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 gap1Delta 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.
Donaton
Monica C V
MC
Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, Belgium.
Holsbeeks
Inge
I
Lagatie
Ole
O
Van Zeebroeck
Griet
G
Crauwels
Marion
M
Winderickx
Joris
J
Thevelein
Johan M
JM
D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems / Q000235:genetics / Q000378:metabolism; D001124:Argininosuccinate Synthase / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D020134:Catalytic Domain / Q000235:genetics; D002956:Citrulline / Q000378:metabolism; D003470:Culture Media; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D015966:Gene Expression Regulation, Fungal; D018698:Glutamic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D020703:ras GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*
D026905:Amino Acid Transport Systems; D003470:Culture Media; C491145:GAP1 protein, S cerevisiae; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D020703:ras GTPase-Activating Proteins; D002956:Citrulline; D018698:Glutamic Acid; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C457179:TPK2 protein, S cerevisiae; D001124:Argininosuccinate Synthase; D009584:Nitrogen