Abstract
BackgroundTranslation 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.ResultsWe 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.ConclusionsWe 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.
Highlights
Translation is an important point of regulation in protein synthesis
Approaches aimed at increasing the susceptibility of S. cerevisiae to puromycin focused on disturbing cell membrane integrity, either by knocking out a component of the ergosterol pathway, or by targeting the pleitropic drug response [19, 20]
Disturbing the sterol synthesis pathway increases susceptibility of K. phaffii to puromycin Yeasts are known to be susceptible only to very high doses of puromycin, but it has been shown in a variety of organisms that usage of extreme doses has distinct effects on cell physiology and translation [6, 9, 20, 22]
Summary
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. 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. 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
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