In Saccharomyces cerevisiae, intracellular glucose levels impact glucose transport and regulate carbon metabolism via various glucose sensors. To investigate mechanisms of glucose sensing, it is essential to know the intracellular glucose concentrations. Measuring intracellular glucose concentrations, however, is challenging when cells are grown on glucose, as glucose in the water phase around cells or stuck to the cell surface can be carried over during cell sampling and in the following attributed to intracellular glucose, resulting in an overestimation of intracellular glucose concentrations. Using lactose as a carryover marker in the growth medium, we found that glucose carryover originates from both the water phase and from sticking to the cell surface. Using a hexokinase null strain to estimate the glucose carryover from the cell surface, we found that glucose stuck on the cell surface only contributes a minor fraction of the carryover. To correct the glucose carryover, we revisited L-glucose as a carryover marker. Here, we found that L-glucose slowly enters cells. Thus, we added L-glucose to yeast cultures growing on uniformly 13C-labeled D-glucose only shortly before sampling. Using GC-MS to distinguish between the two differently labeled sugars and subtracting the carryover effect, we determined the intracellular glucose concentrations among two yeast strains with distinct kinetics of glucose transport to be at 0.89 mM in the wild-type strain and around 0.24 mM in a mutant with compromised glucose uptake. Together, our study provides insight into the origin of the glucose carryover effect and suggests that L-glucose added to the culture shortly before sampling is a possible method that yet has limitations with regard to measurement accuracy.
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