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Unique shaping of the chloroplastic iron-sulfur transfer protein NFU1 sustains dark chlorophyll synthesis and fermentation in Chlamydomonas reinhardtii

ABSTRACT: The sulfur mobilization (SUF) machinery is required for the synthesis of iron-sulfur (Fe- S) clusters and their insertion into client proteins in plastids. The final step relies on several Fe-S cluster transfer proteins, including NIFU-like (NFU) proteins. In this study, we focused on the chloroplastic elongated NFU1 from the green microalga Chlamydomonas reinhardtii. It possesses an N-terminal putative endonuclease domain present in orthologs from many eukaryotic photosynthetic organisms but not from Angiosperms. Using a reverse genetic approach, we demonstrated that CrNFU1 serves as the major maturation factor for several [4Fe-4S]-cluster containing proteins involved in specialized pathways. This includes an atypical hybrid cluster protein and an iron-iron hydrogenase operating in anoxia, as well as the dark-operative protochlorophyllide a oxidoreductase (DPOR) involved in chlorophyll synthesis in the dark. While the N-terminal domain has no in vitro DNA endonuclease activity, it is crucial for CrNFU1 function. Considering that CrNFU1 has retained the capacity to bind nucleic acids and was found associated with chloroplast ribosomes, we propose that the N-terminal domain of CrNFU1 contributes to the co-translational insertion of the [4Fe-4S] cluster(s) in DPOR subunits which are coded by the chloroplast genome. The strong co-occurrence between elongated NFU1 representatives and DPOR supports this intertwined function.

INSTRUMENT(S):

ORGANISM(S): Chlamydomonas Reinhardtii

SUBMITTER: Didier Vertommen

LAB HEAD: Claire Remacle

PROVIDER: PXD070162 | Pride | 2026-07-07

REPOSITORIES: Pride

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			Action	DRS
	EOS1006_A.raw	Raw
	EOS1006_B.raw	Raw
	EOS1006_C.raw	Raw
	EOS1006_D.raw	Raw
	EOS1006_E.raw	Raw

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

Project description: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 located in the cytosol, mitochondria and chloroplasts. After de novo formation on scaffold proteins, Fe-S centers are loaded on client proteins by use of Fe-S cluster 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 HCF101. NFU2 is additionally required for the maturation of the [2Fe-2S] dihydroxyacid dehydratase, important for branched-chain amino acid synthesis. Here, we report that recombinant Arabidopsis thaliana NFU1 is able to solely 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), have been further confirmed by bimolecular fluorescence complementation and in vitro Fe-S cluster transfer experiments. The additional interactions detected with SUFD and SUFA allowed building a model in which NFU1 receives its Fe-S cluster from the SUFBC2D scaffold complex and serves for the maturation of [4Fe-4S] client proteins.

2020-07-06 | PXD015295 | Pride

Fe-S clusters-binding proteins in mitoribosome

Project description:The human mitochondrial ribosome contains three [2Fe-2S] clusters whose assembly pathway, role, and implications for mitochondrial and metabolic diseases are unknown. Here, structure-function correlation studies show that the clusters play structural roles during mitoribosome assembly. To uncover the assembly pathway, we have examined the effect of silencing the expression of Fe-S cluster biosynthetic and delivery factors on mitoribosome stability. We have found that the mitoribosome receives its [2Fe-2S] clusters from the GLRX5-BOLA3. Additionally, the assembly of the small subunit depends on METTL17, recently reported to contain a [4Fe-4S] cluster, which we found to be inserted via ISCA1-NFU1. Consistently, fibroblasts from patients suffering from “multiple mitochondrial dysfunction” syndrome due to mutations in BOLA3 or NFU1 display previously unrecognized attenuation of mitochondrial protein synthesis that contributes to their cellular and pathophysiological phenotypes. Finally, we report that, in addition to their structural role, the mitoribosomal [2Fe-2S] clusters sense changes in the redox environment. In this way, the mitoribosome checks the availability and stability of mitochondrial Fe-S clusters to regulate organellar protein synthesis accordingly.

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WhiB1 transcriptone in response to NO stress

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E. coli monothiol glutaredoxin GrxD replenishes Fe-S clusters to the essential ErpA A-type carrier under low iron stress  

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2024-07-13 | PXD053317 | Pride

IscR of Rhodobacter sphaeroides functions as repressor of genes for iron-sulphur metabolism and represents a new type of iron-sulphur binding protein

Project description:Iron-sulphur (Fe-S) clusters are ensembles of iron and sulphide centres. They are found in all life forms and are important components of many enzymes involved in diverse cellular processes, including respiration, DNA synthesis or gene regulation. However, the increase in oxygen after the emergence of oxygenic photosynthesis created a threat to Fe–S proteins and, consequently, to the organisms relying on them. Therefore, bacteria have evolved mechanisms to maintain a precise intracellular iron concentration. A major role of IscR in R. sphaeroides iron dependent regulation was suggested in a bioinformatic study , which predicted a binding site in the upstream regions of several iron uptake genes. Most known IscR proteins have Fe-S clusters featuring (Cys)3(His)1 ligation. However, IscR proteins from Rhodobacteraceae harbour only one Cys residue and it was considered unlikely that they can ligate an Fe-S cluster. In this study, the role of R. sphaeroides IscR as transcriptional regulator and sensor of the Fe-S cluster status of the cell was analysed. The results provide evidence that R. sphaeroides IscR functions as transcriptional repressor of genes involved in iron metabolism by binding to the predicted DNA binding motif. Furthermore, IscR possesses a unique Fe-S cluster ligation scheme with only a single cysteine involved.

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2021-07-15 | GSE154169 | GEO

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