Molecular mechanisms of iron homeostasis in Saccharomyces cerevisiae

描述（由申请人提供）：铁（Fe）缺乏症是一种影响全球近20亿人的营养障碍。拟议研究的长期目标是了解生物体如何使用酵母酿酒酵母作为模型系统来科普铁缺乏。具体的假设是，mRNA结合蛋白Cth 1和Cth 2协同介导的适应铁限制条件下在S。酿酒酵母通过促进靶向mRNA衰变。这一假说是基于以下观察：1）在缺铁时，Cth 2促进参与代谢途径的许多mRNA的下调，并且cth 2突变细胞在缺铁培养基上生长不良; 2）cth 2菌株上CTH 1基因的缺失进一步加剧了低Fe条件下的生长表型，并且cth 1cth 2突变体不能下调Fe-12过程中靶向周转的几种mRNA。3）伴随着许多mRNA的下调，野生型细胞在低Fe下显示出葡萄糖调节基因的稳态水平增加，但在相同条件下的cth 1cth 2细胞没有。具体目标是： 1.确定Cth 1蛋白对铁限制反应的贡献。将使用传统技术如染色质免疫沉淀（ChIP）、β-半乳糖苷酶报告基因测定、酵母三杂交测定和RNA印迹来确定Cth 1响应铁缺乏的转录激活、mRNA结合和降解促进活性。将进行微阵列分析，以识别响应低Fe的Cth 1和Cth 2的特定靶标。 2.建立缺铁时Cth 1和Cth 2活性对葡萄糖代谢的影响。初步数据表明，铁饥饿酵母表达几个葡萄糖抑制基因，但未能做到这一点的情况下，Cth 1和Cth 2。将使用酶测定以及检查野生型细胞和cth 1cth 2突变体中的磷酸-Snf 1和果糖-1，6-二磷酸酶水平来监测Fe缺乏期间的葡萄糖利用。此外，Cth 1和Cth 2的非mRNA结合等位基因将用于确定它们的衰变促进活性是否是次级葡萄糖饥饿反应所必需的。 铁（Fe）缺乏症是影响全球近20亿人的头号营养障碍。本研究的目的是了解真核生物如何使用遗传上易处理的酵母酿酒酵母（Saccharomyces cerevisiae）调节其代谢以适应铁缺乏的条件，这将有助于更好地了解人类如何在分子水平上科普细胞铁缺乏。

DESCRIPTION (provided by applicant): Iron (Fe) deficiency is a nutritional disorder that affects nearly two billion people worldwide. The long-term goal of the proposed research is to understand how organisms cope with Fe-deficiency using the yeast Saccharomyces cerevisiae as a model system. The specific hypothesis is that the mRNA-binding proteins Cth1 and Cth2 cooperatively mediate the adaptation to Fe-limiting conditions in S. cerevisiae through promoting targeted mRNA decay. This hypothesis is based on the observations that 1) during Fe-deficiency, Cth2 promotes the down regulation of many mRNAs involved in metabolic pathways, and cth2 mutant cells grow poorly on medium lacking Fe; 2) deletion of the CTH1 gene on a cth2 strain further exacerbates the growth phenotype on low Fe conditions and cth1cth2 mutants fail to down regulate several of the mRNAs targeted for turnover during Fe-deficiency; and 3) concomitantly with the down regulation of many mRNAs, wild type cells show increased steady-state levels of glucose-regulated genes under low Fe, but not cth1cth2 cells under the same conditions. The specific aims are to: 1. Establish the contribution of Cth1 protein to the response to Fe limitation. The transcriptional activation, mRNA-binding and degradation-promoting activities of Cth1 in response to Fe-deficiency will be ascertained using traditional techniques such Chromatin ImmunoPrecipitation (ChIP), beta-galactosidase reporter assays, yeast three-hybrid assays and RNA blotting. Microarray analyses will be performed to identify specific targets of Cth1 and Cth2 in response to low Fe. 2. Establish the effects of Cth1 and Cth2 activity on glucose metabolism during Fe-deficiency. Preliminary data suggest that Fe-starved yeast express several glucose-repressed genes, but fail to do so in the absence of Cth1 and Cth2. Glucose utilization during Fe-deficiency will be monitored using enzymatic assays as well as examining phospho-Snfl and fructose-1, 6-bisphosphatase levels in wild type cells and in cth1cth2 mutants. In addition, non-mRNA binding alleles of Cth1 and Cth2 will be used to ascertain whether their decay-promoting activity is necessary for the secondary glucose starvation response. Iron (Fe) deficiency is the number one nutritional disorder affecting nearly two billion people worldwide. The goal of this research is to understand how eukaryotes adjust their metabolism to conditions of Fe-deficiency using the genetically tractable yeast Saccharomyces cerevisiae, which will contribute to a better understanding of how humans cope with cellular Fe-deficiency at the molecular level.