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Quantifying intracellular glucose levels when yeast is grown in glucose media

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 concentration...

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Autores principales: Li, Xiang, Heinemann, Matthias
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10564791/
https://www.ncbi.nlm.nih.gov/pubmed/37816759
http://dx.doi.org/10.1038/s41598-023-43602-z
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author Li, Xiang
Heinemann, Matthias
author_facet Li, Xiang
Heinemann, Matthias
author_sort Li, Xiang
collection PubMed
description 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 (13)C-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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spelling pubmed-105647912023-10-12 Quantifying intracellular glucose levels when yeast is grown in glucose media Li, Xiang Heinemann, Matthias Sci Rep Article 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 (13)C-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. Nature Publishing Group UK 2023-10-10 /pmc/articles/PMC10564791/ /pubmed/37816759 http://dx.doi.org/10.1038/s41598-023-43602-z Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Li, Xiang
Heinemann, Matthias
Quantifying intracellular glucose levels when yeast is grown in glucose media
title Quantifying intracellular glucose levels when yeast is grown in glucose media
title_full Quantifying intracellular glucose levels when yeast is grown in glucose media
title_fullStr Quantifying intracellular glucose levels when yeast is grown in glucose media
title_full_unstemmed Quantifying intracellular glucose levels when yeast is grown in glucose media
title_short Quantifying intracellular glucose levels when yeast is grown in glucose media
title_sort quantifying intracellular glucose levels when yeast is grown in glucose media
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10564791/
https://www.ncbi.nlm.nih.gov/pubmed/37816759
http://dx.doi.org/10.1038/s41598-023-43602-z
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