Inge Holsbeeks

The Gap1 general amino acid permease acts as an amino acid sensor for activation of protein kinase A targets in the yeast Saccharomyces cerevisiae.

Addition of a nitrogen source to yeast (Saccharomyces cerevisiae) cells starved for nitrogen on a glucose‐containing medium triggers activation of protein kinase A (PKA) targets through a pathway that requires for sustained activation both a fermentable carbon source and a complete growth medium (fermentable growth medium induced or FGM pathway). Trehalase is activated, trehalose and glycogen content as well as heat resistance drop rapidly, STRE‐controlled genes are repressed, and ribosomal protein genes are induced. We show that the rapid effect of amino acids on these targets specifically requires the general amino acid permease Gap1. In the gap1Δ strain, transport of high concentrations of l‐citrulline occurs at a high rate but without activation of trehalase. Metabolism of the amino acids is not required. Point mutants in Gap1 with reduced or deficient transport also showed reduced or deficient signalling. However, two mutations, S391A and S397A, were identified with a differential effect on transport and signalling for l‐glutamate and l‐citrulline. Specific truncations of the C‐terminus of Gap1 (e.g. last 14 or 26 amino acids) did not reduce transport activity but caused the same phenotype as in strains with constitutively high PKA activity also during growth with ammonium as sole nitrogen source. The overactive PKA phenotype was abolished by mutations in the Tpk1 or Tpk2 catalytic subunits. We conclude that Gap1 acts as an amino acid sensor for rapid activation of the FGM signalling pathway which controls the PKA targets, that transport through Gap1 is connected to signalling and that specific truncations of the C‐terminus result in permanently activating Gap1 alleles.

Nutrient sensing systems for rapid activation of the protein kinase A pathway in yeast

The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae controls a variety of properties that depend on the nutrient composition of the medium. High activity of the pathway occurs in the presence of rapidly fermented sugars like glucose or sucrose, but only as long as growth is maintained. Growth arrest of fermenting cells or growth on a respiratory carbon source, like glycerol or ethanol, is associated with low activity of the PKA pathway. We have studied how different nutrients trigger rapid activation of the pathway. Glucose and sucrose activate cAMP synthesis through a G-protein-coupled receptor system, consisting of the GPCR Gpr1, the Galpha protein Gpa2 and its RGS protein Rgs2. Glucose is also sensed intracellularly through its phosphorylation. Specific mutations in Gpr1 abolish glucose but not sucrose signalling. Activation of the PKA pathway by addition of a nitrogen source or phosphate to nitrogen- or phosphate-starved cells, respectively, is not mediated by an increase in cAMP. Activation by amino acids is triggered by the general amino acid permease Gap1, which functions as a transporter/receptor. Short truncation of the C-terminus results in constitutively activating alleles. Activation by ammonium uses the ammonium permeases Mep1 and Mep2 as receptor. Specific point mutations in Mep2 uncouple signalling from transport. Activation by phosphate is triggered a.o. by the Pho84 phosphate permease. Several mutations in Pho84 separating transport and signalling or triggering constitutive activation have been obtained.

From feast to famine; adaptation to nutrient availability in yeast

The study of signal transduction in microorganisms has become a major research topic in molecular and cellular biology. In this era, thorough knowledge of microbial physiology is no longer the sole and exclusive interest of academic research. It is now being acknowledged as a major importance for food, feed, and nutritional R&D. Detailed investigation of the mechanisms by which cells respond to environmental stimuli is contributing largely to both our fundamental and applied understanding of microorganisms.