Yeast synthetic drop out medium

For transformations S. boulardii was cultured in yeast-peptone-dextrose (YPD) medium (10 g/L yeast extract, 20 g/L casein peptone and 20 g/L glucose (Sigma Aldrich)). Transformations were streaked on synthetic complete medium without uracil plates (SC U-; 6.7 g/L yeast nitrogen base without amino acids, 1.92 g/L Yeast Synthetic Drop-out Medium without uracil, 10 g/L agar and 20 g/L glucose (Sigma Aldrich)) or SC U- supplemented with geneticin (1.7 g/L yeast nitrogen base without amino acids and ammonium sulphate, 1 g/L monosodium glutamate, 1.92 g/L Yeast Synthetic Drop-out Medium without uracil, 200 mg/L geneticin (G418), 10 g/L agar and 20 g/L glucose (Sigma Aldrich)). In vitro characterisations were cultured in synthetic complete (6.7 g/L yeast nitrogen base without amino acids, 1.6 g/L Yeast Synthetic Drop-out Medium without leucine and 76 mg/L leucine) with one of the following carbon sources as appropriate 20 g/L glucose, 20 g/L fructose, 19 g/L sucrose, 16 g/L inulin or 32.4 g/L sodium acetate (Sigma Aldrich). E. coli was cultured in LB supplemented with 100 mg/L ampicillin sodium salt (Sigma Aldrich). All cultures were incubated at 37 °C and 250 rpm.

S. cerevisiae acquires choline via HNM1. The S. cerevisiae Hnm1 mutants were acquired from Dharmacons. WT and Hnm1 mutant yeasts were grown in Yeast extract-Peptone-Dextrose (YPD) medium at 30 °C overnight before transformation. The pESC-HIS-hMfsd7c plasmid was transformed into WT and Hnm1 mutant yeasts following the previously described protocol.^{43 (link)} After heat shock at 42 °C for 30 min and recovery in YPD for 2 h, the yeasts were spread on 2% agar plates prepared in yeast nitrogen base (YNB) medium (Sigma-Aldrich) supplemented with 2% glucose and yeast synthetic drop-out medium supplements without histidine (Sigma-Aldrich). Positive colonies were picked up randomly and further confirmed by transport assay and western blot.

The glutathione redox state has been tested on 10⁵ cells·mL ⁻¹ grown in SC (0.67% w/v yeast nitrogen base with aminoacids, 2% w/v dextrose; Sigma-Aldrich, St. Louis, MO, USA) medium, treated for 24 h with 50 mg·L⁻¹ of CdS QDs; the cellular extract and glutathione levels were determined as described in Pasquali et al. (2017) [20 (link)]. To determine the mitochondrial morphotype wild type strain, tolerant and sensitive mutants were transformed with the plasmid pYX-142 mtRFP as described by Pasquali et al. (2017) [20 (link)]. Each strain was grown in the selective SC-LEU medium (0.67% w/v yeast nitrogen base w/o aminoacids, 0.16% w/v yeast synthetic drop-out medium supplement w/o leucine, 2% w/v dextrose; Sigma-Aldrich, St. Louis, MO, USA) containing 50 mg·L⁻¹ of CdS QDs, for a 24 h treatment. Microscope images were taken using an Axio Imager 2 (Zeiss, Oberkochen, Germany). The experiments were performed in triplicate. Additional methods related to the growth characteristics data of the wild type strain, and the tolerant and sensitive mutants are reported in the Supplementary Materials.