cAMP in budding yeast: Also a messenger for sucrose metabolism?

S. cerevisiae (or budding yeast) is an important micro-organism for sucrose-based fermentation in biotechnology. Yet, it is largely unknown how budding yeast adapts to sucrose transitions. Sucrose can only be metabolized when the invertase or the maltose machinery are expressed and we propose that the Gpr1p receptor signals extracellular sucrose availability via the cAMP peak to adapt cells accordingly. A transition to sucrose or glucose gave a transient cAMP peak which was maximally induced for sucrose. When transitioned to sucrose, cAMP signalling mutants showed an impaired cAMP peak together with a lower growth rate, a longer lag phase and a higher final OD600 compared to a glucose transition. These effects were not caused by altered activity or expression of enzymes involved in sucrose metabolism and imply a more general metabolic adaptation defect. Basal cAMP levels were comparable among the mutant strains, suggesting that the transient cAMP peak is required to adapt cells correctly to sucrose. We propose that the short-term dynamics of the cAMP signalling cascade detects long-term extracellular sucrose availability and speculate that its function is to maintain a fermentative phenotype at continuously low glucose and fructose concentrations.

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

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 Förster 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 (K0.5 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.