Résumé
Among the three NFU proteins found in plastids, reverse genetic analyses previously indicated that A. thaliana Nfu2 was primarily required for the assembly of iron-sulfur (Fe-S) clusters into photosystem I but also into ferredoxin1,2. Consistently, the spectroscopic characterization of in vitro reconstituted recombinant AtNfu2 indicates that it can indeed assemble one [2Fe-2S] cluster per homotetramer and one [4Fe-4S] cluster per homodimer. Both [2Fe-2S] or [4Fe-4S] cluster-bound forms of Nfu2 are competent cluster donors for apoforms of A. thaliana glutaredoxin S16 or APR1 (adenosine 5’-phosphosulfate reductase 1) respectively3. We now show that the root development of the nfu2 mutant is strongly affected compared to wt plants with a root length decrease of about 70% under standard conditions. While Nfu2 can specifically interact with and transfer a [2Fe-2S] cluster to dihydroxy-acid dehydratase (DHAD) among late-acting Fe-S transfer proteins of the SUF machinery, this observed growth defect is only partially restored by the addition of branched-chain amino acids, suggesting that other targets are responsible for this phenotype. The detailed spectroscopic characterization of the Fe-S center in AtDHAD indicated that it binds a [2Fe-2S] cluster coordinated by at least one non-thiolate ligand which can be replaced by the thiol group of dithiothreitol. Next, the relative function/contribution of the two other plastidial Nfu proteins from A. thaliana, Nfu1 and Nfu3, has been investigated through a genetic approach and compared with Nfu2. While T-DNA insertion lines disrupted for nfu1 did not present any visible phenotype when grown under standard conditions, nfu3 mutants exhibited a growth reduction, even stronger than the one observed in nfu2 mutants. This is accompanied by a decreased photosynthesis efficiency, which is explained by a decrease in PSI amounts and an altered PSI activity as assessed by immunodetection experiments and low temperature fluorescence emission spectra. Finally, using yeast two hybrid experiments, we have explored the interactions between Nfu proteins and other components of the SUF machinery, including HCF101, a protein also known to be required for PSI assembly and PsaC, one of the PSI subunit accommodating Fe-S clusters. Altogether, these results suggest that, in shoots, both Nfu2 and Nfu3 act as late Fe-S transfer proteins, being crucial for PSI biogenesis likely by delivering [4Fe-4S] clusters to HCF101.References1. Touraine B, Boutin JP, Marion-Poll A, Briat JF, Peltier G, Lobréaux S. (2004) Nfu2: a scaffold protein required for [4Fe-4S] and ferredoxin iron-sulphur cluster assembly in Arabidopsis chloroplasts. Plant J. 40, 101-11.2. Yabe T, Morimoto K, Kikuchi S, Nishio K, Terashima I, Nakai M. (2004) The Arabidopsis chloroplastic NifU-like protein CnfU, which can act as an iron-sulfur cluster scaffold protein, is required for biogenesis of ferredoxin and photosystem I. Plant Cell. 16, 993-1007.3. Gao H, Subramanian S, Couturier J, Naik SG, Kim SK, Leustek T, Knaff DB, Wu HC, Vignols F, Huynh BH, Rouhier N, Johnson MK. (2013) Arabidopsis thaliana Nfu2 accommodates [2Fe-2S] or [4Fe-4S] clusters and is competent for in vitro maturation of chloroplast [2Fe-2S] and [4Fe-4S] cluster-containing proteins. Biochemistry. 52, 6633-45.