A novel targeting domain directs essential components of the cytosolic iron–sulfur cluster assembly pathway to the mitochondrion of Toxoplasma parasites

The assembly of iron–sulfur (FeS) clusters for cytosolic and nuclear proteins is essential for eukaryotic cell biology. This assembly is mediated by the Cytosolic Iron–sulfur Assembly (CIA) pathway, which localizes to the cytosol of most eukaryotes. We showed previously that the scaffold protein on which cytosolic FeS clusters assemble localizes to the mitochondrion of the apicomplexan parasite Toxoplasma gondii . The localization and importance of the remainder of the pathway in these parasites, however, remained unclear. Here, we undertake a comprehensive analysis of the CIA pathway in T. gondii parasites. We present evidence that the CIA pathway localizes predominantly to the mitochondrion of the parasite and is essential for parasite survival. We show that the three proteins that make up the CIA Targeting Complex (CTC), which facilitates the transfer of FeS clusters to cytosolic and nuclear client proteins, exhibit dual localization to the mitochondrion and cytosol. We reveal that mitochondrial targeting of the CTC is mediated by a novel loop on the CIA1 protein of the complex, and that this loop is critical for parasite survival. We show that an aromatic amino acid motif in the loop facilitates mitochondrial targeting, and that this loop is functionally conserved in apicomplexans and their closest free-living relatives. Our study provides a comprehensive analysis of the CIA pathway in an important group of intracellular parasites, and elucidates pivotal differences in an otherwise ancient and highly conserved biosynthetic pathway that may reflect an evolutionary fitness advantage conferred on Toxoplasma and related organisms.