NBP35 interacts with DRE2 in the maturation of cytosolic iron-sulfur proteins in Arabidopsis thaliana

Proteins of the cytosolic pathway for iron-sulfur (FeS) cluster assembly are conserved, except that plants lack a gene for CFD1 (Cytosolic FeS cluster Deficient 1). This poses the question of how NBP35 (Nucleotide-Binding Protein 35 kDa), the heteromeric partner of CFD1 in metazoa, functions on its own in plants. Firstly, we created viable mutant alleles of NBP35 in Arabidopsis to overcome embryo lethality of previously reported knockout mutations. RNAi knockdown lines with less than 30% NBP35 protein surprisingly showed no developmental or biochemical differences to wild type. Substitution of Cys14 to Ala, which destabilized the N-terminal Fe4S4 cluster in vitro, caused mild growth defects and a significant decrease in the activity of cytosolic FeS enzymes such as aconitase and aldehyde oxidases. The DNA glycosylase ROS1 was only partially decreased in activity and xanthine dehydrogenase not at all. Plants with strongly depleted NBP35 protein in combination with Cys14 to Ala substitution had distorted leaf development and decreased FeS enzyme activities. To find protein interaction partners of NBP35, a yeast two-hybrid screen was carried out which identified NBP35 and DRE2 (Derepressed for Ribosomal protein S14 Expression). NBP35 is known to form a dimer, and DRE2 acts upstream in the cytosolic FeS protein assembly pathway. The NBP35-DRE2 interaction was not disrupted by Cys14 to Ala substitution. Our results show that NBP35 has a function in the maturation of FeS proteins that is conserved in plants, and is closely allied to the function of DRE2

Chloroplast HCF101 is a scaffold protein for [4Fe-4S] cluster assembly

Oxygen-evolving chloroplasts possess their own iron-sulfur cluster assembly proteins including members of the SUF (sulfur mobilization) and the NFU family. Recently, the chloroplast protein HCF101 (high chlorophyll fluorescence 101) has been shown to be essential for the accumulation of the membrane complex Photosystem I and the soluble ferredoxin-thioredoxin reductases, both containing [4Fe-4S] clusters. The protein belongs to the FSC-NTPase ([4Fe-4S]-cluster-containing P-loop NTPase) superfamily, several members of which play a crucial role in Fe/S cluster biosynthesis. Although the C-terminal ISC-binding site, conserved in other members of the FSC-NTPase family, is not present in chloroplast HCF101 homologues using Mössbauer and EPR spectroscopy, we provide evidence that HCF101 binds a [4Fe-4S] cluster. 55Fe incorporation studies of mitochondrially targeted HCF101 in Saccharomyces cerevisiae confirmed the assembly of an Fe/S cluster in HCF101 in an Nfs1-dependent manner. Site-directed mutagenesis identified three HCF101-specific cysteine residues required for assembly and/or stability of the cluster. We further demonstrate that the reconstituted cluster is transiently bound and can be transferred from HCF101 to a [4Fe-4S] apoprotein. Together, our findings suggest that HCF101 may serve as a chloroplast scaffold protein that specifically assembles [4Fe-4S] clusters and transfers them to the chloroplast membrane and soluble target proteins

RNA silencing of hydrogenase(-like) genes and investigation of their physiological roles in the green alga Chlamydomonas reinhardtii

The genome of the green alga Chlamydomonas reinhardtii encodes two [FeFe]-hydrogenases, HydA1 and HydA2, and the hydrogenase-like protein HYD3. The unique combination of these proteins in one eukaryotic cell allows for direct comparison of their in vivo functions, which have not been established for HydA2 and HYD3. Using an artificial microRNA silencing method developed recently, the expression of HydA1, HydA2 and HYD3 was specifically down-regulated. Silencing of HydA1 resulted in 4-fold lower hydrogenase protein and activity under anaerobic conditions. In contrast, silencing of HydA2 or HYD3 did not affect hydrogen production. Cell lines with strongly (>90%) decreased HYD3 transcript levels grew more slowly than wild-type. The activity of aldehyde oxidase, a cytosolic Fe-S enzyme, was decreased in HYD3-knockdown lines, whereas Fe-S dependent activities in the chloroplast and mitochondria were unaffected. In addition, the HYD3-knockdown lines grew poorly on hypoxanthine, indicating impaired function of xanthine dehydrogenase, another cytosolic Fe-S enzyme. The expression levels of selected genes in response to hypoxia were unaltered upon HYD3 silencing. Together, our results clearly distinguish the cellular roles of HydA1 and HYD3, and indicate that HYD3, like its yeast and human homologues, has an evolutionary conserved role in the biogenesis or maintenance of cytosolic Fe-S proteins

Cytosolic Iron-Sulfur Cluster Assembly (CIA) System: Factors, Mechanism, and Relevance to Cellular Iron Regulation*

FeS cluster biogenesis is an essential process in virtually all forms of life. Complex protein machineries that are conserved from bacteria through higher eukaryotes facilitate assembly of the FeS cofactor in proteins. In the last several years, significant strides have been made in our understanding of FeS cluster assembly and the functional overlap of this process with cellular iron homeostasis. This minireview summarizes the present understanding of the cytosolic iron-sulfur cluster assembly (CIA) system in eukaryotes, with a focus on information gained from studies in budding yeast and mammalian systems