Euglena gracilis is a metabolically flexible, photosynthetic, and adaptable free-living protist of considerable environmental importance and biotechnological value. By label-free liquid chromatography tandem mass spectrometry, a total of 1,786 proteins were identified from the E. gracilis purified mitochondria, representing one of the largest mitochondrial proteomes so far described. Despite this apparent complexity, protein machinery responsible for the extensive RNA editing, splicing, and processing in the sister clades diplonemids and kinetoplastids is absent. This strongly suggests that the complex mechanisms of mitochondrial gene expression in diplonemids and kinetoplastids occurred late in euglenozoan evolution, arising independently. By contrast, the alternative oxidase pathway and numerous ribosomal subunits presumed to be specific for parasitic trypanosomes are present in E. gracilis. We investigated the evolution of unexplored protein families, including import complexes, cristae formation proteins, and translation termination factors, as well as canonical and unique metabolic pathways. We additionally compare this mitoproteome with the transcriptome of Eutreptiella gymnastica, illuminating conserved features of Euglenida mitochondria as well as those exclusive to E. gracilis. This is the first mitochondrial proteome of a free-living protist from the Excavata and one of few available for protists as a whole. This study alters our views of the evolution of the mitochondrion and indicates early emergence of complexity within euglenozoan mitochondria, independent of parasitism.

• Keywords
• Euglena gracilis, Euglenozoa, mitochondria, proteome, protist,
• MeSH
• Euglena gracilis metabolism   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Proteome * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Mitochondrial Proteins MeSH
• Proteome * MeSH

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

• MeSH
• Humans MeSH
• Mitochondrial Proteins metabolism   MeSH
• Proteome analysis   metabolism   MeSH
• Protozoan Proteins metabolism   MeSH
• Life Cycle Stages physiology   MeSH
• Trypanosoma brucei brucei growth & development   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Mitochondrial Proteins MeSH
• Proteome MeSH
• Protozoan Proteins MeSH