Dissection of the Molecular Details of Fe Deficiency Acclimation

描述（由申请人提供）：铁（Fe）是多种蛋白质活性所需的丰富蛋白质辅因子，因此对于从DNA合成到呼吸的多种细胞功能至关重要。因此，细胞必须确保这些Fe依赖性蛋白质可获得足够的Fe供应，但同时必须避免细胞中过量的Fe，这可能导致细胞毒性反应。虽然我们对铁稳态的理解已经受益于超过半个世纪的研究在许多生物体中，有关细胞内铁运输的分子细节是缺乏的。Merchant小组已经开发了莱茵衣藻作为参考生物，用于研究铁营养不良背景下的铁代谢。据估计，世界上大约三分之一的人口患有症状性铁缺乏症，多种人类疾病是由铁稳态的失调引起的，了解这些机制至关重要。一个特征不足的反应是当细胞外铁不可用时，从铁蛋白中回收铁。这一过程如何在分子水平上进行调节尚不清楚。该项目的目标是确定铁限制期间铁在细胞内的运输途径，并发现负责完成和调节铁循环的蛋白质。该项目的具体目标有三个方面。首先，将对细胞进行生化分级，并确定在Fe-充足与-缺乏条件下Fe-利用和Fe-储存隔室之间的Fe分布，特别是在碳源利用（呼吸与光合作用）的背景下。第二，反向遗传学将被用来表征参与铁调控基因预测参与铁循环。通过确定这些蛋白质的亚细胞位置以及这些基因如何受Fe调控来辅助表征。第三，一个经典的遗传筛选将被实施，以发现新的组成部分，铁稳态，可能不会在基因或mRNA水平上进行调节，因此，已经逃脱了转录组研究的通知。选择这些目标为理解细胞中受调控的Fe运输机制奠定了先例，表征了已知由Fe限制诱导的基因，并发现了参与适应Fe限制的新基因。此外，该项目旨在提供各种技术的培训，并获得使用参考真核生物的专业知识。

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.