Jennifer Staudacher1,2, Corinna Rebnegger1,2, Brigitte Gasser3,4. 1. Christian Doppler Laboratory for Growth-decoupled Protein Production in Yeast, Department of Biotechnology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria. 2. Institute of Microbiology and Microbial Biotechnology, Department of Biotechnology, BOKU University of Natural Resources and Life Sciences, Muthgasse 18, 1190, Vienna, Austria. 3. Christian Doppler Laboratory for Growth-decoupled Protein Production in Yeast, Department of Biotechnology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria. brigitte.gasser@boku.ac.at. 4. Institute of Microbiology and Microbial Biotechnology, Department of Biotechnology, BOKU University of Natural Resources and Life Sciences, Muthgasse 18, 1190, Vienna, Austria. brigitte.gasser@boku.ac.at.
Abstract
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.
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.
Authors: Qian Wang; Timothy R Chan; Robert Hilgraf; Valery V Fokin; K Barry Sharpless; M G Finn Journal: J Am Chem Soc Date: 2003-03-19 Impact factor: 15.419