Dissecting the molecular interactions of mitochondrial glutaredoxin S15 in plants

• Applicant: Professor Dr. Andreas Meyer
• Subject Area: Plant Physiology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 406708156

Several metabolic pathways and cellular processes in plants depend on the functioning of iron-sulfur (Fe-S) proteins, whose cofactors are assembled through dedicated assembly machineries present in the cytosol, plastids and mitochondria. To cite only a few examples, Fe-S proteins are present in the photosynthetic and respiratory electron transfer chains and they are needed for sulfur and nitrogen assimilation, or co-enzyme synthesis such as biotin and lipoic acid. In plants as in other organisms, the incorporation of Fe-S clusters into proteins requires first the de novo assembly of iron-sulfur clusters (ISCs) onto scaffold proteins and their transfer to acceptor proteins via the action of several maturation factors, and among those class II glutaredoxins (GRXs). The recent demonstration that GRXS15 coordinates an [2Fe-2S] cluster using glutathione molecules which can be transferred to an acceptor protein and that null mutants for the mitochondrial GRXS15 in Arabidopsis are embryo-lethal provided clear evidence that GRXS15 is an essential component of the ISC transfer machinery. This finding opens a new avenue towards molecular understanding of how mitochondrial Fe-S proteins are assembled. The proposed project aims primarily at dissecting the role of GRXS15 in the transfer of ISCs to target proteins in Arabidopsis. The biochemical, spectroscopic and structural analysis of GRXS15 holoforms obtained by in vitro anaerobic reconstitution will allow determining the oligomerisation status and nature of the assembled ISCs. On the basis of structural alignments and using targeted mutagenesis and combinatorial approaches, we will generate new knowledge on the structure-function relationship of this GRX and of proteins of the same class. The properties of these variants will be determined (i) by analysing their ability to bind an ISC and its lability, (ii) by finely examining protein-protein interactions with known or newly identified partner proteins, (iii) by performing heterologous expression in a yeast mutant deficient in mitochondrial Grx5 and (iv) by assessing their possible redox properties using in vitro activity assays with roGFP2 and oxidation sensitivity tests. Following earlier work, grxs15 null mutants partially rescued through expression of heterologous GRXs or mutated GRXS15 are expected to display distinct developmental and physiological phenotypes. Thus, it will be investigated whether these phenotypes are related to specific Fe-S enzyme defects and whether a bottleneck in mitochondrial ISC transfer is restricted to mitochondrial target proteins or whether it also affects cytosolic metalloenzymes. This could be because a yet unknown mitochondrial sulfur-containing compound is known to be exported for the maturation of cytosolic and nuclear proteins, and because synthesis of the molybdenum cofactor found in several cytosolic Fe-S enzymes requires a mitochondrial Fe-S enzyme.

• DFG Programme: Research Grants
• International Connection: France
• Partner Organisation: Agence Nationale de la Recherche / The French National Research Agency
• Cooperation Partner: Professor Dr. Nicolas Rouhier