RESUMO
Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.
Assuntos
Liases de Carbono-Enxofre/metabolismo , Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/enzimologia , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/genética , Ferredoxinas/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Sinais de Localização Nuclear , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética , Ligação Proteica , Multimerização Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , FrataxinaRESUMO
Fe/S clusters are part of the active site of many enzymes and are essential for cell viability. In eukaryotes the cysteine desulfurase Nfs (IscS) donates the sulfur during Fe/S cluster assembly and was thought sufficient for this reaction. Moreover, Nfs is indispensable for tRNA thiolation, a modification generally required for tRNA function and protein synthesis. Recently, Isd11 was discovered as an integral part of the Nfs activity at an early step of Fe/S cluster assembly. Here we show, using a combination of genetic, molecular, and biochemical approaches, that Isd11, in line with its strong association with Nfs, is localized in the mitochondrion of T. brucei. In addition to its involvement in Fe/S assembly, Isd11 also partakes in both cytoplasmic and mitochondrial tRNA thiolation, whereas Mtu1, another protein proposed to collaborate with Nfs in tRNA thiolation, is required for this process solely within the mitochondrion. Taken together these data place Isd11 at the center of these sulfur transactions and raises the possibility of a connection between Fe/S metabolism and protein synthesis, helping integrate two seemingly unrelated pathways.