A yeast FRET biosensor enlightens cAMP signalling

Abstract

The cAMP-PKA signalling cascade in budding yeast regulates adaptation to changing environments. Many questions remain about the function of cAMP dynamics, largely because no robust method for in vivo cAMP measurements exists for yeast. Here we developed yEPAC, a FRET-based biosensor for cAMP measurements in yeast. We show that this biosensor can be used in flow cytometry, allowing for highthroughput single cell-level quantification that complements microscopy measurements. We quantified both steady-state cAMP levels and dynamic changes in response to sudden nutrient transitions. Steadystate cAMP levels were found to correlate with growth rate (rather than glycolytic flux) and were independent of extracellular sensing. During transitions, a cAMP-peak differentiates between different carbon source transitions, and is generated only when switching from non-or slowly-fermentable sugars to rapidly fermentable ones. Furthermore, generation of the cAMP peak is mediated by a combination of extracellular sensing and metabolism; deficits in either results in a largely diminished response. Moreover, the cAMP peak follows Weber's law; its height scales with the relative, and not the absolute, change in glucose. This means that cells respond to small changes in extracellular glucose when the initial level is low, but ignore these changes when this level is high. Lastly, we speculate that the cAMP peak height conveys information about prospective growth rates, especially for fermentable sugars. In conclusion, our yEPAC-sensor makes possible new avenues for understanding yeast physiology, signalling and metabolic adaptation.

Molecular Targets and Biological Functions of cAMP Signaling in Arabidopsis

“…In animals and yeast, cellular cAMP levels display dynamic changes [ 206 , 209 ], as well as rigorous feedback control [ 210 , 211 ]. In fact, cAMP triggers changes in its own concentration [ 20 ].…”

An improved ATP FRET sensor for yeast shows heterogeneity during nutrient transitions

“…We hypothesised that the baseline drift and the pH sensitivity are caused by the fluorescent proteins, since the donor mseCFP is poorly characterised and mVenus is known to be pH sensitive and not photostable 25 . Based on this, we undertook improvement of AT1.03 by changing the fluorescent proteins to ymTq2Δ11 (donor) and tdTomato (acceptor) as a more reliable and pH-insensitive FRET pair (Botman et al, in submission 33 ). Replacements of mseCFP for ymTq2Δ11 and cp173-mVenus for tdTomato resulted in yAT1.03.…”

Nutrient sensing and cAMP signaling in yeast: G-protein coupled receptor versus transceptor activation of PKA

“…In order to monitor cAMP levels upon nutrient addition in a temporally-resolved manner, we have used the EPAC cAMP sensor as optimized by the lab of Bas Teusink [18]. Yeast cells expressing the cAMP sensor were grown in the absence of glucose, using glycerol as the sole carbon source.…”

“…All strains and plasmids used in this work are summarized in Table 1 and 2, respectively. For FRET measurements, the wild type strain was transformed with the mTurq2del-EPACdDEPCD-tdTomato plasmid (URA3) kindly provided by Bas Teusink (VU Amsterdam) [18]. For the GST-pull down experiments, the gap1∆ strain was transformed with a plasmid expressing HA-tagged Gap1.…”

“…In 2017, the first functional S. cerevisiae variant of the EPAC sensor was developed, based on the original EYFP-EPAC2-ECFP construct of Nikolaev et al [17]. Lately, the research group of Bas Teusink has developed an alternative cAMP sensor for S. cerevisiae based on the mammalian sensor of Klarenbeek et al [16], using mTurquoise2del as FRET donor and tdTomato as FRET acceptor [18]. The major advantage of this latest sensor is the fact that the fluorescent proteins used are less pH sensitive, within the physiological intracellular pH-range of the yeast cells, enabling these sensors to report the cAMP concentration more reliably and independent of changes in intracellular pH.…”