An Arabidopsis iron transporter as a model for regulated endocytosis in plants – TRAFIRT

Iron is an essential element for all organisms. Iron uptake is a strictly tuned process relying mostly on the control of iron transporters, at the transcriptional and post-transcriptional levels in yeast and mammals respectively. Plant were long thought to regulate their iron uptake machinery dependent on the IRT1 root iron transpoter at the transcriptional level. However, recent evidences suggest a post-transcriptional regulation likely affecting IRT1 protein localization and stability, through iron-regulated endocytosis. Only a few examples of regulated endocytosis exist in plants, and our understanding of the plant endocytic pathway is scarce. This proposal aims at : (i) elucidating how iron controls its own uptake through regulated endocytosis and degradation of IRT1 in the model plant Arabidopsis thaliana, and (ii) at establishing a system to study the poorly described plant endocytic pathway and endomembrane system. To this end, we use an integrated approach of cell biology, forward and reverse genetics, molecular and biochemical methods. First, we will reveal the differential stability of IRT1 on plant grown under various iron regimes. In parallel, we will visualize in vivo the trafficking of IRT1 in the cell by monitoring the influence of trafficking inhibitors and co-localization with known markers of endosomes to highlight the pathways used by IRT1 on its way to its degradation site. The second objective of the project is to identify proteins involved in IRT1 trafficking and turnover. First, a yeast two hybrid screen identified putative regulators of IRT1 trafficking that are currently analyzed by reverse genetics and cell biology approaches. In parallel, we will identify proteins involved directly or indirectly in the regulation of IRT1 stability and trafficking. FIT is a transcription factor required for stabilization of IRT1. Using both a forward genetic suppressor screen of the chlorotic fit mutant, and FIT chromatin-immunoprecipitation coupled to microarray analyses, we will identified proteins required for FIT-mediated stabilization of IRT1. Both strategies will focus on candidates with a role in protein modification and trafficking based on the knowledge gained from yeast and mammals. Finally, we will use IRT1 as a model to better describe the complex plant endomembrane system, which is likely composed of many sub-populations of each compartment. IRT1 will be systematically co-localized with a set of hundreds of lines corresponding to proteins predicted by bioinformatics to be located in the endomembrane system, and thereby representing the vast majority of putative sub-populations of each compartment found in the cell.