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

Area of Science:

• Biochemistry
• Molecular Biology
• Yeast Genetics

Background:

• Traditional glucose uptake assays utilize isotope-labeled sugars, posing risks of radioactive waste and experimenter exposure.
• Characterizing sugar transporter kinetics is crucial for understanding cellular metabolism and developing therapeutic strategies.

Purpose of the Study:

• To develop an isotope-free, fluorometric method for determining the kinetic parameters of sugar transporters.
• To establish a real-time assay for measuring sugar uptake rates in Saccharomyces cerevisiae.

Main Methods:

• Utilized a pH-sensitive green fluorescent protein (pHluorin) as a genetically encoded biosensor.
• Measured the real-time cytosolic pH decrease in starved yeast cells following a glucose pulse.
• Employed the hexose transporter-deficient (hxt0) yeast strain EBY.VW4000 for transporter characterization.

Main Results:

• Demonstrated that the rate of cytosolic pH decrease is directly dependent on the rate of glucose uptake.
• Successfully employed the pHluorin biosensor to quantify sugar transporter velocity in real time.
• Validated the method's utility in a well-established yeast expression system for kinetic analysis.

Conclusions:

• The developed fluorometric assay provides a safe and efficient alternative to isotope-based methods for sugar transporter characterization.
• This approach enables accurate determination of kinetic parameters for hexose transporters.
• The method is applicable to a wide range of sugar transporters within a versatile yeast system.