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Benoît Revel

Identification of proteins involved in the fate of uranium in the plant Arabidopsis thaliana and the yeast Saccharomyces cerevisiae

Published on 20 January 2021
Thesis presented January 20, 2021

Uranium is a naturally-occurring radionuclide in the Earth’s crust. It is mainly redistributed in the environment by anthropogenic activities such as uranium and phosphate mining, nuclear industry, military activities and soil fertilization. Uranium may locally accumulate to concentrations that can lead to potential risks for ecosystems, agrosystems, and ultimately human health. Indeed, this radionuclide is chemotoxic and potentially radiotoxic (natural uranium has low specific activity) for all living organisms. Even if uranium is not essential for plants, it is taken up from the soil, incorporated into the biomass, and eventually, can enters the food chain. Thus, contamination of soils by uranium and its absorption by plants represent a significant health risk for humans. The aim of my PhD project is to improve the knowledge of the molecular mechanisms governing the fate of uranium in plant. This project is divided into two parts: the first aims to identify uranium transporters, the second to identify proteins that could be cellular targets for uranium.
In the first part, we used the yeast Saccharomyces cerevisiae as a unicellular eucaryote model that allows us to overpass constraints related to study complex multicellular organisms as higher plants. This organism is particularly relevant to identify metal transporters in plants because of the great conservation of genetic heritage between these two organisms. Thus, we have identified, for the first time, a transporter capable of transporting uranium in a living organism. First, a metabolism-dependent uranium transport was highlighted and characterized. Then, competitive experiments with essential metals allowed us to identify calcium, iron and copper absorption pathways as potential routes for uranium uptake. A study of various mutants revealed that Δmid1, Δcch1 and Δftr1 mutants, affected in calcium (MID1/CCH1 channel) and Fe3+ (FTR1) transport respectively, also exhibited highly reduced uranium uptake. Ectopic expression of the MID1 gene into the Δmid1 mutant restored uranium uptake levels of the wild type strain demonstrating that the MID1/CCH1 calcium channel is involved in uranium absorption process in yeast.
In the second part, by using metalloproteomic approaches combining column chromatographic fractionation analyses, protein identification by mass spectrometry (LC-MS/MS) and metal quantification by induced coupled plasma mass spectrometry (ICP-MS), we identified 53 candidate proteins for uranium binding in vivo. One of these proteins, GRP7, was overproduced and further biochemically characterized in more detail. After demonstrating its ability to bind uranium in vitro, we initiated its structural analysis by structural nuclear magnetic resonance (NMR) to identify uranium binding sites.
Taken together, this new fundamental knowledge could lead to the development of several biotechnological approaches to control uranium pollution in the environment.

Uranium, Plants, Stress, Yeast, Proteins