Purification and biochemical characterization of Arabidopsis At-NEET, an ancient iron-sulfur protein, reveals a conserved cleavage motif for subcellular localization

•At-NEET is modified by post-translational cleavage in Arabidopsis.•At-NEET is localized in the chloroplast stroma.•Plant CISDs contain a conserved cleavage motif of V-[R/K]↓A-E.•The consensus cysteine residues of At-NEET are essential for coordination of 2Fe-2S clusters. CDGSH iron-sulfur domain-containing proteins (CISDs) are newly discovered proteins with electron-accepting and electron-donating moieties. Although the CISDs of plants and animals show high sequence similarity in their CDGSH domain at the C-terminus, their N-terminal peptides have low sequence homology. Here, we show that At-NEET, a recently identified Arabidopsis CISD, contains a cleavable N-terminal peptide for chloroplast targeting, which is different from the uncleavable N-terminal peptide of mammal CISDs for mitochondrial outer membrane localization. Using affinity purification to isolate endogenous At-NEET, we identified a consensus sequence for the chloroplast transit peptide cleavage site of V-[R/K]↓A-E in At-NEET as well as other plant CISDs. Moreover, chloroplast subfractionation and immunogold labeling experiments showed that At-NEET localizes to the stroma of chloroplast. In addition, biochemical characterization revealed that At-NEET contains a conserved Cys(3)-His(1) ligand in the CDGSH domain, which is essential for coordination of 2Fe-2S clusters and protein folding. Our findings suggest that plant and animal CISDs contain an evolutionarily conserved CDGSH domain. However, they show different subcellular localization patterns that may result in distinct physiological functions.