg8 (green) and vacuole (red, stained with FM4‐64). WT, Δopi3 and Δcho2 cells expressing GFP‐Atg8 were grown to log phase in SD‐URA medium and shifted to SD‐N for 3 h. Cells were observed by confocal microscopy before (SD) and during nitrogen starvation (SD‐N; left panel). Scale bar 1 μm. Right panel—quantification of cells with or without GFP inside vacuoles. Statistical analysis was done by ANOVA multiple comparisons test‐Sidak's, compared with WT (****P ≤ 0.0001; ns, not significant), error bars represent SEM of at least 3 independent experiments. Number of cells analyzed for each strain and condition: SD (WT (n = 446), Δcho2 (n = 272), Δopi3 (n = 272)), SD‐N (WT (n = 398), Δcho2 (n = 224), Δopi3 (n = 213)).
  2. CPY maturation assay: precursor CPY (pCPY) is transported to the vacuole and processed into a mature form (mCPY) in the vacuolar lumen. WT, Δopi3 and Δcho2 cells, as well as Δpep4 cells as negative control for CPY maturation, all expressing GFP‐Atg8, were grown to log phase in SD‐URA, and shifted to SD‐N for 3 h. Cells were harvested at the indicated time points and subjected to western blotting (Fig EV2B). Quantification of CPY maturation (mCPY/(mCPY+pCPY)) is shown for samples in log phase (SD) and starvation (3 h SD‐N). Statistical analysis was done by ANOVA multiple comparison test‐Dunnett's, compared with WT (****P ≤ 0.0001, ns, not significant), error bars represent SEM of at least 3 independent experiments.
  3. WT, Δopi3 and Δcho2 cells were grown to log phase in SD medium, stained for 30 min on ice with FM4‐64, washed 3 times with cold SD, and observed at indicated time points after wash by widefield microscopy (Fig EV2C). Quantification of cells with stained vacuoles (%), statistical analysis was done by Sidak's multiple comparison test compared with WT (ns, not significant), error bars represent SEM of two independent exp. Number of cells analyzed for each strain and time points; (WT: 0 min (n = 170), 15 min (n = 186), 30 min (n = 130), 40 min (n = 210), 50 min (n = 352)), (Δcho2: 0 min (n = 195), 15 min (n = 191), 30 min (n = 126), 40 min (n = 98), 50 min (n = 150)), (Δopi3: 0 min (n = 148), 15 min (n = 200), 30 min (n = 224), 40 min (n = 221), 50 min (n = 216)).
  4. WT, Δcho2 and Δopi3 cells were grown to log phase in SD‐URA medium and shifted to SD‐N for 3 h. Cells were harvested after 3 h of starvation, fixed, embedded and observed by EM. V‐vacuole, N‐nucleus. Scale bar 1 μm.
  5. PC levels were quantified by shotgun lipidomics in WT, Δopi3 and Δcho2 cells expressing GFP‐Atg8 during log phase in SD‐URA. Statistical analysis was done by ANOVA multiple comparisons test‐Dunnett's, compared with WT (**P ≤ 0.005, ****P ≤ 0.0001), error bars represent SEM of 3 independent experiments.