Kofaktory obsahující železo u anaerobního parazita Giardia intestinalis

Železo je nepostradatelným prvkem téměř pro všechny organismy. Nejčastěji je v buňce přítomné jako součást železitosirných (FeS) klastrů a hemu. Díky těmto kofaktorům mají proteiny schopnost katalyzovat enzymatickou reakci, přenášet elektrony a plyny a nebo detekovat signál. Mnoho FeS a hemových proteinů se účastní dobře prostudovaných drah, jako je fotosyntéza nebo dýchací řetězec. Nicméně o syntéze FeS klastrů v různých buněčných kompartmentech nebo o roli nově objevených FeS nebo hemo-proteinů se stále mnoho informací neví. Velmi málo je známo především o tom, jak jsou u anaerobně žijících protistů FeS klastry formovány nebo jak je využíván hem. V těchto organismech se totiž dráhy dýchacího řetězce či fotosyntézy nevyskytují. Rozhodli jsme se proto zaměřit na železo obsahující kofaktory u anaerobního parazita Giardia intestinalis. Tento organismus prošel dramatickou reduktivní evolucí, jejímž výsledkem je vznik jednoho z nejmenších eukaryotických genomů a nejvíce redukované formy mitochondrie - mitosomu. Charakterizovali jsme některé komponenty mitochondriální (ISC) a cytoplasmatické (CIA) FeS klastrovací dráhy. Pomocí proteomické analýzy jsme nejprve detekovali komponenty dráhy ISC v mitosomu. Poté jsme analyzovali přítomnost a buněčnou lokalizaci CIA proteinů. V genomu G. intestinalis jsme...Iron is an essential element in nearly all organisms. It is present mainly as a component of iron sulfur (FeS) clusters or as a heme iron. These cofactors enable proteins to transfer electrons or diatomic gasses, signal sensing and enzyme catalysis. Numerous FeS and heme depending proteins are involved in photosynthesis and respiratory chain pathways, which are well described processes. However, there is still much to learn about more recently discovered pathways such as formation of FeS clusters in various cell compartments and about roles of novel FeS or heme proteins. Particularly, only limited information is available about how FeS clusters are assembled or how heme is used in anaerobic protists, in which cytochrome-dependent respiration and photosynthesis does not occur. We decided to focus on iron cofactors in anaerobic parasite Giardia intestinalis. This organism undergone dramatic reductive evolution that resulted in formation of one of the smallest eukaryotic genome and the most reduced form of mitochondria, the mitosome. We characterized some components of mitochondrial (ISC) and cytoplasmic (CIA) FeS assembly machineries. We have detected ISC components in mitosome by proteomic analysis. Furthermore we investigated the presence and subcellular localization of CIA proteins in Giardia. In...Katedra parazitologieDepartment of ParasitologyPřírodovědecká fakultaFaculty of Scienc

Vestiges of the Bacterial Signal Recognition Particle-Based Protein Targeting in Mitochondria

The main bacterial pathway for inserting proteins into the plasma membrane relies on the signal recognition particle (SRP), composed of the Ffh protein and an associated RNA component, and the SRP-docking protein FtsY. Eukaryotes use an equivalent system of archaeal origin to deliver proteins into the endoplasmic reticulum, whereas a bacteria-derived SRP and FtsY function in the plastid. Here we report on the presence of homologs of the bacterial Ffh and FtsY proteins in various unrelated plastid-lacking unicellular eukaryotes, namely Heterolobosea, Alveida, Goniomonas, and Hemimastigophora. The monophyly of novel eukaryotic Ffh and FtsY groups, predicted mitochondrial localization experimentally confirmed for Naegleria gruberi, and a strong alphaproteobacterial affinity of the Ffh group, collectively suggest that they constitute parts of an ancestral mitochondrial signal peptide-based protein-targeting system inherited from the last eukaryotic common ancestor, but lost from the majority of extant eukaryotes. The ability of putative signal peptides, predicted in a subset of mitochondrial-encoded N. gruberi proteins, to target a reporter fluorescent protein into the endoplasmic reticulum of Trypanosoma brucei, likely through their interaction with the cytosolic SRP, provided further support for this notion. We also illustrate that known mitochondrial ribosome-interacting proteins implicated in membrane protein targeting in opisthokonts (Mba1, Mdm38, and Mrx15) are broadly conserved in eukaryotes and nonredundant with the mitochondrial SRP system. Finally, we identified a novel mitochondrial protein (MAP67) present in diverse eukaryotes and related to the signal peptide-binding domain of Ffh, which may well be a hitherto unrecognized component of the mitochondrial membrane protein-targeting machinery

Analysis of diverse eukaryotes suggests the existence of an ancestral mitochondrial apparatus derived from the bacterial type II secretion system

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion

The Minimal Proteome in the Reduced Mitochondrion of the Parasitic Protist Giardia intestinalis

The mitosomes of Giardia intestinalis are thought to be mitochondria highly-reduced in response to the oxygen-poor niche. We performed a quantitative proteomic assessment of Giardia mitosomes to increase understanding of the function and evolutionary origin of these enigmatic organelles. Mitosome-enriched fractions were obtained from cell homogenate using Optiprep gradient centrifugation. To distinguish mitosomal proteins from contamination, we used a quantitative shot-gun strategy based on isobaric tagging of peptides with iTRAQ and tandem mass spectrometry. Altogether, 638 proteins were identified in mitosome-enriched fractions. Of these, 139 proteins had iTRAQ ratio similar to that of the six known mitosomal markers. Proteins were selected for expression in Giardia to verify their cellular localizations and the mitosomal localization of 20 proteins was confirmed. These proteins include nine components of the FeS cluster assembly machinery, a novel diflavo-protein with NADPH reductase activity, a novel VAMP-associated protein, and a key component of the outer membrane protein translocase. None of the novel mitosomal proteins was predicted by previous genome analyses. The small proteome of the Giardia mitosome reflects the reduction in mitochondrial metabolism, which is limited to the FeS cluster assembly pathway, and a simplicity in the protein import pathway required for organelle biogenesis

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Abstract: Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways

Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Abstract: Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways

Iron containing cofactors in anaerobic parasite Giardia intestinalis

Iron is an essential element in nearly all organisms. It is present mainly as a component of iron sulfur (FeS) clusters or as a heme iron. These cofactors enable proteins to transfer electrons or diatomic gasses, signal sensing and enzyme catalysis. Numerous FeS and heme depending proteins are involved in photosynthesis and respiratory chain pathways, which are well described processes. However, there is still much to learn about more recently discovered pathways such as formation of FeS clusters in various cell compartments and about roles of novel FeS or heme proteins. Particularly, only limited information is available about how FeS clusters are assembled or how heme is used in anaerobic protists, in which cytochrome-dependent respiration and photosynthesis does not occur. We decided to focus on iron cofactors in anaerobic parasite Giardia intestinalis. This organism undergone dramatic reductive evolution that resulted in formation of one of the smallest eukaryotic genome and the most reduced form of mitochondria, the mitosome. We characterized some components of mitochondrial (ISC) and cytoplasmic (CIA) FeS assembly machineries. We have detected ISC components in mitosome by proteomic analysis. Furthermore we investigated the presence and subcellular localization of CIA proteins in Giardia. In..