The cellular trafficking of Fe-S clusters in plants – Fe-S traffic

A functional genomic strategy was used for the characterization of all the members of each family, combining approaches of genetics and physiology (study of extinction lines of Arabidopsis thaliana), cell biology (study the subcellular localization of proteins and in vivo interactions (in yeast or in planta system)) and biochemistry and structural biology (study of protein-protein interactions, characterization of the three dimensional structure of proteins alone or in complex, partner isolation and identification).

The different experiences performed in yeast and in planta indicate that ISCA proteins interact with each other, likely forming ISCA2-4 and ISCA3-4 hetero-complexes. Furthermore we have found a physical interaction with NFU mitochondrial proteins, a result which is not described in the literature. In contrast to observations made in yeast or mammals, the study of mutant plants indicates that NFU and ISCA transfer proteins are essential in plants.

Understanding the cellular and molecular mechanisms controlling iron metabolism and more specifically the maturation of Fe-S proteins is a prerequisite to develop a strategy for overcoming situations of deficiency or excess iron for plants. Many human diseases are caused by Fe-S protein dysfunction and these mechanisms appear conserved between organisms. By understanding the molecular origin of these defects, the results could therefore help to find ways to fight against these diseases.

At mid-term of this project, the results were valued by two oral presentations made by one of the partners in international conferences and the setting of new international collaborations. Two publications are already in preparation.

Several metabolic pathways and cellular processes in plants depend on the functioning of iron-sulfur (Fe-S) proteins, whose cofactor is assembled through dedicated assembly machineries. To cite only a few examples, Fe-S proteins are needed for photosynthesis, respiration, sulfur and nitrogen assimilation, co-enzyme synthesis and by similarity with other eukaryotes, for DNA repair and replication or ribosome biogenesis. In plants as in other organisms, the incorporation of Fe-S clusters into proteins requires first the de novo assembly of Fe-S clusters onto scaffold proteins and then the transfer of these preformed clusters to acceptor proteins via the action of several chaperones and/or so-called carrier proteins. Using a combination of genetic, physiological, biochemical and structural approaches, the general objective of this research proposal is to understand precisely the molecular mechanisms controlling the second step, i.e., the delivery of Fe-S clusters from scaffold proteins to final acceptors, in the context of the chloroplastic and mitochondrial Fe-S cluster assembly machineries. Whereas the majority of the proteins required to assemble Fe-S clusters in the cells have likely been identified, the in vivo roles of many components, essentially those involved in Fe-S cluster trafficking, remain to be clarified. Owing to the fact that there are several dozens of Fe-S proteins but relatively few scaffold proteins in cells, carrier proteins are essential sentinels ensuring the correct and specific distribution of the different types of Fe-S clusters to acceptor client proteins. This project focuses on the Nfu and A-type carrier protein families which are assumed, from current working models, to be the major contributors for the trafficking of Fe-S clusters. Incidentally, in addition to providing improved knowledge on the global functioning of these biogenesis systems, the designed experimental program, which combines in vitro and in vivo approaches, should bring crucial information about the cellular network and regulatory mechanisms coordinating the concerted action of the different components.