Using the AKAR3-EV biosensor to assess Sch9- & PKA-signalling in budding yeast

Budding yeast uses the well-conserved TORC1-Sch9 and cAMP-PKA signalling pathways to regulate adaptations to changing nutrient environments. Dynamic and single-cell measurements of the activity of these two cascades will improve our understanding of cellular adaptation of yeast. Here, we employed the AKAR3-EV biosensor developed for mammalian cells to measure the cellular phosphorylation status determined by Sch9 and PKA activity in budding yeast. Using various mutant strains and inhibitors, we show that AKAR3-EV robustly measures the Sch9- 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 highly heterogeneous for mannose. The Sch9 and PKA pathways have a relatively high affinity for glucose (K0.5of 0.24 mM) under glucose derepressed conditions. Lastly, steady-state FRET levels of AKAR3-EV seem to be independent of growth rates, suggesting that the Sch9- and PKA-dependent phosphorylation activity 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.