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

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.

Translation initiation factor eIF1A rescues hygromycin B sensitivity caused by deleting the carboxy‐terminal tail in the GPN‐loop GTPase Npa3

“…Here we took advantage of the puromycin-based assay, which is an affordable non-radioactive system for protein labeling, to evaluate directly in vivo translation rate in Npa3 mutant cells. Previous studies suggest that specific S. cerevisiae strains are amenable to puromycin-based assays [68,69]. In this study, we demonstrated that, in our specific conditions, the puromycin system employed to directly assess translation in yeast is functional (Fig.…”

Protein production dynamics and physiological adaptation of recombinant Komagataella phaffii at near-zero growth rates

“…A possible explanation for higher secretion rates in methanol conditions would be that growth on this carbon source is natively accompanied by very high expression of methanol utilization genes such as Aox1 [ 59 ], and methanol-grown cultures might therefore generally sustain high translation activity. However, in a recent study, translation activity at intermediate growth rates was comparable between methanol and glucose-limited conditions [ 60 ]. Another explanation would be that expression of the recombinant gene remained higher in the P AOX1 -based system, which, however, would be very difficult to compare directly.…”

The Unkempt RNA binding protein reveals a local translation program in centriole overduplication

“…OPP is a puromycin analog that enters the ribosome acceptor site and is incorporated into nascent polypeptides. This modification can be fluorescently labeled to mark sites of translation within the cell 50 . RPE-1 cells were S phase arrested, induced for Plk4 overexpression for 16 hours, labeled with OPP, and co-stained with Cep131.…”