Yeast complementation assays and growth curves

A pRS426-MET25 vector was used as the backbone for a plasmid containing the entire cDNA sequence of TgACS1. The plasmid was synthesized (GeneCust) by cloning the predicted transcribed sequence from TgACS1 (which was obtained from Toxodb.org, and codon optimized for heterologous expression in yeasts) using EcoRI and KpnI restriction sites. All strains were transfected either with this plasmid of the pRS426-MET25 vector lacking TgACS1 as a control.

The isogenic S. cerevisiae strains used in this study (i.e. WT: YB332: MATa ura3 leu2 his3A200 ade2 lys2-801, and faa1∆faa4∆: YB525: MATa ura3 leu2 his3D200 ade2 lys2–801 faa1∆::HIS3; faa4∆::LYS2) were kindly provided by Dr. Juliette Jouhet [Commissariat aux Energies Atomiques et aux Energies Alternatives (CEA), Grenoble, France] and have been described earlier (50, 51). After transfection, all the strain of the present study were grown on synthetic medium containing 6.7 g/L of YNB (MP Biomedicals), 0.77 g/L of CSM-URA (i.e. Complete Supplement Mixture-Uracil, MP Biomedicals) and g/L of glucose, as described. In complementation experiments, the media were supplemented with 22.5 mM cerulenin (Sigma-Aldrich), 100 mM FFA, and 0.2% (vol/vol) final concentration of Tween 20 (Sigma-Aldrich). The different FFAs were dissolved in 100% Tween 20 at a concentration of 50 mM. Cerulenin was dissolved in acetone at 45 mM. As controls, media without FFAs and/or cerulenin were also supplemented with 0.2% (vol/vol) Tween 20 and/or acetone.

The isogenic S. cerevisiae strains used in this study (i.e. WT (YB332: MATa ura3 leu2 his3A200 ade2 lys2-801) and faa1∆faa4∆ (YB525: MATa ura3 leu2 his3D200 ade2 lys2–801 faa1∆::HIS3; faa4∆::LYS2) were kindly provided by Dr. Juliette Jouhet (Commissariat aux Energies Atomiques et aux Energies Alternatives (CEA), Grenoble, France) and have been described earlier (52, 53). After transfection, all the strains described in the present work were grown on synthetic medium containing 6.7 g/L of YNB (MP Biomedicals), 0.77 g/L of CSM-URA (MP Biomedicals) and g/L of glucose, as described. In complementation experiments, the media were supplemented with 22.5 mM cerulenin (Sigma-Aldrich), 100 mM FFA, and 0.2% (vol/vol) final concentration of Tween 20 (Sigma-Aldrich). The different FFAs were dissolved in 100% Tween 20 at a concentration of 50 mM. Cerulenin was dissolved in acetone at 45 mM. As controls, media without FFAs and/or cerulenin were also supplemented with 0.2% (vol/vol) Tween 20 and/or acetone.

Three clones per transformation were used for complementation tests. For each experiment, one colony was inoculated in 6 mL of CSM-URA + glucose (2%) and grown overnight at 30°C at 180 rpm. Growth assays were performed in 96-well microplates (Thermo Scientific Nunc MicroWell 96-Well, Nunclon Delta-treated, Flat-Bottom Microplate) filled with 200 mL of culture in glucose medium supplemented or not by FA or cerulenin and set at an OD600: optical Density (OD₆₀₀= 0.1). The microplate was incubated at 30°C in an Infinite M2000 PRO microplate reader (Tecan) set for plate orbital shake for 10 s and absorbance measurements (OD₆₀₀) every 20 min, with four measurements per well. OD₆₀₀ readings from experimental wells minus the original OD₆₀₀ values resulted in the ∆OD₆₀₀ values shown in Fig. S5.

We transfected the pRS426-MET25-TgACS1 plasmid into a yeast strain lacking a peroxisomal-CoA synthetase, ∆PCS60 (17, 35). These experiments were also performed in Tween 20, with and without 12 µg/mL doxorubicin, a cancer drug inducing lipid peroxidation and peroxisome proliferation in yeast cells (23, 60). OD₆₀₀ was calculated as described above.