Donaton Monica C V, Holsbeeks Inge, Lagatie Ole, Van Zeebroeck Griet, Crauwels Marion, Winderickx Joris, Thevelein Johan M
Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, Belgium.
Mol Microbiol. 2003 Nov;50(3):911-29. doi: 10.1046/j.1365-2958.2003.03732.x.
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.
在含葡萄糖的培养基上，向缺乏氮源的酵母（酿酒酵母）细胞中添加氮源，会通过一条需要可发酵碳源和完整生长培养基才能持续激活的途径（可发酵生长培养基诱导或FGM途径），触发蛋白激酶A（PKA）靶标的激活。海藻糖酶被激活，海藻糖和糖原含量以及耐热性迅速下降，STRE控制的基因被抑制，核糖体蛋白基因被诱导。我们发现，氨基酸对这些靶标的快速作用特别需要通用氨基酸通透酶Gap1。在gap1Δ菌株中，高浓度L-瓜氨酸的转运速率很高，但海藻糖酶未被激活。氨基酸的代谢并非必需。Gap1中转运减少或缺陷的点突变体也显示出信号传导减少或缺陷。然而，鉴定出两个突变，S391A和S397A，它们对L-谷氨酸和L-瓜氨酸的转运和信号传导有不同影响。Gap1 C末端的特定截短（例如最后14或26个氨基酸）并未降低转运活性，但在以铵作为唯一氮源生长期间，也导致了与PKA活性持续较高的菌株相同的表型。Tpk1或Tpk2催化亚基中的突变消除了过度活跃的PKA表型。我们得出结论，Gap1作为一种氨基酸传感器，可快速激活控制PKA靶标的FGM信号通路，通过Gap1的转运与信号传导相关联，并且C末端的特定截短会导致Gap1等位基因永久激活。
