Figure 2.

Re-localized Psd1 constructs are stable and functional

(A) Schematic of chimeric constructs. MTS (mitochondrial targeting signal) and TM (transmembrane domain) of residues are indicated. Psd subunits β, α, and LGS motif are shown. All constructs have a 3XFLAG tag at the C-terminus.

(B) The indicated strains were pre-cultured at 30°C in YPD, and after isolation of whole-cell extracts, the α and β subunits of Psd1 were analyzed by immunoblotting. Pic1 served as a loading control (n = 3).

(C) The indicated strains pre-cultured at 30°C in YPD were spotted onto SCD with (+) or without (−) 2 mM ethanolamine and incubated at 30°C for 3 days (n = 3).

(D) Mitochondrial phospholipids from the indicated strains were labeled overnight in rich lactate medium spiked with ³²Pi and separated by TLC. The migration of phosphatidylcholine (PC), phosphatidylinositol (PI), PS, phosphatidylglycerol (PG), PE, phosphatidic acid (PA), and CL is indicated (n = 6).

(E) The relative abundance of PE was determined for each strain (mean ± SEM for n = 6). Statistical differences (2 symbols p ≤ 0.01; 3 symbols p ≤ 0.001 compared to ::WT (∗) or psd2Δpsd1Δ (#)) were calculated by unpaired Student's t-test (for both ::Yme1 comparisons) or Mann-Whitney rank-sum test (all the rest).

(F) Serial dilutions of the indicated strains were spotted onto SCD with or without 2 mM ethanolamine, synthetic complete lactate (SC Lactate), or synthetic complete ethanol-glycerol (SCEG) plates and incubated at 30°C or 37°C for the indicated duration (n = 3).