Using the AKAR3-EV biosensor to assess Sch9p-and PKA-signalling in budding yeast

The AKAR3-EV biosensor measures the signalling activity of two major kinases -PKA and Sch9- in single living budding yeast cells. Budding yeast uses the TORC1-Sch9p and cAMP-PKA signalling pathways to regulate adaptations to changing nutrient environments. Dynamic and single-cell measurements of the activity of these cascades will improve our understanding of the cellular adaptation of yeast. Here, we employed the AKAR3-EV biosensor developed for mammalian cells to measure the cellular phosphorylation status determined by Sch9p and PKA activity in budding yeast. Using various mutant strains and inhibitors, we show that AKAR3-EV measures the Sch9p- and PKA-dependent phosphorylation status in intact yeast cells. At the single-cell level, we found that the phosphorylation responses are homogenous for glucose, sucrose, and fructose, but heterogeneous for mannose. Cells that start to grow after a transition to mannose correspond to higher normalized Forster resonance energy transfer (FRET) levels, in line with the involvement of Sch9p and PKA pathways to stimulate growth-related processes. The Sch9p and PKA pathways have a relatively high affinity for glucose (K-0.5 of 0.24 mM) under glucose-derepressed conditions. Lastly, steady-state FRET levels of AKAR3-EV seem to be independent of growth rates, suggesting that Sch9p- and PKA-dependent phosphorylation activities are transient responses to nutrient transitions. We believe that the AKAR3-EV sensor is an excellent addition to the biosensor arsenal for illuminating cellular adaptation in single yeast cells.

Automated summary

The AKAR3-EV biosensor measures the activity of PKA and Sch9 kinases in single yeast cells, providing insights into the cellular adaptations of yeast to changing nutrient environments. The biosensor was able to accurately measure the phosphorylation status in intact yeast cells, with heterogeneous responses observed for different sugars. The study highlights the importance of the AKAR3-EV sensor in understanding cellular adaptation in single yeast cells.