Abstract
A major signal transduction pathway regulating cell growth and many associated physiological properties as a function of nutrient availability in the yeast Saccharomyces cerevisiae is the protein kinase A (PKA) pathway. Glucose activation of PKA is mediated by G-protein coupled receptor (GPCR) Gpr1, and secondary messenger cAMP. Other nutrients, including nitrogen, phosphate and sulfate, activate PKA in accordingly-starved cells through nutrient transceptors, but apparently without cAMP signaling. We have now used an optimized EPAC-based fluorescence resonance energy transfer (FRET) sensor to precisely monitor in vivo cAMP levels after nutrient addition. We show that GPCR-mediated glucose activation of PKA is correlated with a rapid transient increase in the cAMP level in vivo, whereas nutrient transceptor-mediated activation by nitrogen, phosphate or sulfate, is not associated with any significant increase in cAMP in vivo. We also demonstrate direct physical interaction between the Gap1 amino acid transceptor and the catalytic subunits of PKA, Tpk1, 2 and 3. In addition, we reveal a conserved consensus motif in the nutrient transceptors that is also present in Bcy1, the regulatory subunit of PKA. This suggests that nutrient transceptor activation of PKA may be mediated by direct release of bound PKA catalytic subunits, triggered by the conformational changes occurring during transport of the substrate by the transceptor. Our results support a model in which nutrient transceptors are evolutionary ancestors of GPCRs, employing a more primitive direct signaling mechanism compared to the indirect cAMP second-messenger signaling mechanism used by GPCRs for activation of PKA.
Highlights
For all living organisms, but especially for microorganisms, nutrients are a major determinant for cell growth and survival
We show that G-protein coupled receptor (GPCR)-mediated glucose activation of protein kinase A (PKA) is correlated with a rapid transient increase in the cyclic adenosine monophosphate (cAMP) level in vivo, whereas nutrient transceptormediated activation by nitrogen, phosphate or sulfate, is not associated with any significant increase in cAMP in vivo
Our results support a model in which nutrient transceptors are evolutionary ancestors of GPCRs, employing a more primitive direct signaling mechanism compared to the indirect cAMP second-messenger signaling mechanism used by GPCRs for activation of PKA
Summary
But especially for microorganisms, nutrients are a major determinant for cell growth and survival. Any change in nutrient availability and composition in the extracellular environment triggers rapid adaptation mechanisms within the cells to properly adjust cellular properties. With respect to the yeast Saccharomyces cerevisiae, the presence of fermentable sugar in the extracellular medium has major effects on cell growth and physiology. On the other hand, when yeast grows on non-fermentable carbon sources, they proliferate more slowly and show the opposite phenotypes of cells growing on fermentable sugar [1]. To maintain the high PKA phenotype typical for fermenting cells, a complete fermentable growth medium is required. When fermenting cells are starved for any other essential nutrient, they will arrest in the G1 phase of the cell cycle, enter the stationary phase G0 and acquire a low GPCR versus transceptor activation of PKA
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