2.3. l-lactate Uptake Assay

A previously established protocol was used [7]. Briefly, yeast cells were grown overnight to an OD₆₀₀ of 1.0 ± 0.1, harvested by centrifugation at 4000× g, 4 °C for 5 min, washed once with ice-cold water, and resuspended to an OD₆₀₀ of 50 ± 5 in the buffer of interest: 50 mM HEPES/Tris (pH 6.8), 50 mM MES/Tris (pH 5.8), 50 mM citric acid/Tris (pH 4.8 and pH 3.8). For radiolabeled l-lactate uptake, 20 µL of 5 mM l-lactate spiked with 0.04 µCi of ¹⁴C-labeled l-lactate (Hartmann Analytics, Braunschweig, Germany) were added to 80 µL of cell suspension in a 1.5 mL tube to yield a final concentration of 1 mM l-lactate. The reaction was stopped by adding 1 mL of ice-cold water, transferring the suspension to a GF/C glass microfiber filter membrane (Whatman/Sigma-Aldrich, Darmstadt, Germany) and removing the supernatant by vacuum filtration. The filters were washed with 7 mL of ice-cold water and transferred to scintillation vials containing 3 mL of scintillation cocktail (Quicksafe A; Zinsser Analytic) for counting (Packard TriCarb liquid scintillation counter, Perkin Elmer Inc., Waltham, MA, USA). Single-exponential (uptake data) and sigmoidal fittings (pH-dependent capacity) were calculated after subtraction of the background from non-expressing yeast cells (SigmaPlot version 11.0). Measurements were done in triplicate (independent yeast cultures) with three technical replicates each, and error bars denote the standard error of the mean (SEM).