Mechanisms and regulation of intracellular iron trafficking in Chlamydomonas reinhardtii

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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.