Iron-sulfur (Fe-S) clusters are universal co-factors embedded in proteins within which they promote electron transfer, catalytic activities and protein folding (1). Proteins carrying such cofactors (Fe-S client apoproteins) support a large panel of fundamental biological functions. To supply them with Fe-S clusters, cells have evolved towards the use of multicomponent Fe-S assembly and delivery machineries in various cellular compartments. They include the ISC (Iron-Sulfur Cluster) machinery in proteobacteria and in eukaryotic mitochondria, the SUF (Sulfur Utilization Factor) machinery in plastids, and the eukaryotic CIA (Cytosolic Iron–sulfur Assembly) machinery dedicated to cytosolic and nuclear client apoproteins (2-3). Because of the importance of Fe-S client proteins in many cellular processes, our collaborative project aims at identifying essential components of these machineries and their specific roles in these pathways. Here we focus on HCF101 proteins, a small class of atypical P-Loop NTPases belonging to restricted and divergent eukaryotic phyla, including plant, algae and apicomplexan parasites. The Arabidopsis HCF101 was the first identified as a late-acting Fe-S carrier protein in the SUF machinery (4-5) and shown to be essential for photosynthesis by targeting the [4Fe-4S] PsaC photosystem I (PSI) subunit (6). Here we show that AtHCF101 is also essential for the biogenesis of the NDH (NADP dehydrogenase) complex in plastid, by targeting two Fe-S proteins of the NDH subcomplex A (7). The Apicomplex parasite Toxoplasma gondii (Tg) also possesses an ancestral plastid (apicoplast) but its photosynthetic function has been lost during evolution, questioning the function of TgHCF101. Contrary to plants, we found that TgHCF101 localizes in the cytosol where it interacts with ABCE1, a highly conserved [4Fe-4S] protein required for eukaryotic translation initiation and ribosome biogenesis (8). In conclusion, although HCF101 proteins share a highly conserved three-domain structure, they have evolved specialized functions in distinct Fe-S assembly machineries in plants and Apicomplex parasites.