The plastidial Arabidopsis thaliana NFU1 protein binds and delivers [4Fe-4S] clusters to specific client proteins

Abstract

Proteins incorporating iron–sulfur (Fe-S) co-factors 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, transfer proteins load Fe-S clusters onto client proteins. Among the plastidial representatives of these transfer proteins, NFU2 and NFU3 are required for the maturation of the [4Fe-4S] clusters present in photosystem I subunits, acting upstream of the high-chlorophyll fluorescence 101 (HCF101) protein. NFU2 is also required for the maturation of the [2Fe-2S]-containing dihydroxyacid dehydratase, important for branched-chain amino acid synthesis. Here, we report that recombinant Arabidopsis thaliana NFU1 assembles one [4Fe-4S] cluster per homodimer. Performing co-immunoprecipitation experiments and assessing physical interactions of NFU1 with many [4Fe-4S]-containing plastidial proteins in binary yeast two-hybrid assays, we also gained insights into the specificity of NFU1 for the maturation of chloroplastic Fe-S proteins. Using bimolecular fluorescence complementation and in vitro Fe-S cluster transfer experiments, we confirmed interactions with two proteins involved in isoprenoid and thiamine biosynthesis, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase, respectively. An additional interaction detected with the scaffold protein SUFD enabled us to build a model in which NFU1 receives its Fe-S cluster from the SUFBC2D scaffold complex and serves in the maturation of specific [4Fe-4S] client proteins. The identification of the NFU1 partner proteins reported here more clearly defines the role of NFU1 in Fe-S client protein maturation in Arabidopsis chloroplasts among other SUF components.

Functional relationships of three NFU proteins in the biogenesis of chloroplastic iron‐sulfur clusters

“…As described previously, the coding region of NFU1 includes a plastid transit peptide (1‐69 AAs), a redox‐active NFU domain (90‐156 AAs) with the conserved CXXC motif, and a redox‐inactive NFU domain (168‐227 AAs) at the C‐terminus (Figure 4a). Fluorescent protein tagging and confocal microscopic analysis showed that NFU1 is targeted to the chloroplast (Léon et al., 2003; Roland et al., 2020). NFU1 was proposed to bind and deliver 4Fe‐4S clusters and possibly 3Fe‐4S clusters to recipient apoproteins (Roland et al., 2020).…”

“…Fluorescent protein tagging and confocal microscopic analysis showed that NFU1 is targeted to the chloroplast (Léon et al., 2003; Roland et al., 2020). NFU1 was proposed to bind and deliver 4Fe‐4S clusters and possibly 3Fe‐4S clusters to recipient apoproteins (Roland et al., 2020). Therefore, we expressed the 6xHis‐tagged NFU1 protein in Escherichia coli strain Rosetta 2 (DE3) and purified the recombinant NFU1 protein with nickel‐charged agarose resin under native and aerobic conditions.…”

“…It was proposed that NFU1 may bind and deliver iron‐sulfur clusters (i.e., 4Fe‐4S and 3Fe‐4S) to recipient apoproteins (Roland et al., 2020). Using spectroscopic analysis, we found that the affinity‐purified recombinant NFU1 protein had a broad absorption peak at ~410 nm (Figure 4b), a feature of 4Fe‐4S and 3Fe‐4S clusters (Kennedy et al., 1984; Nakamaru‐Ogiso et al., 2002).…”

“…Full‐length NFU1, NFU2, and NFU3 contain a plastid transit peptide, a redox‐active NFU domain with a conserved CXXC motif (C stands for cysteine, X stands for any amino acid), and a redox‐inactive NFU domain (Léon et al., 2003; Lu, 2018; Przybyla‐Toscano et al., 2018; Yabe et al., 2004). The chloroplast localization of NFU1, NFU2, and NFU3 had been demonstrated by fluorescent protein tagging and confocal microscopic analysis (Léon et al., 2003; Nath et al., 2017; Roland et al., 2020).…”

Sulphur availability modulates Arabidopsis thaliana responses to iron deficiency

“…In plants, Fe-S proteins are mainly involved in electron transfer reactions (e.g. within the photosynthetic and respiratory electron transport chains in chloroplasts and mitochondria, respectively) or in the catalysis of oxidation-reduction reactions [2][3][4][5]. The biosynthesis of cysteine also connects Fe and S homeostasis, since the desulphuration of cysteine to alanine is a reaction in the assembly process of Fe-S clusters [2].…”