Fe-S clusters are ubiquitous cofactors of proteins involved in a variety of essential cellular processes. The biogenesis of Fe-S clusters in the cytosol and their insertion into proteins is accomplished through the cytosolic iron-sulphur protein assembly (CIA) machinery. The early- and middle-acting modules of the CIA pathway concerned with the assembly and trafficking of Fe-S clusters have been previously characterised in the parasitic protist Trypanosoma brucei. In this study, we applied proteomic and genetic approaches to gain insights into the network of protein-protein interactions of the late-acting CIA targeting complex in T. brucei. All components of the canonical CIA machinery are present in T. brucei including, as in humans, two distinct CIA2 homologues TbCIA2A and TbCIA2B. These two proteins are found interacting with TbCIA1, yet the interaction is mutually exclusive, as determined by mass spectrometry. Ablation of most of the components of the CIA targeting complex by RNAi led to impaired cell growth in vitro, with the exception of TbCIA2A in procyclic form (PCF) trypanosomes. Depletion of the CIA-targeting complex was accompanied by reduced levels of protein-bound cytosolic iron and decreased activity of an Fe-S dependent enzyme in PCF trypanosomes. We demonstrate that the C-terminal domain of TbMMS19 acts as a docking site for TbCIA2B and TbCIA1, forming a trimeric complex that also interacts with target Fe-S apo-proteins and the middle-acting CIA component TbNAR1.

• MeSH
• Cytosol metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Protein Conformation MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Iron-Sulfur Proteins chemistry   metabolism   MeSH
• Protozoan Proteins chemistry   metabolism   MeSH
• Trypanosoma brucei brucei growth & development   metabolism   MeSH
• Trypanosomiasis metabolism   parasitology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Iron-Sulfur Proteins MeSH
• Protozoan Proteins MeSH

The majority of established model organisms belong to the supergroup Opisthokonta, which includes yeasts and animals. While enlightening, this focus has neglected protists, organisms that represent the bulk of eukaryotic diversity and are often regarded as primitive eukaryotes. One of these is the "supergroup" Excavata, which comprises unicellular flagellates of diverse lifestyles and contains species of medical importance, such as Trichomonas, Giardia, Naegleria, Trypanosoma and Leishmania. Excavata exhibits a continuum in mitochondrial forms, ranging from classical aerobic, cristae-bearing mitochondria to mitochondria-related organelles, such as hydrogenosomes and mitosomes, to the extreme case of a complete absence of the organelle. All forms of mitochondria house a machinery for the assembly of Fe-S clusters, ancient cofactors required in various biochemical activities needed to sustain every extant cell. In this review, we survey what is known about the Fe-S cluster assembly in the supergroup Excavata. We aim to bring attention to the diversity found in this group, reflected in gene losses and gains that have shaped the Fe-S cluster biogenesis pathways.

• Keywords
• Evolution, Excavata, Fe–S cluster, Mitochondria,
• MeSH
• Eukaryota cytology   metabolism   MeSH
• Mitochondria metabolism   MeSH
• Iron-Sulfur Proteins metabolism   MeSH
• Iron metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Iron-Sulfur Proteins MeSH
• Iron MeSH

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.

• MeSH
• Active Transport, Cell Nucleus MeSH
• Cell Nucleus metabolism   MeSH
• Ferredoxins metabolism   MeSH
• Frataxin MeSH
• Nuclear Localization Signals MeSH
• Carbon-Sulfur Lyases chemistry   genetics   metabolism   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondria metabolism   MeSH
• Molecular Sequence Data MeSH
• Protein Multimerization MeSH
• Nuclear Matrix-Associated Proteins chemistry   genetics   metabolism   MeSH
• Iron-Binding Proteins metabolism   MeSH
• Protozoan Proteins chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Trypanosoma brucei brucei enzymology   genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• cysteine desulfurase MeSH Browser
• Ferredoxins MeSH
• Nuclear Localization Signals MeSH
• Carbon-Sulfur Lyases MeSH
• Mitochondrial Proteins MeSH
• Nuclear Matrix-Associated Proteins MeSH
• Iron-Binding Proteins MeSH
• Protozoan Proteins MeSH

The anaerobic intestinal pathogen Giardia intestinalis does not possess enzymes for heme synthesis, and it also lacks the typical set of hemoproteins that are involved in mitochondrial respiration and cellular oxygen stress management. Nevertheless, G. intestinalis may require heme for the function of particular hemoproteins, such as cytochrome b5 (cytb5). We have analyzed the sequences of eukaryotic cytb5 proteins and identified three distinct cytb5 groups: group I, which consists of C-tail membrane-anchored cytb5 proteins; group II, which includes soluble cytb5 proteins; and group III, which comprises the fungal cytb5 proteins. The majority of eukaryotes possess both group I and II cytb5 proteins, whereas three Giardia paralogs belong to group II. We have identified a fourth Giardia cytb5 paralog (gCYTb5-IV) that is rather divergent and possesses an unusual 134-residue N-terminal extension. Recombinant Giardia cytb5 proteins, including gCYTb5-IV, were expressed in Escherichia coli and exhibited characteristic UV-visible spectra that corresponded to heme-loaded cytb5 proteins. The expression of the recombinant gCYTb5-IV in G. intestinalis resulted in the increased import of extracellular heme and its incorporation into the protein, whereas this effect was not observed when gCYTb5-IV containing a mutated heme-binding site was expressed. The electrons for Giardia cytb5 proteins may be provided by the NADPH-dependent Tah18-like oxidoreductase GiOR-1. Therefore, GiOR-1 and cytb5 may constitute a novel redox system in G. intestinalis. To our knowledge, G. intestinalis is the first anaerobic eukaryote in which the presence of heme has been directly demonstrated.

• MeSH
• Cytochromes b5 chemistry   metabolism   MeSH
• Cytoplasm metabolism   MeSH
• Giardia chemistry   metabolism   MeSH
• Heme metabolism   MeSH
• Molecular Sequence Data MeSH
• Protozoan Proteins chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Protein Transport MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytochromes b5 MeSH
• Heme MeSH
• Protozoan Proteins MeSH

In yeast (Saccharomyces cerevisiae) and animals, the sulfhydryl oxidase Erv1 functions with Mia40 in the import and oxidative folding of numerous cysteine-rich proteins in the mitochondrial intermembrane space (IMS). Erv1 is also required for Fe-S cluster assembly in the cytosol, which uses at least one mitochondrially derived precursor. Here, we characterize an essential Erv1 orthologue from the protist Trypanosoma brucei (TbERV1), which naturally lacks a Mia40 homolog. We report kinetic parameters for physiologically relevant oxidants cytochrome c and O(2), unexpectedly find O(2) and cytochrome c are reduced simultaneously, and demonstrate that efficient reduction of O(2) by TbERV1 is not dependent upon a simple O(2) channel defined by conserved histidine and tyrosine residues. Massive mitochondrial swelling following TbERV1 RNA interference (RNAi) provides evidence that trypanosome Erv1 functions in IMS protein import despite the natural absence of the key player in the yeast and animal import pathways, Mia40. This suggests significant evolutionary divergence from a recently established paradigm in mitochondrial cell biology. Phylogenomic profiling of genes also points to a conserved role for TbERV1 in cytosolic Fe-S cluster assembly. Conversely, loss of genes implicated in precursor delivery for cytosolic Fe-S assembly in Entamoeba, Trichomonas, and Giardia suggests fundamental differences in intracellular trafficking pathways for activated iron or sulfur species in anaerobic versus aerobic eukaryotes.

• MeSH
• Cytochromes c chemistry   MeSH
• Phylogeny MeSH
• Gene Knockdown Techniques MeSH
• Kinetics MeSH
• Oxygen chemistry   MeSH
• Mitochondrial Proteins chemistry   genetics   MeSH
• Mitochondria enzymology   ultrastructure   MeSH
• Evolution, Molecular MeSH
• Mutagenesis, Site-Directed MeSH
• Oxidation-Reduction MeSH
• Oxidants MeSH
• Oxidoreductases chemistry   genetics   MeSH
• Protozoan Proteins chemistry   genetics   MeSH
• RNA Interference MeSH
• Protein Folding MeSH
• Amino Acid Substitution MeSH
• Protein Transport MeSH
• Trypanosoma brucei brucei cytology   enzymology   MeSH
• Mitochondrial Swelling MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytochromes c MeSH
• Oxygen MeSH
• Mitochondrial Proteins MeSH
• Oxidants MeSH
• Oxidoreductases MeSH
• Protozoan Proteins MeSH
• sulfhydryl oxidase MeSH Browser