Aw, Yi Tong VincentSeidi, AzadehHayward, JenniLee, JiwonMakota, VictorMelanie, Rugvan Dooren, Giel2023-02-070950-382Xhttp://hdl.handle.net/1885/285059Iron-sulfur (Fe-S) clusters are prosthetic groups on proteins that function in a range of enzymatic and electron transfer reactions. Fe-S cluster synthesis is essential for the survival of all eukaryotes. Independent Fe-S cluster biosynthesis pathways occur in the mitochondrion, plastid, and cytosolic compartments of eukaryotic cells. Little is known about the cytosolic Fe-S cluster biosynthesis in apicomplexan parasites, the causative agents of diseases such as malaria and toxoplasmosis. NBP35 serves as a key scaffold protein on which cytosolic Fe-S clusters assemble, and has a cytosolic localization in most eukaryotes studied thus far. Unexpectedly, we found that the NBP35 homolog of the apicomplexan Toxoplasma gondii (TgNBP35) localizes to the outer mitochondrial membrane, with mitochondrial targeting mediated by an N-terminal transmembrane domain. We demonstrate that TgNBP35 is critical for parasite proliferation, but that, despite its mitochondrial localization, it is not required for Fe-S cluster synthesis in the mitochondrion. Instead, we establish that TgNBP35 is important for the biogenesis of cytosolic Fe-S proteins. Our data are consistent with TgNBP35 playing a central and specific role in cytosolic Fe-S cluster biosynthesis, and imply that the assembly of cytosolic Fe-S clusters occurs on the cytosolic face of the outer mitochondrial membrane in these parasites.Research School of Biology; Australian Research Council, Grant/Award Number: DP110103144; National Health and Medical Research Council, Grant/Award Number: GNT1182369application/pdfen-AU© 2020 John Wiley & Sons LtdApicomplexairon–sulfur clustermitochondriaToxoplasma202010.1111/mmi.146512021-12-02