1. Glucose pulse experiment

pHluorin calibration was performed as described by Smits et al. [8] with some modifications. CEN.PK2‐1C (MATa; his3Δ1; leu2‐3112; ura3‐52; trp1‐289; MAL2‐8c; SUC2) was the strain used in the titration. Cells from a single colony were cultivated in bath conditions at 30 °C and 200 rmp in 50 mL of minimal medium containing 6.8 g·L⁻¹ Yeast Nitrogen Base (Sigma‐Aldrich, Stl. Louis, MO, USA), 20 mg·L⁻¹ L‐histidine (Sigma‐Aldrich), 60 mg·L⁻¹ L‐leucine (SERVA Electrophoresis GmbH, Heidelberg, Germany), 20 mg·L⁻¹ L‐tryptophan (Sigma‐Aldrich), 1% v/v ethanol (VWR International, Radnor, PA, USA) and 1% v/v glycerol (Sigma‐Aldrich). The OD_600nm of the culture was adjusted to 1.5 with YNB buffer (containing 6.8 g·L⁻¹ Yeast Nitrogen Base) to a final volume of 40 mL. Cells were washed in 10 mL of the same buffer, followed by 1 min centrifugation at 4000 rpm at room temperature. Cells were collected and resuspend in PBS 10 mm containing 100 µg·mL⁻¹ of digitonin (150 µL of the solution was used per OD_600nm unit), followed by an incubation of 10 min at 30 °C to allow permeabilization. After incubation cells were collected by centrifugation (5 min at 4000 rpm and 4 °C) and resuspended in PBS 10 mm to an OD_600nm = 1.5. The cell suspension was split into tubes (3 mL) and centrifuged twice to collect the PBS (5 min, 4000 rpm followed by 1 min, 4000 rpm at 4 °C). Cells were washed in 1 mL of citric acid/Na₂HPO₄ in a range of pH 4.86–8.41 (1 min, 4000 rpm at 4 °C) and resuspended in the same pH buffer. For the titration, cells were diluted to an OD_600nm = 0.55 in the final pH buffer, and fluorescence was measured in a 96 well black polystyrene clear‐bottom microplate (Greiner Bio‐One International GmbH, Kremsmünster, Austria). 100 µL of the cell suspension was added per well. Fluorescence excitation was provided at 390 and 470 nm (10 nm bandwidth), and emission was measured at 510 nm using a FLUOstar Omega microplate reader (BMG LABTECH GmbH). The wild‐type strain was used to correct for background fluorescence. The ratio R390/470 was calculated and plotted against the corresponding pH.

CEN.PK2‐1C cells from a single colony were cultivated in bath conditions at 30 °C and 200 rmp in YNB medium containing 6.8 g·L⁻¹ Yeast Nitrogen Base, 20 mg·L⁻¹ L‐histidine, 60 mg·L⁻¹ L‐leucine, 20 mg·L⁻¹ L‐tryptophan and 10.2 g·L⁻¹ potassium hydrogen phthalate (VWR International) and supplemented with 111 mm galactose (Sigma‐Aldrich). The pH of the media was adjusted to 5 with KOH (Sigma‐Aldrich). Cells were diluted and collected in mid‐log phase for the pulse experiment. The glucose pulse was performed by the addition of 111 mm glucose (final concentration; Boom BV, Meppel, Netherlands) to the galactose pregrown cells. Excitation at 390 and 470 nm (10 nm bandwidth) was used and emission was measured at 510 nm in a FLUOstar Omega microplate reader. The wild‐type strain was used to correct for background fluorescence. The ratio R390/470 was calculated and converted to pH based on the In situ pHluorin calibration above described.