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A label-free real-time method for measuring glucose uptake kinetics in yeast.

ABSTRACT: Glucose uptake assays commonly rely on the isotope-labeled sugar, which is associated with radioactive waste and exposure of the experimenter to radiation. Here, we show that the rapid decrease of the cytosolic pH after a glucose pulse to starved Saccharomyces cerevisiae cells is dependent on the rate of sugar uptake and can be used to determine the kinetic parameters of sugar transporters. The pH-sensitive green fluorescent protein variant pHluorin is employed as a genetically encoded biosensor to measure the rate of acidification as a proxy of transport velocity in real time. The measurements are performed in the hexose transporter-deficient (hxt0) strain EBY.VW4000 that has been previously used to characterize a plethora of sugar transporters from various organisms. Therefore, this method provides an isotope-free, fluorometric approach for kinetic characterization of hexose transporters in a well-established yeast expression system.

SUBMITTER: Schmidl S

PROVIDER: S-EPMC7811510 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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A label-free real-time method for measuring glucose uptake kinetics in yeast.

Schmidl Sina S Iancu Cristina V CV Reifenrath Mara M Choe Jun-Yong JY Oreb Mislav M

FEMS yeast research 20210101 1

Glucose uptake assays commonly rely on the isotope-labeled sugar, which is associated with radioactive waste and exposure of the experimenter to radiation. Here, we show that the rapid decrease of the cytosolic pH after a glucose pulse to starved Saccharomyces cerevisiae cells is dependent on the rate of sugar uptake and can be used to determine the kinetic parameters of sugar transporters. The pH-sensitive green fluorescent protein variant pHluorin is employed as a genetically encoded biosensor ...[more]

PMID: 33338229

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