Amino Acid Sensing and Signaling in Yeast

描述（由申请人提供）：真菌的生存和发病需要感知和适应新环境的能力。细胞外氨基酸水平的变化可能预示着“新”环境的到来，从而导致特定信号通路的诱导和营养物质的获取。已知Ssy1-Ptr3-Ssy5 （SPS）氨基酸感知途径参与了这种环境适应。本提案的长期目标是了解真核细胞中SPS信号传导的分子机制，以酿酒酵母为模式生物。在细胞外氨基酸存在的情况下，ssy5依赖的两种转录因子Stp1和Stp2的内蛋白水解过程导致促进氨基酸摄取的氨基酸转运蛋白的表达。SPS氨基酸感应途径存在于几种致病性念珠菌中，是动物模型中白色念珠菌致病性所必需的。我们的初步数据显示，在葡萄糖限制条件下，sps靶基因表达的意外激活需要Gcn2， Gcn2是在缺乏氨基酸的细胞中激活通用氨基酸控制（GAAC）反应的激酶关键，但不需要Gcn4， Gcn4是GAAC反应所需的转录因子。在经历氨基酸饥饿的细胞中，Gcn2通过一种不寻常的机制增加GCN4 mRNA的翻译，该机制涉及GCN4 mRNA先导序列中的小上游开放阅读框（uORF）。这种独特的

DESCRIPTION (provided by applicant): The ability to sense and adapt to new environments is required for fungal survival and pathogenesis. Changes in the level of extracellular amino acids may signal a 'new' environment, which results in induction of specific signaling pathways and acquisition of nutrients. The Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway is known to be involved in this environmental adaptation. The long-term objective of this proposal is to understand the molecular mechanisms underlying SPS signaling in eukaryotic cells using Saccharomyces cerevisiae as a model organism. In the presence of extracellular amino acids, Ssy5-dependent endoproteolytic processing of two transcription factors Stp1 and Stp2 leads to expression of amino acid transporters that facilitate amino acid uptake. The SPS amino acid sensing pathway exists in several pathogenic Candida species and is required for pathogenicity of C. albicans in an animal model. Our preliminary data have revealed unexpected activation of SPS-target gene expression under glucose limitation conditions that requires Gcn2, a kinase key to activation of the General Amino Acid Control (GAAC) response in cells deprived of amino acids, but not Gcn4, a transcription factor required for the GAAC. In cells experiencing amino acid starvation,Gcn2 increases the translation of GCN4 mRNA via an unusual mechanism involving small upstream open reading frames (uORF) in the leader sequence of GCN4 mRNA. This unique protein translational control is conserved in mammalian cells, with ATF4/5 as the counterpart of yeast GCN4. Although uORFs are predicted to exist in 10-25% eukaryotic genes, only a few uORFs have been studied in detail. Herein, we describe specific aims that we will use to study this newly discovered link between extracellular and intracellular amino acid sensing pathways through the common regulator Gcn2 using yeast genetics, cell biology and biochemical tools. We will also characterize the mechanism underlying Ubc4/5-dependent degradation of processed Stp2 and the role of Stp2 degradation in pathway regulation. These studies will provide novel insights into mechanisms underlying amino acid sensing and signaling in yeast.