Mechanisms and regulation of intracellular iron trafficking in Chlamydomonas reinhardtii

铁是所有光合作用生物体的重要微量营养素。莱茵衣藻通过由铁还原酶(FRE1)和铁氧合酶-铁渗透酶(FOX1-FTR1)组成的高亲和力吸收系统吸收铁。此外，莱茵梭菌细胞通过IRT1/IRT2转运蛋白系统吸收铁(可能还有其他金属)。虽然铁进入藻细胞的机制已被研究和建立，但目前尚不清楚铁如何在细胞质和细胞器之间运输以及如何调节其同化和运输。最近对缺铁的莱茵哈蒂氏弧菌的转录组分析发现，几个候选的细胞内铁运输和调控的转录水平增加。我们推测，这些蛋白质在细胞器之间的铁运输和铁稳态的调节中起着重要作用。通过结合反向遗传学和生化方法，我们将分析这些蛋白在莱茵梭菌中的功能。同时，将采用转录组学(RNA-Seq)、蛋白质组学(LC-MS/MS)和金属含量测定(ICP-MS)对这些蛋白质的整个细胞和分离的细胞器进行野生型和击倒突变体的检测。这将使我们能够更好地了解莱茵梭菌细胞内铁的分布和铁稳态的调节。此外，还将采用下拉试验，以确定调控莱茵哈迪尔铁虫缺铁反应的转录因子。

Iron is an important micronutrient for all photosynthetic organisms. Chlamydomonas reinhardtii, assimilate iron via a high affinity uptake system which comprises a ferric-reductase (FRE1) and a ferroxidase-ferric-permease system (FOX1-FTR1). Additionally, C. reinhardtii cells assimilate iron (and possibly other metals) via IRT1/IRT2 transporter system. While mechanisms of iron import into algal cells have been studied and established, mechanisms of how iron is transported between cytoplasm and organelles and how its assimilation and trafficking are regulated are currently unknown. Recent transcriptome analysis of iron-deficient C. reinhardtii identified increased transcript levels of several candidates for intracellular iron trafficking and regulation. We hypothesize that these proteins play an important role in the transport of iron between organelles and in the regulation of iron homeostasis. By combining reverse genetics and biochemical approaches we will analyze the function of these proteins in C. reinhardtii. In parallel, transcriptomics (RNA-Seq), proteomics (LC-MS/MS) and determination of metal content (ICP-MS) on whole cells and isolated organelles from wild-type and knock-down mutants of these proteins will be employed. This will allow us to better understand the intracellular iron distribution and the regulation of iron homeostasis in C. reinhardtii. In addition, a pull-down assay will be employed with the aim to identify the transcription factor responsible for the regulation of iron deficiency responses in C. reinhardtii.