Background: Proteins incorporating iron-sulfur (Fe-S) cofactors are required for a plethora of metabolic processes. Their maturation depends on three Fe-S cluster assembly machineries in plants, located in the cytosol, mitochondria and chloroplasts. After de novo formation on scaffold proteins, Fe-S clusters are loaded onto client proteins by use of transfer proteins. Among plastidial representatives of this latter category, NFU2 and NFU3 are required for the maturation of the [4Fe-4S] clusters found in photosystem I subunits, acting upstream of the high-chlorophyll fluorescence 101 (HCF101) protein. NFU2 is additionally required for the maturation of the [2Fe-2S] dihydroxyacid dehydratase, important for branched-chain amino acid synthesis.

Question: NFU1 is a paralog of NFU2 and NFU3. In this study we aimed at determining if NFU1 has either an accessory or very specific function or a functional redundancy with the other Fe-S transfer protein(s).

Findings: Here, we report that recombinant Arabidopsis thaliana NFU1 is able to assemble a [4Fe-4S] cluster per homodimer. We also provide insights into the specificity of NFU1 for the maturation of chloroplastic Fe-S proteins by performing co-immunoprecipitation experiments and assaying the physical interaction of NFU1 with many [4Fe-4S]-containing plastidial proteins using binary yeast two-hybrid assays. Interactions with two proteins involved in isoprenoid and thiamine biosynthesis, respectively 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (ISPG) and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (THIC), were further confirmed by bimolecular fluorescence complementation and in vitro Fe-S cluster transfer experiments. The additional interaction detected with the scaffold protein SUFD allowed building a model in which NFU1 receives its Fe-S cluster from the SUFBC2D scaffold complex and serves for the maturation of specific [4Fe-4S] client proteins.