Geneticin

Synthetic media used in this study contains 6.7 g/l yeast nitrogen base with ammonium sulfate (Conda Pronadisa #1545) and either 2% glucose, 2% ethanol, 3% glycerol, or 0.2% oleic acid (Sigma) +0.1% Tween 80, with complete amino acid mix (oMM composition, Hanscho et al, 2012 (link)), unless written otherwise; when Hygromycin or Geneticin antibiotics were used, media contains 0.17 g/l yeast nitrogen base without Ammonium Sulfate (Conda Pronadisa #1553) and 1 g/l of monosodium glutamic acid (Sigma‐Aldrich #G1626) instead of yeast nitrogen base with ammonium sulfate. When mentioned, 500 mg/l Hygromycin B (Formedium), 500 mg/l Geneticin (G418; Formedium), and 200 mg/l Nourseothricin (WERNER BioAgents “ClonNat”) were used.

Yeast were grown in rich yeast extract peptone dextrose (YPD) medium (2% glucose, 2% peptone, 1% yeast extract) or synthetic complete (SC) minimal medium (2% glucose, yeast nitrogen base supplemented with appropriate amino acid and base dropout mixtures; Formedium, Norfolk, UK) for maintaining of plasmids. Cells were routinely grown overnight to early/mid-log phase (OD₆₀₀≤1.0) prior to experimental procedures, unless otherwise stated. To minimise nutritional challenges prior to starvation experiments, very low density cultures (OD₆₀₀=0.1) were adhered to concavalin A-treated coverslips for time-lapse microscopy. For glucose-starvation experiments, rich and minimal media were supplemented with 2% raffinose instead of glucose, but were otherwise identical. Geneticin (Formedium), used at a concentration of 250 μg/ml in rich media, and methotrexate (Alfa Aesar), used at a working concentration of 20 mM, were prepared in SC minimal medium as described previously (MacDonald and Piper, 2015 (link)). Expression of proteins from the CUP1 promoter was achieved by addition of 50–100 µM CuCl₂ to the medium for at least 1 h prior to downstream analysis.