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Treatment with surfactants enables quantification of translational activity by O-propargyl-puromycin labelling in yeast

BACKGROUND: Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in yeast. Recently, O-propargyl-puromycin (OPP) labelling has been established for mammalian cells, but unmodified yeasts a...

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Autores principales: Staudacher, Jennifer, Rebnegger, Corinna, Gasser, Brigitte
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056590/
https://www.ncbi.nlm.nih.gov/pubmed/33879049
http://dx.doi.org/10.1186/s12866-021-02185-3
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author Staudacher, Jennifer
Rebnegger, Corinna
Gasser, Brigitte
author_facet Staudacher, Jennifer
Rebnegger, Corinna
Gasser, Brigitte
author_sort Staudacher, Jennifer
collection PubMed
description BACKGROUND: Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in yeast. Recently, O-propargyl-puromycin (OPP) labelling has been established for mammalian cells, but unmodified yeasts are unsusceptible to puromycin. RESULTS: We could increase susceptibility by using a Komagataella phaffii strain with an impaired ergosterol pathway (erg6Δ), but translation measurements are restricted to this strain background, which displayed growth deficits. Using surfactants, specifically Imipramine, instead, proved to be more advantageous and circumvents previous restrictions. Imipramine-supplemented OPP-labelling with subsequent flow cytometry analysis, enabled us to distinguish actively translating cells from negative controls, and to clearly quantify differences in translation activities in different strains and growth conditions. Specifically, we investigated K. phaffii at different growth rates, verified that methanol feeding alters translation activity, and analysed global translation in strains with genetically modified stress response pathways. CONCLUSIONS: We set up a simple protocol to measure global translation activity in yeast on a single cell basis. The use of surfactants poses a practical and non-invasive alternative to the commonly used ergosterol pathway impaired strains and thus impacts a wide range of applications where increased drug and dye uptake is needed. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12866-021-02185-3.
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spelling pubmed-80565902021-04-20 Treatment with surfactants enables quantification of translational activity by O-propargyl-puromycin labelling in yeast Staudacher, Jennifer Rebnegger, Corinna Gasser, Brigitte BMC Microbiol Methodology Article BACKGROUND: Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in yeast. Recently, O-propargyl-puromycin (OPP) labelling has been established for mammalian cells, but unmodified yeasts are unsusceptible to puromycin. RESULTS: We could increase susceptibility by using a Komagataella phaffii strain with an impaired ergosterol pathway (erg6Δ), but translation measurements are restricted to this strain background, which displayed growth deficits. Using surfactants, specifically Imipramine, instead, proved to be more advantageous and circumvents previous restrictions. Imipramine-supplemented OPP-labelling with subsequent flow cytometry analysis, enabled us to distinguish actively translating cells from negative controls, and to clearly quantify differences in translation activities in different strains and growth conditions. Specifically, we investigated K. phaffii at different growth rates, verified that methanol feeding alters translation activity, and analysed global translation in strains with genetically modified stress response pathways. CONCLUSIONS: We set up a simple protocol to measure global translation activity in yeast on a single cell basis. The use of surfactants poses a practical and non-invasive alternative to the commonly used ergosterol pathway impaired strains and thus impacts a wide range of applications where increased drug and dye uptake is needed. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12866-021-02185-3. BioMed Central 2021-04-20 /pmc/articles/PMC8056590/ /pubmed/33879049 http://dx.doi.org/10.1186/s12866-021-02185-3 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis 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/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Methodology Article
Staudacher, Jennifer
Rebnegger, Corinna
Gasser, Brigitte
Treatment with surfactants enables quantification of translational activity by O-propargyl-puromycin labelling in yeast
title Treatment with surfactants enables quantification of translational activity by O-propargyl-puromycin labelling in yeast
title_full Treatment with surfactants enables quantification of translational activity by O-propargyl-puromycin labelling in yeast
title_fullStr Treatment with surfactants enables quantification of translational activity by O-propargyl-puromycin labelling in yeast
title_full_unstemmed Treatment with surfactants enables quantification of translational activity by O-propargyl-puromycin labelling in yeast
title_short Treatment with surfactants enables quantification of translational activity by O-propargyl-puromycin labelling in yeast
title_sort treatment with surfactants enables quantification of translational activity by o-propargyl-puromycin labelling in yeast
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056590/
https://www.ncbi.nlm.nih.gov/pubmed/33879049
http://dx.doi.org/10.1186/s12866-021-02185-3
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