Dissection of the Molecular Details of Fe Deficiency Acclimation

说明(申请人提供)：铁(Fe)是一种丰富的蛋白质辅因子，对多种蛋白质的活性都是必需的，因此对从DNA合成到呼吸作用的许多细胞功能都是必不可少的。因此，细胞必须确保这些铁依赖的蛋白质有足够的铁供应，但同时必须避免细胞中过量的铁，这可能会导致细胞毒性反应。尽管我们对铁稳态的了解得益于对许多生物体半个多世纪的研究，但关于细胞内铁运输的分子细节尚不清楚。Merchant小组已经开发出莱茵衣藻作为在铁营养不良的情况下研究铁代谢的参考生物。据估计，世界上大约三分之一的人口患有症状性铁缺乏，多种人类疾病是由铁稳态调节不当引起的，了解这些机制是至关重要的。一种特征不足的反应是，当细胞外铁不可用时，从可支配蛋白质中回收铁。这一过程是如何在分子水平上调节的尚不清楚。该项目的目标是确定铁限制期间细胞内铁的运输途径，并发现负责完成和调节铁循环的蛋白质。这个项目的具体目标有三个方面。首先，将对细胞进行生化分离，并确定铁在富铁和缺铁条件下的铁利用和铁储存之间的分配，特别是在碳源利用(呼吸作用和光合作用)的背景下。其次，反向遗传学将被用来描述铁调节基因的特征，这些基因被预测参与铁循环。确定这些蛋白质的亚细胞位置以及铁是如何调控这些基因的，将有助于确定这些蛋白质的特性。第三，将实施经典的遗传筛选，以发现铁稳态的新成分，这些成分可能不在基因或mRNA水平上受到调控，因此逃脱了转录组研究的注意。选择这些目的是为了为了解细胞内铁的调节运输机制、鉴定已知的铁限制诱导的基因以及发现参与适应铁限制的新基因奠定先例。此外，该项目旨在提供广泛的技术培训，并获得与真核生物参照生物合作的专门知识。

DESCRIPTION (provided by applicant): Iron (Fe) is an abundant protein cofactor required for the activity of a myriad of proteins and consequently is essential for numerous cellular functions ranging from DNA synthesis to respiration. Therefore, the cell must ensure that a sufficient supply of Fe is available to these Fe-dependent proteins but, at the same time, excess Fe in the cell, which can lead to cytotoxic reactions, must be avoided. Although our understanding of Fe homeostasis has benefited from over half a century of study in numerous organisms, the molecular details concerning intracellular Fe trafficking are lacking. The Merchant group has developed Chlamydomonas reinhardtii as a reference organism for studying Fe metabolism in the context of poor Fe nutrition. As it is estimated that roughly one-third of the world's population suffers from symptomatic Fe deficiency and multiple human diseases are caused by mis-regulation of Fe homeostasis, understanding these mechanisms is crucial. One under-characterized response is the recycling of Fe from dispensable proteins when extracellular Fe is unavailable. How this process is regulated at the molecular level is not known. The goal of this project is to identify the trafficking pathways of Fe within the cell during Fe-limitation and discover proteins responsible for accomplishing and regulating Fe recycling. The specific aims of this project are three-fold. First, cells will be biochemically fractionated and distribution of Fe between Fe-utilizing and Fe-storage compartments in Fe-replete vs. -deficient conditions will be determined, especially in the context of carbon source utilization (respiration vs. photosynthesis). Second, reverse genetics will be employed to characterize the involvement of Fe-regulated genes predicted to be involved in Fe recycling. Characterization will be aided by determining the subcellular location of these proteins and how these genes are regulated by Fe. Third, a classical genetic screen will be implemented to discover novel components of Fe homeostasis that may not be regulated at the gene or mRNA level and, therefore, have escaped the notice of transcriptome studies. These aims were chosen to set a precedent for understanding the mechanism of regulated Fe trafficking in the cell, characterize genes known to be induced by Fe-limitation and discover novel genes involved in acclimating to Fe-limitation. In addition, this project has been designed to provide training with a wide-range of techniques and to gain expertise in working with a reference eukaryotic organism.