Figure 5.

Supplements for distinct ER-resident phospholipid biosynthetic pathways decrease accumulation of glycosylated nonfunctional Psd1

(A) Pre-cultures of the indicated strains were inoculated in YPD medium supplemented with 1% (v/v) Tergitol (−), 0.5mM lyso-phosphatidylethanolamine in 1% (v/v) Tergitol (L), or 2mM ethanolamine (E). Following overnight growth at 30°C, cell extracts were harvested and immunoblotted for Psd1 β, Psd1 α, Cho1, and Kar2; Aac2 acted as loading control (n = 5).

(B) The relative abundance of LGS mutant Psd1 ER β-α and Mito β-α was determined from yeast analyzed in (A) (mean ± SEM, n = 5).

(C) Pre-cultures of the indicated strains were inoculated in YPD medium alone or supplemented with 2mM ethanolamine (E) or 2mM choline (C). Following overnight growth at 30°C, cell extracts were harvested and immunoblotted as listed.

(D) The relative abundance of LGS mutant Psd1 ER β-α and Mito β-α was determined from yeast analyzed in (C) (mean ± SEM, n = 5). For (B) and (D), statistical differences (3 symbols p ≤ 0.001; 4 symbols p ≤ 0.0001) compared to psd2Δpsd1Δ were calculated by one-way ANOVA with Dunnett's multiple comparison test.

(E) Cellular phospholipids from the indicated strains grown in YPD alone or supplemented with choline, ethanolamine, or lyso-phosphatidylethanolamine were labeled overnight with ¹⁴C-Acetate and separated by TLC (n = 6).

(F) Cellular PE abundance was determined for each strain in each growth condition (mean ± SEM for n = 6). Statistical differences (1 symbol p ≤ 0.05; 3 symbols p ≤ 0.001) relative to growth in YPD alone were determined by one-way ANOVA with Holm-Sidak pairwise comparison (for :WT samples) or one-way ANOVA by ranks (:LGS samples).

(G) Steady-state abundance of Kar2 and Aac2 in indicated strains grown in absence or presence of lyso-PE or ethanolamine relative to psd2Δpsd1Δ:WT yeast grown in YPD alone (mean ± SEM for n = 4).

(H) Steady-state abundance of Kar2 and Aac2 in indicated strains grown in absence or presence of ethanolamine or choline relative to psd2Δpsd1Δ::WT yeast grown in YPD alone (mean ± SEM for n = 5). For (G) and (H), statistical differences (1 symbol p ≤ 0.05; 2 symbols p ≤ 0.01; 3 symbols p ≤ 0.001; 4 symbols p ≤ 0.0001) compared to WT in YPD alone (∗) or YPD alone for a given strain (#) were calculated with unpaired Student's t-test.